Protected: Factors Associated with Neonatal Arterial Hypertension: Case and Control Study

Abstract

Background. Neonatal high blood pressure has been diagnosed more frequently in recent years, and its impact extends to adulthood. However, the knowledge gaps on associated factors, diagnosis, and treatment are challenging for medical personnel. The incidence of this condition varies depending on neonatal conditions. Patients in the Newborn Unit are at increased risk of developing high blood pressure. The persistence of this condition beyond the neonatal stage increases the risk of cardiovascular disease and chronic kidney disease in childhood and adulthood.
Methodology. A case-control study was carried out. It included hospitalized patients with neonatal hypertension as cases. Three controls were randomly selected for each case and matched by gestational age. The variables were analyzed based on their nature. Multivariate analysis was performed using a multivariate conditional regression model to identify variables associated with the outcome. Finally, the model was adjusted for possible confounders.
Results. 37 cases were obtained and matched with 111 controls. In the univariate analysis, heart disease (OR 2.86; 95% CI 1.22-6.71), kidney disease (OR 7.24; 95% CI 1.92-28.28), bronchopulmonary dysplasia (OR 6.62; 95% CI 1.42-50.82) and major surgical procedures (OR 3.71; 95% CI 1.64-8.39) had an association with neonatal arterial hypertension. Only the latter maintained this finding in the multivariate analysis (adjusted OR 2.88; 95% CI 1.14-7.30). A significant association of two or more comorbidities with neonatal arterial hypertension was also found (OR 3.81; 95% CI 1.53-9.49).
Conclusions. The study analyzed the factors related to high blood pressure in hospitalized neonates, finding relevant associations in the said population. The importance of meticulous neonatal care and monitoring of risk factors such as birth weight and major surgeries is highlighted.

Keywords: Hypertension, Prematurity, Bronchopulmonary Dysplasia, Epigenetics, Neonate, Prematurity, Kidney Disease, Blood Pressure

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Protected: Peritoneal Dialysis Network in North-East Italy: Survey About the Peritoneal Catheter Exit-Site Infection Management and Comparison with ISPD Guidelines

Abstract

Introduction. The Triveneto Peritoneal Dialysis (PD) Network aims to bring together doctors and nurses who deal with PD in a collaborative network in which to exchange mutual knowledge and optimize the use of this method of replacing renal function. A topic of particular interest was the management of peritoneal catheter exit-site infection, given the recent publication of the new guidelines of the International Society of Peritoneal Dialysis (ISPD).
Materials and methods. The survey concerned the criteria for carrying out nasal swab and exit-site, management of exuberant granulation tissue “Proud Flesh”, treatment of exit-site infection (ESI), use of silver dressings, the role of subcutaneous tunnel ultrasound and cuff shaving.
Results. All PD centers in the North-East Italy area have joined the survey with at least one operator per centre. There was a wide variability between the indications for performing the exit-site swab. In the presence of ESI, the prevalent approach is that of oral systemic empiric therapy associated (20.0%) or less (28.9%) with topical therapy, and then adapting it in a targeted manner to the culture examination.
Discussion. From the discussion of the survey emerged the importance of the ESI as an outcome indicator, which allows us to verify whether our clinical practice is in line with the reference standards. It is essential to know and base our activity on what is indicated in national and international guidelines and to document the events that occur in the patient population of each dialysis unit.

Keywords: Peritoneal Dialysis, Exit-site management, Catheter-related Infections, Survey

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Protected: Executive Dysfunction in Patients Undergoing Chronic Haemodialysis Treatment: A Possible Symptom of Vascular Dementia

Abstract

Introduction. Patients undergoing chronic haemodialysis (HD) treatment have an 8-10 times higher risk of experiencing stroke events and developing cognitive impairment. The high vascular stress they are subjected to may be the basis for the development of vascular dementia (VaD).
Objective. The aim of the study is to investigate the executive functions, typically impaired in VaD, of patients undergoing chronic haemodialysis treatment.
Method. HD patients were recruited from the U.O.C. of Nephrology and Dialysis (ASP Ragusa). Risk factors for VaD were collected and then the Frontal Assessment Battery (FAB) was administered.
Results. 103 HD patients were included (males = 63%, age 66 ± 14 years). Risk factors for VaD included a high percentage of patients with anaemia (93%), hypertension (64%) and coronary artery disease (68%).  The cognitive data obtained via FAB show a percentage of 55% deficit scores. All risk factors found a significant association with cognitive scores. Anemia, hypertension, intradialytic hypotension, coronary artery disease, and homocysteine are negative predictors of executive function integrity.
Conclusions. More than half of the patients had deficit scores on the FAB. Reduced cognitive flexibility, high sensitivity to interference, poor inhibitory control and impaired motor programming with the dominant hand were evident. In conclusion, a marked impairment of the executive functions, generally located in the frontal lobes of the brain, was detected in the HD patient, which could be a symptom of a dementia of a vascular nature.

Keywords: hemodialysis, cognitive, impairment, vascular, dementia

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Protected: Clinical implications of serum anti-PLA2R levels and glomerular PLA2R deposits in primary membranous nephropathy

Abstract

Introduction. The clinical implications of serum anti-PLA2R with glomerular PLA2R deposits in primary membranous nephropathy (PMN) is scarcely reported. Hence the study was designed to demonstrate the prevalence of serum anti-PLA2R levels and PLA2R staining in glomeruli in PMN and the clinical implications of the two parameters.
Objectives.

  1. Investigate the prevalence of anti PLA2R positivity in PMN.
  2. Ascertain correlation between serum anti-PLA2R levels and glomerular staining for PLA2R with clinical and lab parameters in PMN.

Patients and Methods. Fifty PMN patients during the period from October 2017 to December 2018 were included. Labs were done and eGFR was calculated as per MDRD 6. Anti-PLA2R titres were done in all patients. Titres more than 20 RU/ml were considered positive. Glomerular staining for PLA2R was graded on fresh frozen tissue by immunofluorescence technique.
Results. Anti-PLA2R antibody positivity and glomerular PLA2R deposition was observed in 42% (21/50) and 86% (43/50) patients respectively. 79.3% (23/29) had positive glomerular PLA2R deposition with negative serum anti PLA2R. Positive correlation were observed between serum PLA2R antibody and serum creatinine (p = 0.0001) and urine protein-creatinine ratio levels with tissue PLA2R staining grades (p = 0.04). Negative association was found between serum albumin (p = 0.026) and tissue PLA2R staining grades.
Conclusion. Serum anti-PLA2R wasn’t a sensitive marker of primary membranous nephropathy in our study group emphasising the need to consider a compendium of serological markers for diagnosis of primary membranous nephropathy and to rely more on glomerular deposition of PLA2R as a better clinical indicator for PMN.

Keywords: anti-PLA2R, Membranous Nephropathy, Glomerular PLA2R deposits, Tissue PLA2R staining, Nephrotic Syndrome

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Advanced Care Planning (ACP) and Hemodialysis: a Pilot Project for the Application of Italian Law 219/2017 in Dialysis Units

Abstract

The law 219/2017 is the first Italian law about advanced care planning (ACP). ACP is an important part of the therapeutic relationship between patients and doctors: thanks to ACP patients can think and discuss about end of life decisions, considering clinical aspects, but also psychological, cultural, social and ethical issues. Patients prepare themselves in advance because of the possibility of future cognitive impairment, can identify a surrogate decision maker and make end-life decisions according to their goals and values.

End-stage kidney disease (ESRD) is often characterized by important symptoms, psychological suffering and social disadvantage, and patients affected by ESRD often have slow physical and cognitive decline. Despite this, access to palliative care is reduced for these patients as compared to patients affected by other end-stage organ failures. This is the reason why we want to explore the possibility of applying APC to ESRD patients.

This pilot study, regarding three patients from the Dialysis Unit of ASST Crema in Italy, has been conducted to verify the applicability of the law 219/2017 in Dialysis Units. It shows that we have to deeply investigate this issue from both sanitary workers’ and patients’ and families’ points of view. We need more studies with a larger number of patients and a longer period of follow-up, but we also need to teach sanitary workers how to approach APC and to teach people what APC is and why it’s so important for everyone.

Keywords: advanced care planning, end-stage kidney disease, dialysis

Sorry, this entry is only available in Italian.

Introduzione

Che cos’è la pianificazione condivisa delle cure (PCC)?

Per “pianificazione condivisa delle cure” si intende un processo che si svolge all’interno di una relazione di fiducia tra il paziente e il personale sanitario, in cui si illustrano al paziente e alle persone a lui vicine la situazione attuale di malattia, le possibilità di cura e la prognosi e si riflette in anticipo in merito alle decisioni relative al fine vita tenendo inevitabilmente conto, oltre che degli aspetti clinici, di quelli psicologici e della dimensione culturale, sociale, spirituale ed etica del paziente [1].

Nel corso di questo processo il paziente si prepara alla propria eventuale, futura, incapacità di autodeterminarsi (e quindi di acconsentire o meno alle cure proposte), può identificare un fiduciario ed esplicita ai curanti le proprie indicazioni per le fasi di incapacità e/o per il fine vita in linea con i propri valori ed i propri obbiettivi [2].

In sintesi, la PCC permette al paziente di esprimere che cosa significhi per lui vivere e morire bene [2, 3]. 

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Apheresis Techniques for the Treatment of Hyperbilirubinemia in the Nephrology Unit

Abstract

Therapeutic apheresis is an important hematological and nephrological method for conditions with altered plasma composition. It is also indicated for the removal of protein-bound molecules, such as bilirubin. Several techniques can remove these compounds, such as the extracorporeal circulation molecular adsorption system (MARS), plasma exchange (PEX), and plasma adsorption and perfusion (PAP). Here we report our experience in the comparison between MARS, PEX and PAP, since current guidelines do not specify which method is the most appropriate and under which circumstances it should be used.

The choice of technique cannot be based on the desired plasma bilirubin concentration, since these three techniques show similar results with a similar final outcome (exitus). In fact, PAP, PEX and MARS significantly reduce bilirubin levels, but the degree of reduction is not different among the three. Furthermore, the three techniques do not differ in the rate of cholinesterase change, while less reduction of liver transaminases was found by using PAP.

MARS should be preferred in the case of renal involvement (hepatorenal syndrome with hyperbilirubinemia). PAP has the advantage of being simple and inexpensive. PEX remains an option when emergency PAP is not available, but the risk of using blood products (plasma and albumin) must be considered.

Keywords: Molecular Adsorbent Recirculating System, MARS, Plasma Adsorption Perfusion, plasma exchange

Sorry, this entry is only available in Italian.

Introduzione: inquadramento storico

La gialla discolorazione della pelle e occhi, lo yearkon ebraico, è una dei mali che la Bibbia riporta qualora non si ascolti la voce di Dio (Deuteronomio 28, 21). Già nel 500 a.C. se ne riconosce la causa nella ostruzione o costipazione del fegato, come riportato dal medico bizantino Alessandro di Tralles, e, infatti, la bile gialla è uno dei quattro umori identificati da Ippocrate di Cos (morto nel 377 a.C.).

Verso la fine del 1800 lo sviluppo di tecniche biochimiche applicate alla clinica porta alla identificazione della bilirubina (fra gli altri anche Virchow diede importanti contributi).

Nel 1933 Zimmermann e Yannet scoprono che il Kernicterus (una degenerazione dei gangli della base in corso di ittero, descritta da Christian Georg Schmorl nel 1904 e prima da Johannes Orth nel 1875) è causato dalla deposizione di bilirubina a livello cerebrale [1].

La bilirubina è lipofila, può attraversare la barriera emato-cerebrale. Gerard Odell nel 1959 [2] e poi il celebre Gilbert nel 1973 [3] dimostrano allora che l’albumina plasmatica riduce i livelli plasmatici di bilirubina grazie a un potente legame fra i due.

Nonostante la coeva introduzione della emodialisi, queste tecniche non si presentano come utili a rimuovere la bilirubina, proprio a causa del forte legame con la albumina, e solo la dialisi peritoneale presenta di una qualche utilità al proposito nei lontani anni ’60 [4].

Nei primi anni ’50 un biochimico dell’Università di Harvard (Boston), Edwin Cohn, usando un pool di plasma umano, sviluppò una procedura su larga scala per la purificazione dell’albumina come alternativa al plasma liofilizzato per i soldati feriti. La “centrifuga di Cohn” fu, di fatto, un antenato della plasmaferesi [5]. 

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The Outpatient Activity of the Onconephrology Clinic of Cremona in the First Semester of 2023

Abstract

Despite the rapidly growing area of onconephrology in the last decade, nephropathic patients have been rarely involved in clinical trials of cancer therapy, particularly in the case of chronic kidney disease (CKD) stage 4 (CKD4) or stage 5 (CKD5). We could offer better therapeutic opportunities to our patients thanks to the Onconephrology Clinic and the Multidisciplinary group, in which a dedicated team of specialists guarantees the highest level of possible care. In this paper, we analysed the activity of the first Italian OnconephrologyClinic, twelve years after its foundation. We studied retrospectively a cohort of 174 patients referred to our center in the last six months (from 11/01/2023 to 12/07/2023), with a total of 262 visits (40 first visits). We highlight a prevalence of moderated or advanced kidney disease, in contrast with the literature, which is probably the result of a transversal II level clinic with different specialists involved. Furthermore, in patients with a prolonged follow-up, we observed a progressive better attention to every kidney involvement, particularly in patients in active cancer therapy, by the oncologist colleagues. We observed a reduction of treatment withdrawals due to kidney toxicity, thanks to a multidisciplinary approach and experienced-based management. On the other side, we highlight also a delayed addressing of patients with acute kidney injury (AKI), which often results in chronic kidney damage. This could be related to a delayed identification of the reduced renal function, which is difficult to correctly value in patients with cancer.

Keywords: onconephrology, cancer therapy, nephrectomy, kidney function

Sorry, this entry is only available in Italian.

Introduzione

L’onconefrologia, come testimoniato dal crescente numero di articoli pubblicati negli ultimi dieci anni e dal fiorire di congressi dedicati, nonché dalla nascita di ambulatori a impronta multidisciplinare in molte regioni italiane, è una branca super-specialistica che suscita sempre più interesse nel mondo nefrologico. La ricerca in ambito oncologico genera nuovi farmaci con una rapidità a cui il nefrologo non è uso: nell’ultimo decennio, dopo le terapie a bersaglio molecolare, sono stati introdotti nuovi antimetaboliti, gli inibitori dei chekpoint immunitari, i farmaci coniugati, nonché protocolli di combinazione spesso comprendenti platini e/o molecole a eliminazione o tossicità renale. Per il nefrologo che si accosta a questo ambito, l’aggiornamento continuo sul panorama farmacologico non è sufficiente. Infatti, più del 70% degli studi registrativi, a partire dalla fase II, non comprendono pazienti affetti da insufficienza renale cronica, specie se moderato-severa [1, 2] nonostante le linee guida dell’EMA [3] circa il disegno degli studi di farmacocinetica; inoltre, la letteratura offre pochi studi di farmacocinetica in fase di post marketing. A complicare le cose, non vi è uniformità negli strumenti usati negli studi registrativi né per quanto riguarda i criteri “renali” di esclusione, né per la valutazione della funzione renale. Per questi motivi, la mancanza di un nefrologo dedicato frequentemente preclude a questo gruppo di pazienti possibilità terapeutiche che non dovrebbero essergli negate, vista sia l’elevata incidenza di tumori nella popolazione con CKD che l’eccesso di mortalità per neoplasia dei pazienti nefropatici sia in terapia conservativa [4, 5] che in trattamento dialitico sostitutivo [6] rispetto alla popolazione generale. 

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Correlation of Beta Trace Protein Levels with Serum Creatinine-Based Estimated Glomerular Filtration Rate Equations in Chronic Kidney Disease

Abstract

Background. Estimated GFR (eGFR) is calculated using serum creatinine (SCr) based equations which have their own limitations. Novel biomarkers like beta trace protein (BTP) are studied for eGFR estimation. The aim of this study is to determine the serum levels of BTP in healthy controls and chronic kidney disease (CKD) cases and to find out the correlation of BTP levels with that of SCr and SCr-based eGFR formulas.
Methods. The control group comprised of 20 healthy adults. The cases comprised of 20 patients each in CKD stages 3, 4, and 5, categorized based on eGFR calculated using MDRD formula. Baseline characteristics of the study population were recorded. BTP was measured by ELISA (Enzyme Linked Immunosorbent Assay) method and SCr by modified Jaffe’s method. The statistical analyses were performed with the SPSS for Windows, version 16.0.
Results. The median value of blood urea nitrogen (BUN) in the cases was 26.50 mg/dL (IQR 19.25-37) and for control it was 9.5 mg/dL (IQR 8-12). The median value of SCr in the cases was 2.75 mg/dL (IQR 1.725-4.45) and in the controls, it was 0.7mg/dL (IQR 0.6 -0.8). The median value of BTP in cases was 6389.25 ng/ml (IQR 5610.875-10713.75) and in controls, it was 1089.5 ng/ml (IQR 900.5-1309.75).
Conclusion. Serum BTP levels correlated with SCr levels and renal function. We could establish the relationship between the two biomarkers, SCr and BTP, and derive a regression equation.

Keywords: Beta trace protein, estimated GFR, CKD, correlation

Introduction

Any structural or functional abnormalities of the kidneys that persist beyond 3 months irrespective of the etiology is defined as chronic kidney disease (CKD) [1]. In clinical practice, renal function is assessed indirectly using serum creatinine (SCr) values and SCr-based glomerular filtration rate (GFR) equations. The ‘gold standard’ for GFR estimation, the inulin clearance cannot be used routinely, because of the difficult and cumbersome nature of the procedure. Serum cystatin C based equations have also been recently developed [2, 3]. Notable equations are Cockroft-Gault formula, Chronic Kidney Disease Epidemiology (CKD-EPI) equation and Modification of Diet in Renal Disease Study (MDRD) equation [46]. However, each equation has its own inherent limitations such as a lack of accuracy, precision, and variations with ethnicity [78].

Factors such as protein intake and muscle mass determine SCr levels. Due to lower muscle mass, SCr levels are lower in women and in older individuals. SCr level has a curvilinear relationship with GFR because of its tubular secretion in mild-to-moderate degrees of renal failure. A SCr value of 1.5 mg/dl could represent a GFR of anywhere between 30-90 ml/min. Such wide range of eGFR is not acceptable and hence, SCr-based assays are not considered ideal. The need for better biomarkers need not be overemphasized.

Beta Trace Protein (BTP) is a monomeric low-molecular-weight glycoprotein with 168 amino acids. The molecular mass of BTP is estimated to be between 23000 and 29000 daltons. It depends on N-glycosylation at three positions in its structure. BTP mRNA is expressed in the choroid plexus, pachymeninges, and oligodendrocytes. BTP synthesis occurs in testes, epididymis, and heart in small quantities. The elimination of BTP is by glomerular filtration. It is reabsorbed by the proximal tubular cells and actively degraded within the lysosomes. The t- 1/2 of BTP is about 1.2 hours [912].

Hence, BTP-based GFR equations could mitigate the limitations of SCr-based equations for GFR estimation. No studies on correlation of serum BTP levels with SCr-based eGFR are available yet from South Asian population.

 

Methods

This study was done as a cross-sectional study at Sri Ramachandra Institute of Higher Education and Research (SRIHER), after obtaining Institutional ethics committee clearance (Ethics Reg No: CSPMED/19/NOV/57/170). Informed consent in written form was obtained from all study participants prior to this study.
The control group was comprised of 20 healthy male and female adults. The ‘cases’ group comprised of 20 patients each in CKD stages 3, 4, and 5. The CKD stages were categorized based on eGFR calculated using MDRD formula. Baseline characteristics of the cases and controls such as age, gender, body mass index (BMI), and blood pressure were recorded. Based on the SCr levels, eGFR was estimated for both the cases and controls using existing formulae, MDRD and CKD-EPI. Blood pressure was measured using aneroid sphygmomanometer.
Renal failure patients of > 18 years of age in CKD stages 3, 4 and 5, who visited nephrology outpatient department were recruited for this study. Controls were healthy males and females who had attended master health check-up facility in the hospital. The study excluded paediatric and adolescent population, pregnant women and transplant recipients. A venous blood sampling was carried out by trained phlebotomist.
The quantitative estimation of urea was determined by Kinetic UV / Urease – GLDH method. The quantitative estimation of SCr was determined by modified Jaffe’s method. Both the assays were done in Beckman Coulter AU 5800 and AU 680 Automated Clinical Chemistry Analyzers. BTP was assayed by immunometric (sandwich) ELISA from Cayman chemicals – Prostaglandin D synthase lipocalin type human ELISA kit, Item no: 10007684.

Statistical analyses

The statistical analyses were performed with the Statistical Package for the Social Sciences (SPSS) statistical software package for Windows, version 16.0. Shapiro-Wilks test was used as test for normality. Mean and standard deviation were calculated for the age and BMI. Independent sample-t test was performed and a two-tailed ‘p’ value of < 0.05 was considered statistically significant. For parameters such as BUN, SCr, MDRD equation based eGFR, CKD-EPI equation based eGFR, serum BTP levels, systolic and diastolic BP that followed non-normal distribution, median and interquartile range (IQR) were calculated. Mann-Whitney U test and Kruskal-Wallis test were used for parameters of non-normal distribution and ‘p’ value of < 0.05 was considered statistically significant. One way ANOVA was performed for parameters of normal distribution and ‘p’ value of < 0.05 was considered statistically significant. Correlation analysis between BTP and BUN, Creatinine, MDRD eGFR, CKD-EPI eGFR, SBP and DBP was done by Spearman correlation in the study cohort and ‘p’ value of < 0.001 was considered statistically significant.

 

Results

This study cohort (n=80) included 48 males (60%) and 32 females (40%). The mean age of controls was 40.3±10.702 years and that of CKD cases was 55.53±15.285 years (p <0.05). The mean BMI of controls was 25.150±3.618 kg/m2 and that of cases was 26.88±5.297 kg/m2 (p 0.407). The median and IQR for systolic blood pressure (SBP) in the cases was 140 mm Hg (IQR 130 -140) and in the controls was 110 mm Hg (IQR 100-110). The median diastolic blood pressure (DBP) in the cases was 80 mm Hg (IQR 80-90), and in the controls it was 70 mm Hg (IQR 70-80). The blood pressure was significantly higher in all stages of CKD when compared to the control group (p <0.001).
The median value and IQR of BUN in the cases was 26.50 mg/dL (19.25-37) and in the controls was 9.5 mg/dL (8-12). The median value of SCr in the cases was 2.75 mg/dL with IQR (1.725-4.45), and in the controls it was 0.7 mg/ dL (IQR 0.6 – 0.8). The median value of eGFR using MDRD equation for cases was 23.950 ml/ min/1.73m2 (IQR 12.825-37.5), and for controls it was 105.6 ml/ min/1.73 m2 (IQR 100.8-115.875). Median value of GFR estimated using CKD-EPI for cases was 23.5 ml/min/1.73m2 (IQR 12-38.5) and for controls, it was 112.5 ml/min/1.73m2 (106.25-116.5) (Table I). BUN and SCr were significantly increased in the cases than in controls, as expected (p < 0.001). MDRD eGFR and CKD-EPI eGFR were significantly decreased in the cases than in controls (p <0.001).

Parameter CKD (n=60) Control (n=20) p-value

BUN

(mg/dL)

26.50

(19.25-37)

9.5

(8-12)

<0.001**

Creatinine

(mg/dL)

2.75

(1.725-4.45)

0.7

(0.6-0.8)

<0.001**

MDRD eGFR

(ml/min/1.73m2)

23.950

(12.825-37.5)

105.6

(100.8-115.875)

<0.001**

CKD-EPI eGFR

(ml/min/1.73m2)

23.5

(12-38.5)

112.5

(106.25-116.5)

<0.001**
Table I. Comparison of BUN, Creatinine, MDRD eGFR, and CKDEPI eGFR between chronic kidney disease patients (CKD group) and control group (normal healthy individuals).
Data represented as median with interquartile range. Comparison done via Mann-Whitney U test. *Statistically significant (p<0.05) * *Statistically significant (p<0.001)

The median value and IQR of BTP in ‘cases’ were 6389.25 ng/ml (5610.875-10713.75), and in controls it was 1089.5 ng/ml (900.5-1309.75). Serum BTP levels were significantly increased in all CKD stages when compared with the control group (p <0.001) (Table II).

Parameter CKD (n=60) Control (n=20) p-value

BTP

(ng/ml)

6389.25

(5610.875-10713.75)

1089.5

(900.5-1309.75)

<0.001**
Table II. Comparison of Beta trace protein (BTP) between chronic kidney disease patients (CKD group) and control group (normal healthy individuals).
Data represented as Median with interquartile range. Comparison done via Mann Whitney U test. *Statistically significant (p<0.05) * *Statistically significant (p<0.001)

The parameters were compared among groups CKD 3, CKD 4, CKD 5, and control group. Normal distribution was seen for parameters BMI, BTP, MDRD eGFR and CKD-EPI eGFR. Non-normal distribution was seen for parameters age, BUN, SCr, SBP and DBP. The median and IQR of BUN in CKD Stage 3 group was 19.5 mg/dl (15.25-22.5), in CKD Stage 4 group was 26 mg/dL (19.5-32), in CKD Stage 5 group was 38.5 mg/dL (34.25 -45) and in control was 9.5 mg/dL (8-12). The values are statistically significant, (p < 0.001) as calculated by Kruskal-Wallis test. BUN levels were significantly higher in all stages of CKD when compared with the control group. There was significant difference between stage 3 and stage 5 CKD cohorts, but significance was not demonstrated between stage 3 and stage 4, and stage 4 and stage 5 groups. The median and IQR of SCr in CKD stage 3 group was 1.6 mg/dL (1.4-1.775), in CKD stage 4 group was 2.75 mg/dL (2.43-3.15), in CKD stage 5 group was 5.2 mg/dL (4.35 – 5.775), and in control group it was 0.7 mg/dL (0.6-0.8). The values are statistically significant, (p < 0.001) as calculated by Kruskal-Wallis test. SCr levels were significantly higher in all stages of CKD when compared with the control group. There was significant difference in SCr levels between CKD stage 3 and 5 and also between CKD stage 4 and 5. There was no significance seen between stage 3 and stage 4 CKD cohorts.
The mean BMI of CKD stage 3 group was 27.24 ± 5.13 kg/m2, CKD stage 4 group was 27.63 ± 6.32 kg/m2, CKD stage 5 group was 25.77 ± 4.33 kg/m2, and in control group it was 25.82 ± 3.62 kg/m2. There was no statistical significance among the groups for BMI. The mean MDRD eGFR of CKD stage 3 group was 42.86 ± 7.42 ml/ min/1.73m2, CKD stage 4 group was 22.84 ± 4.37 ml/ min/ 1.73m2, CKD stage 5 group was 10.41 ± 2.63 ml/ min/1.73m2, and in control group it was 107.25 ± 8.88 ml/min/1.73m2. The mean CKD-EPI eGFR of CKD stage 3 group was 45.8 ± 9.73 ml/min/1.73m2, CKD stage 4 group was 23.75 ± 4.62 ml/ min/1.73m2, CKD stage 5 group was 10.3 ± 2.56 ml/min/1.73m2, and in control group it was 112.35 ± 8.29 ml/min/1.73m2. There was statistical significance among the groups for eGFR by MDRD and CKD-EPI equations. The mean BTP levels of CKD stage 3 was 5222.33 ± 900.15 ng/ml, CKD stage 4 was 6234.23 ±575.31 ng/ml, CKD stage 5 was 11628.3 ± 1695.39 ng/ml, and in control group was 1130.35 ± 314.42 ng/ml. BTP levels were significantly higher in the CKD groups when compared to the control group. BTP levels were significantly higher in CKD stage 5 when compared to CKD stage 3 and CKD stage 4. BTP levels were significantly higher in CKD stage 4 when compared to CKD stage 3 (Table III). 

Parameter CKD3 (n=20) CKD4 (n=20) CKD 5 (n=20) Control

(n=20)

p-value

BMI

(Kg/m2)

27.24

± 5.13

27.63

± 6.32

25.77

± 4.33

25.82

± 3.62

0.522

MDRD eGFR

(ml/min/1.73m2)

42.86

± 7.42

22.84

± 4.37

10.41

± 2.63

107.25

± 8.88

<0.001**

CKD-EPI eGFR

(ml/min/1.73m2)

45.8

± 9.73

23.75

± 4.62

10.3

± 2.56

112.35

± 8.29

<0.001**

BTP

(ng/ml)

5222.33 ± 900.15 6234.23

± 575.31

11628.3

± 1695.39

1130.35 ± 314.42 <0.001**
Table III. Comparison of BMI, MDRD eGFR, CKD-EPI eGFR and BTP among the groups CKD stage 3, CKD stage 4, CKD stage 5 and control group. Data represented as Mean ± standard deviation (SD). Comparison done by One way ANOVA test. *Statistically significant (p<0.05) * *Statistically significant (p<0.001).

The correlation analysis between BTP and BUN, SCr, MDRD eGFR and CKD-EPI eGFR was done by Spearman correlation in the study cohort (n=80) (Table IV). In the correlation analysis between BTP and BUN, the correlation coefficient (R-value) was 0.835, implying a positive correlation between the two parameters. The correlation was statistically significant (p <0.001). The correlation coefficient (R-value) for correlation analysis between BTP and SCr was 0.917 with p<0.001, implying a statistically significant positive correlation between these two parameters. In the correlation analysis between BTP and eGFR based on MDRD equation, the correlation coefficient (R-value) was –0.913 (p<0.001) and R-value for BTP and eGFR based on CKD-EPI equation was –0.909 (p<0.001). This showed a statistically significant negative correlation between eGFR based on MDRD equation and eGFR based on CKD-EPI equation with BTP.

Regression Equation

Using SCr values and BTP levels, we could establish the relationship between the two biomarkers and derive a regression equation.

Serum creatinine = 0.014 +0.422 × BTP / 1000

BTP scores over serum creatinine values in CKD stages 3, 4, and 5. BTP detects CKD (stage 3 and above) above the cut-off of 2818.5 ng/ml when compared to SCr (Figure 1).

Figure 1. Linear regression analysis between serum creatinine and BTP.
Figure 1. Linear regression analysis between serum creatinine and BTP.Fig

Discussion

Low-molecular-weight-proteins such as BTP and beta-2 microglobulin (B2M) are recently studied as new endogenous markers for GFR estimation [912]. BTP has been investigated for assessment of GFR in adult and paediatric population.

Pöge et al. developed eGFR equations from 85 adult kidney transplant recipients with mean age of 50 years and mean GFR 39 ml/min/1.73 m2. They developed two equations, which required SCr and blood urea levels in addition to BTP levels [13].

                   GFR1 = 89.85 × BTP-0.5541 × urea-0.3018

                   GFR2 = 974.31× BTP-0.2594 × serum creatinine-0.647

In another similar study, White et al. developed two GFR estimation equations from 163 adult kidney transplant recipients with mean age of 53±12 years and mean GFR of 59±23 ml/min/1.73 m2. Male population constituted 67% and whites 90% in this study cohort. They included a correction factor for females in their equation [14].

                   GFR1 = 112.1 × BTP-0.662× urea-0.280 × (0.880 if female)

                   GFR2 = 167.8 × BTP-0.758 × serum creatinine-0.204 × (0.871 if female)

Bhavsar et al. studied the role of BTP for predicting hypertensive CKD progression to ESRD in African Americans. They compared BTP levels and cystatin C with measured GFR using iothalamate clearance. 246 individuals reached ESRD and at the end of follow-up period of 102 months, it was demonstrated that BTP levels fared better in predicting ESRD when compared to other markers [15]. In a similar study by Katharina-Susanne Spanaus et al. in primary nondiabetic CKD, the investigators demonstrated that the diagnostic performance of all three biomarkers ‒ SCr, serum cystatin C, and serum BTP levels ‒ for detecting even minor degrees of deterioration of renal function was good. The study was done in 177 patients with 7 years follow-up. All 3 markers provided similar risk prediction for progression to CKD [16]. Foster et al. demonstrated that the use of multiple biomarkers improves risk prediction in individuals with moderate CKD. Serum levels of BTP and B2M might contribute in predicting additional risk information beyond conventional eGFR equations. The study was prospective cohort study, which included 3,613 adults from the Chronic Renal Insufficiency Cohort (CRIC) study. The mean age of the study cohort was 57.9 years. Females constituted 45%, diabetics 51.9% and non-Hispanic Blacks 41% of the study cohort [17].
Clinical investigations did not establish the superiority of BTP over conventional markers consistently. Natalie Ebert et al. in their study in 566 elderly (more than 70 years of age) participants concluded that BTP did not outperform serum creatinine and cystatin C levels in estimating GFR [18]. Karin Werner et al. demonstrated the superiority of GFR estimation using combined SCr and serum cystatin C levels over GFR estimation done using BTP and B2M levels. They concluded this from their validation study in 126 elderly participants aged between 72 years and 98 years with a mean measured GFR (mGFR) of 54 ml/min/1.73 m2. The mGFR was done using iohexol clearance [19].
Studies similar to those carried out in adults using BTP for GFR estimation were conducted in children by Abbinkwt al., Benlarmi et al., and Witzel et al. [2022]. It is to be noted that none of these BTP based equations have been validated in a large sample size for GFR estimation. Hence, clinicians still rely on SCr and SCr-based eGFR equations.
Measurement of BTP is done by nephelometric method or by ELISA [23]. Published studies on BTP have used mostly nephelometry-based assays. Bhavsar et al. in their study on the role of BTP for predicting hypertensive CKD progression used nephelometric assay for BTP estimation. [15] Nephelometric BTP estimation was also used by Katharina Susanne Spanaus et al. in primary nondiabetic CKD, where the investigators demonstrated that the diagnostic performance of all three biomarkers ‒ SCr, serum cystatin C, or serum BTP ‒ for detecting even minor degrees of deterioration of renal function was good [16]. The same nephelometric BTP assay was used by Lesley A. Inker and his colleagues when estimating GFR using BTP and B2M in CKD and dialysis population [24]. Natalie Ebert et al. and Karin Werner et al. used the same techniques for BTP estimation in their studies [18]. In our study, BTP levels were estimated using ELISA.
There was a significant positive correlation between BUN and SCr with BTP levels. Hebaha et al. have also reported a positive correlation among these parameters in their prospective cohort study comprising 40 Type II Diabetes mellitus patients and 10 controls [22]. Significant negative correlation between MDRD eGFR and CKD-EPI eGFR with BTP was seen in our study (Table IV).

Parameter Correlation coefficient (R-value) with BTP p-value
BUN 0.835 <0.001**
Creatinine 0.917 <0.001**
MDRD eGFR -0.913 <0.001**
CKD-EPI eGFR -0.909 <0.001**
Table IV. Correlation analysis between Beta trace protein (BTP) and BUN, Creatinine, MDRD eGFR and CKD-EPI eGFR. Spearman correlation; Correlation coefficient expressed as R-value. *Statistically significant (p<0.05) * *Statistically significant (p<0.001)

Although several studies on GFR estimation using serum BTP levels had been done in the West, no studies are available from South Asia. Even studies demonstrating the correlation between SCr and SCr-based eGFR with serum BTP levels are not yet available. This might be due to the fact that ‘normal’ values of BTP are not yet arrived and the values obtained from the studies have not been validated in a larger population to be accepted universally.
Our study has several advantages. To our knowledge, this is the first study of BTP level estimation using ELISA method in an Indian population. In our study, BTP levels correlated well with SCr levels and SCr-based eGFR formulas in CKD. We could derive a regression equation using SCr and BTP levels. Hence, serum BTP levels may be a useful and reliable biomarker for identifying the magnitude of renal dysfunction in CKD patients.
The main limitation of our study is that it is a single centre study and study population were only from Indian ethnicity. BTP levels were assayed by ELISA method only. BTP estimation using nephelometry in addition to ELISA technique would have added more value to the study.
Having demonstrated the correlation of BTP with SCr and SCr-based eGFR formulas, we recommend validating this study findings in a larger cohort. Development of an eGFR formula using BTP levels with or without SCr, cystatin C and B2M could be attempted. BTP can also be studied as a biomarker for adverse clinical outcomes including cardiovascular diseases in early stages of CKD [25]. Urinary BTP levels and its correlation with early GFR impairment in cases of acute kidney injury (AKI) and CKD could be explored in the future [26].

 

Conclusion

Serum BTP levels, estimated by ELISA correlates with SCr levels and renal function. As the renal function deteriorates in advanced stages of CKD, there is corresponding increase in serum BTP levels. A relationship between the two biomarkers, SCr and BTP was established and a regression equation was derived. BTP detects CKD (stage 3 and above) above the cut-off of 2818.5 ng/ml when compared to SCr. Serum BTP levels could be superior to SCr levels in diagnosing CKD in stages 3 to 5. Serum BTP is a potential biomarker for GFR estimation and could overcome the limitations of SCr-based eGFR equations.

 

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New Therapeutic Strategies in the Treatment of CKD Anemia: Hypoxia-Induced Factor Prolyl-Hydroxylase Inhibitors

Abstract

The link between chronic renal failure and anemia has been known for more than 180 years, negatively impacting the quality of life, cardiovascular risk, mortality, and morbidity of patients with chronic kidney disease (CKD). Traditionally, the management of anemia in CKD has been based on the use of replacement martial therapy, vitamin therapy, and the use of erythropoiesis-stimulating agents (ESAs). In recent years, alongside these consolidated therapies, new molecules known as hypoxia-induced factor prolyl-hydroxylase inhibitors (HIF-PHIs) have appeared. The mechanism of action is expressed through an increased transcriptional activity of the HIF gene with increased erythropoietin production. The drugs currently produced are roxadustat, daprodustat, vadadustat, molidustat, desidustat, and enarodustat; among these only roxadustat is currently approved and usable in Italy. The possibility of oral intake, pleiotropic activity on martial and lipidic metabolism, and the non-inferiority compared to erythropoietins make these drugs a valid alternative to the treatment of anemia associated with chronic kidney disease in the nephrologist practice.

Keywords: CKD, Anemia, Erythropoietin, HIF, Roxadustat

Sorry, this entry is only available in Italian.

Introduzione

Il link che intercorre tra malattia renale cronica (MRC) e anemia è ormai noto da più di 180 anni [1]. La Kidney Disease: Improving Global Outcomes (KDIGO) definisce l’anemia come la presenza di valori di emoglobina sierica (Hb) < 13,0 g/dl per gli uomini e < 12,0 g/dl per le donne non in stato di gravidanza.

Ad oggi è ormai consolidato come al progredire della malattia renale incrementi anche la prevalenza di anemia, condizione che impatta negativamente sulla qualità di vita, sul rischio cardiovascolare, sulla mortalità e sulla morbidità di pazienti già con rischio aumentato per tutte queste condizioni [24].

L’anemia associata a malattia renale cronica è determinata da differenti meccanismi patogenetici. Oltre alla diminuita capacità del rene di produrre l’eritropoietina (EPO), si è visto come l’attività delle tossine uremiche sia capace di inibire i meccanismi deputati all’eritropoiesi e diminuire la sopravvivenza degli eritrociti. Accanto a questi meccanismi si aggiungono le alterazioni del metabolismo marziale, dove l’eccesso di epcidina risulta il principale attore impattando negativamente sull’assorbimento dietetico del ferro e sulla mobilizzazione dei suoi depositi corporei [5].

Tradizionalmente la gestione dell’anemia in corso di MRC si è basata sull’uso di terapia marziale di rimpiazzo e sull’uso di agenti stimolanti l’eritropoiesi (erythropoiesis-stimulating agents, ESAs) [68]. È proprio da questi ultimi agenti che, a partire dagli anni ’80, i pazienti hanno avuto il maggior beneficio tramite una riduzione dei sintomi relativi all’astenia e la liberazione dalla dipendenza di emotrasfusioni e delle correlate complicanze, tra cui sovraccarico marziale, infezioni e sensibilizzazioni che potenzialmente inficiavano le possibilità di trapianto. Secondo le principali linee guida, in pazienti con MRC, la presenza di valori di emoglobina compresi tra 9-10 g/dl necessita di correzione mediante la somministrazione di eritropoietine, fino al raggiungimento i valori compresi tra 11-12 g/dl, con una personalizzazione della terapia a seconda dei casi [2].

Se da un lato i benefici derivanti da tali terapie sono molteplici, bisogna considerare la presenza di effetti avversi. I pazienti sottoposti a terapia con ESAs presentano un aumentato rischio di ipertensione, convulsioni e coagulazione dell’accesso vascolare per emodialisi [9, 10]. Inoltre, gli ESAs non si sono dimostrati efficaci nel ridurre gli aventi avversi correlati all’anemia (mortalità, eventi cardiovascolari non fatali, ipertrofia ventricolare sinistra e progressione della malattia renale) in studi prospettici controllati e randomizzati [1113].
Recenti studi in pazienti affetti da CKD in emodialisi e pre-dialisi dimostrano un aumento del rischio di morte, di eventi avversi cardiovascolari ed ictus [14, 15]. Infine, questi agenti sono stati associati a progressione di malattie maligne e a morte in pazienti affetti da neoplasia [16].

La terapia marziale si colloca accanto alla terapia con ESAs nel trattamento dell’anemia secondaria a CKD. Il ferro è fondamentale non solo per implementare i depositi, ma anche per rendere più efficace l’azione degli ESAs [17, 18].

La progressiva riduzione di efficacia degli ESAs parallelamente alle preoccupazioni relative ai potenziali eventi avversi di questi farmaci, hanno progressivamente portato allo sviluppo di nuovi farmaci che presentassero una migliore sicurezza generale e cardiovascolare, superando l’iporeattività degli ESAs associata all’infiammazione.

Tra i nuovi approcci terapeutici compaiono gli inibitori del dominio prolil idrossilasi del fattore inducibile da ipossia (HIF-PHIs).

 

Meccanismo molecolare e farmacodinamica

I fattori indotti dall’ipossia (Hypoxia-inducible factors ‒ HIFs) sono dei fattori di trascrizione regolati dalla quantità di ossigeno presente nell’ambiente cellulare. Il fattore HIF fu originariamente identificato nel 1991 da Semenza e collaboratori [19].

HIF è costituito da una subunità α sensibile all’ossigeno e da una subunità β, formando una struttura etero dimerica [20].

In condizioni di normo ossigenazione cellulare, la subunità α di HIF viene idrossilata da una prolil-idrossilasi (PHD) con conseguente ubiquitinazione da parte della E3 ubiquitina ligasi (processo facilitato dal legame con la proteina di Von Hippel-lindau (VHL)) e successiva degradazione proteasomica. In condizioni di ipossiemia, invece, l’attività di PHD è ridotta e ciò consente la sopravvivenza e la traslocazione nucleare di HIF α dopo la sua dimerizzazione con la subunità β [21] determinando l’attivazione trascrizionale genetica.

Sono stati identificati tre distinti sottotipi di HIF-α: HIF-1α, HIF-2α e HIF-3α. HIF-1α è espresso in quasi tutte le cellule e la sua attività nucleare determina la trascrizione di numerosi geni coinvolti nel metabolismo energetico, glucidico e marziale, nell’angiogenesi e nell’infiammazione [22]. HIF-2α è principalmente espresso da cellule simil-fibroblastiche presenti nell’interstizio renale e dalle cellule endoteliali, anche se studi successivi hanno dimostrato la sua espressione negli epatociti, cardiomiociti, pneumociti e cellule gliali [23, 24]. Esso è principalmente coinvolto nella regolazione della produzione di eritropoietina (EPO) e nel trasporto marziale [25]. La funzione del sottotipo HIF-3α non è nota, ma si pensa possa essere coinvolta nella regolazione dell’espressione genica degli altri due sottotipi [23].

Mentre il rene rappresenta la principale fonte fisiologica di produzione di EPO nell’età adulta, il fegato risulta essere il sito principale di sintesi durante lo sviluppo embrionale. In ogni caso, nell’adulto, il fegato mantiene la sua capacità di sintesi in risposta ad una moderata o severa ipossia o in caso di attivazione farmacologica del fattore HIF [26]. Infatti, similmente al rene, il fegato risponde in presenza di ipossia severa incrementando il numero di epatociti EPO secernenti localizzati attorno alle vene centrali [27]. Va inoltre considerato come l’espressione di mRNA per EPO è stata riscontrata anche nelle cellule cerebrali, polmonari, cardiache, nel midollo emopoietico, nella milza e nel tratto riproduttivo [28].

Da quanto suddetto, la capacità dei farmaci che vengono qua descritti di determinare una correzione dei valori di emoglobina nel paziente con malattia renale cronica è legata all’inibizione delle prolil-idrossilasi (PHD) che, non potendo determinare l’ubiquitinazione e conseguentemente la degradazione del fattore HIF, fa sì che quest’ultimo possa traslocare nel nucleo ed avviare i processi trascrizionali descritti (Figura 1).

Figura 1. In questa figura è rappresentata la regolazione del fattore HIF in condizioni di normossia e ipossia, modulata dall’azione dei farmaci inibitori la prolil idrossilasi (PH) coinvolta nella degradazione proteasomica del fattore nucleare di trascrizione HIF.
Figura 1. In questa figura è rappresentata la regolazione del fattore HIF in condizioni di normossia e ipossia, modulata dall’azione dei farmaci inibitori la prolil idrossilasi (PH) coinvolta nella degradazione proteasomica del fattore nucleare di trascrizione HIF.

In tal modo si genera un’incrementata produzione di EPO che a livello midollare determinerà lo stimolo all’eritropoiesi. Dagli studi effettuati, l’attività di tali farmaci, però, non si limita solo all’incremento dell’EPO. L’azione sul fegato garantisce anche una soppressione dell’epcidina, un’aumentata espressione di ceruloplasmina, transferrina e recettori della transferrina, con incremento dell’assorbimento e biodisponibilità del ferro [2932], effetto sinergico con l’incremento dell’EPO nel correggere l’anemia in pazienti con MRC che, com’è noto, presentano uno stato infiammatorio cronico con carenza marziale cronica e frequente resistenza alla terapia con EPO [33].

Allo stato attuale sono disponibili diverse molecole inibitrici della prolil-idrossilasi del fattore ipossia indotto per il trattamento dell’anemia nel paziente con MRC dipendente o non dipendente da emodialisi. Tra queste si annoverano: roxadustat, daprodustat, vadadustat, molidustat, desidustat ed enarodustat. Il roxadustat ha ricevuto l’approvazione dalla EMA e dall’AIFA nel 2021, mentre l’FDA ha approvato il daprodustat nei primi giorni del febbraio 2023.

 

Farmacocinetica

Questi farmaci vengono assunti per via orale, il loro assorbimento è indipendente dalla presenza di cibo, ma può essere limitato dalla presenza di chelanti a scambio ionico. Dopo l’assorbimento subiscono un metabolismo di primo passaggio a livello epatico ad opera del citocromo P-450 e della uridina difosfato grucoronil transferasi. Il metabolita attivo circola nel plasma legato per il 99% a proteine plasmatiche per cui la sua biodisponibilità non è influenzata dal trattamento emodialitico.

L’eliminazione avviene con urine e feci in massima parte come metaboliti, in minima parte come farmaco non modificato [34].

 

Effetti collaterali

Al pari degli ESAs, anche gli HIF-PHIs possono presentare potenziali eventi avversi dipendenti da dose e farmacocinetica.

Poiché i fattori responsabili della produzione di eritropoietina sono altamente sensibili all’ipossia rispetto ad altri bersagli HIF (quali ad esempio il VEGF) [35], gli HIF-PHIs sono in grado di ottenere effetti pro-eritropoietici a dosi che non elicitano un più ampio spettro di risposte di HIF nei pazienti con CKD, compresa la stimolazione di pathway VEGF-dipendenti [36].

Gli eventi avversi gravi (SAE), riportati negli studi di fase 3, non sono stati considerati correlati al farmaco e rientravano nell’intervallo delle frequenze attese di SAE nei pazienti con CKD.

Tuttavia, le informazioni sulla prescrizione della Japanese Pharmaceutical and Medical Devices Agency includono un avviso di sicurezza relativo al rischio potenziale di tromboembolia, infarto cerebrale e miocardico, embolia polmonare e trombosi venosa profonda e degli accessi vascolari con HIF-PHIs [37]. Una maggiore incidenza di eventi tromboembolici (11,3% vs. 3,9%) è stata riportato con roxadustat rispetto a darbepoetina alfa nell’analisi di sicurezza degli studi aggregati di fase 3 nei pazienti in emodialisi [37].

I dati preliminari di 3 studi con roxadustat in pazienti dializzati non hanno riportato alcun aumento del rischio di mortalità per tutte le cause e per roxadustat rispetto a epoetina alfa, mentre il rischio insufficienza cardiaca o angina instabile, che richiede ospedalizzazione, è stato significativamente ridotto per roxadustat vs epoietin alfa [38]. Questo potrebbe essere dovuto ai potenziali effetti pleiotropici da parte di HIF-PHIs quali roxadustat e daprodustat che si sono dimostrati in grado di ridurre i livelli sierici di colesterolo totale, LDL e trigliceridi [3941]. L’effetto ipolipemizzante potrebbe essere spiegato dall’aumento dell’assorbimento HIF-dipendente delle lipoproteine e dalla riduzione della sintesi del colesterolo attraverso una maggiore degradazione della 3-idrossi-3-metil-glutaril-CoA reduttasi [42, 43].

L’iperkaliemia è un evento avverso segnalato frequentemente con roxadustat negli studi cinesi di fase 3 sia nei pazienti con CKD non dipendenti da dialisi (NDD-CKD) che in quelli in trattamento emodialitico (DD-CKD) [4447] e in studi di fase 2 in pazienti trattati con altri HIF-PHIs [48, 49]. Un ulteriore evento avverso segnalato in pazienti in terapia con roxadustat non in dialisi è l’acidosi metabolica, sebbene i meccanismi alla base non siano chiari, riportata nel 12% dei casi [45]. Vi sono poi da prendere in considerazione potenziali eventi avversi correlati agli effetti pro-angiogenici degli HIF-PHIs. In particolare, in pazienti con retinopatia vascolare [48], non vi è stato alcun aumento nell’incidenza di emorragia retinica, edema maculare o cambiamenti nella pressione intraoculare o nell’acuità visiva negli studi clinici con roxadustat o daprodustat [49, 50].

Poiché l’attivazione dell’HIF è evidente in molti tumori (soprattutto quando, in crescita, sperimentano l’ipossia e cooptano la via dell’HIF per l’adattamento metabolico e l’angiogenesi) sono state avanzate preoccupazioni relative alle capacità di questi prodotti di promuovere la crescita tumorale o facilitare le metastasi [51].  Tuttavia, ad oggi, gli studi sugli animali non hanno mostrato alcuna evidenza che l’esposizione prolungata agli HIF-PHI sia pro-oncogenica [52, 53]. A tal proposito sono necessarie osservazioni a lungo termine nell’uomo ed attualmente non ne è raccomandato l’uso in pazienti con storia di neoplasia a causa dell’esclusione negli studi effettuati di pazienti con neoplasie maligne attive o in anamnesi più recente di 2-5 anni.

Altre preoccupazioni includono il potenziale rischio di ipertensione arteriosa polmonare (poiché l’attivazione di HIF aumenta il tono vascolare nelle arterie polmonari [54, 55], eventi tromboembolici che sono stati osservati in pazienti con policitemia di Chuvash [5658], promozione della crescita delle cisti renali [56], eventi avversi sul metabolismo del glucosio e del fegato [59] e gli eventi avversi sulle calcificazioni vascolari e sui livelli dell’FGF23 [60, 61].

 

Trial e risultati

Roxadustat

Molteplici sono gli studi che hanno valutato l’efficacia ed il profilo di roxadustat sia in pazienti con malattia renale cronica non dipendenti da dialisi (NDD-CKD) che in pazienti dialisi-dipendenti o incipienti (DD- CKD, ID-CKD), ESA naive o già in trattamento con ESAs.

Nel 2019 Chen N. e collaboratori hanno pubblicato I risultati di due trial clinici condotti in Cina. In uno si valutava l’efficacia in 154 pazienti con MRC non in trattamento dialitico, randomizzati 2:1 a ricevere il farmaco o placebo. Nel gruppo trattamento si osservava un incremento di emoglobina di 1,9 g/dl a differenza del gruppo placebo in cui si osservava una riduzione di 0,4 g/dl. Inoltre, nel gruppo trattato si riducevano significativamente anche i livelli di epcidina e colesterolo [62]. Nel secondo trial invece gli autori hanno avviato terapia con roxadustat in pazienti in trattamento dialitico che da sei mesi effettuavano terapia con epoietina alfa e li hanno confrontati con un gruppo di pazienti che proseguivano terapia con EPO. In questo studio si evidenziava la non inferiorità del roxadustat rispetto all’epoietina alfa nel mantenere valori di emoglobina a target. Paragonato ad epoetina alfa, roxadustat determinava un incremento del valore di transferrina, una stabilità della sideremia ed un minor calo della saturazione della transferrina. In ultimo la riduzione del colesterolo e dell’epcidina era maggiore nel gruppo trattato con roxadustat [63].

Una pooled analysis su studi che hanno coinvolto pazienti in trattamento emodialitico in terapia con ESA randomizzati a proseguire EPO o assumere roxadustat (ROCKIES, PYRENEES, SIERRAS, HIMALAYAS) ha mostrato come roxadustat non sia inferiore all’eritropoietina nel correggere e mantenere i valori di emoglobina con profilo di sicurezza cardiovascolare simile [64].

Una pooled analysis riguardante gli studi ALPS, ANDES, OLYMPUS, in pazienti con MRC, non in trattamento emodialitico, randomizzati a ricevere roxadustat o placebo, ha evidenziato come il primo sia più efficace del placebo nell’incrementare i valori di emoglobina, con minore frequenza di trasfusioni e con stesso profilo di sicurezza o eventi avversi [65].

Nel 2021 Barratt J. e collaboratori hanno confrontato roxadustat con darbepoietina alfa in uno studio di non inferiorità che ha coinvolto 616 pazienti con MRC non in dialisi (DOLOMITES trial) [66]. I risultati mostravano come roxadustat avesse la stessa capacità della darbepoietina nel mantenere stabile i valori di emoglobina per 104 settimane. Nel 2022 Fishbane e collaboratori in un trial su 2133 pazienti in trattamento dialitico riscontravano una capacità di roxadustat di incrementare i valori di emoglobina con stessa incidenza di eventi avversi quando paragonato ad epoietina alfa (ROCKIES study) [67].

Daprodustat

Anche per questo farmaco vari sono i trial in letteratura che hanno analizzato, in confronto con le molecole di eritropoietina più comunemente usate nella pratica clinica, la capacità di controllare nel tempo i valori di emoglobina e gli eventi avversi correlati.

Sono stati disegnati ed effettuati due trial clinici i cui risultati sono stati pubblicati nel 2021 (ASCEND-D ed ASCEND-ND trial) che si sono rivolti a pazienti in trattamento dialitico e non in trattamento dialitico (MRC G3-G5) rispettivamente.

Nel primo trial venivano randomizzati 2964 pazienti in trattamento dialitico sostitutivo a ricevere daprodustat o ESAs (epoietina alfa in pazienti in trattamento emodialitico, darbepoietina alfa in pazienti in trattamento peritoneo dialitico) per 52 settimane. I valori medi di emoglobina, la somministrazione di ferro endovenoso, gli effetti sulla pressione arteriosa e gli eventi avversi, non differivano nei due gruppi di trattamento [68].

Nel trial ASCEND-ND, che ha arruolato quasi 4000 partecipanti, sono stati randomizzati pazienti con MRC non in trattamento emodialitico e in terapia con eritropoietine, a ricevere daprodustat o darbepoietina alfa. I risultati derivanti dall’indagine garantivano il mantenimento dei valori di emoglobina in entrambi i gruppi con stessi eventi avversi [69].

Nel 2022 è stata pubblicata una metanalisi coinvolgente 8 dei principali trial clinici indaganti daprodustat e 8245 pazienti. I risultati mostrano che, in comparazione con ESAs, daprodustat mantiene la stessa efficacia nell’incrementare i valori di emoglobina, riduce in misura maggiore i livelli di epcidina e gli eventi cardiovascolari maggiori [70].

Vadadustat

I due trial clinici principali che hanno valutato vadadustat come farmaco in grado di mantenere adeguati valori di emoglobina sono stati PRO2TECT e INNO2VATE, rivolti rispettivamente a pazienti con malattia renale cronica in trattamento conservativo ed emodialitico.

Il PRO2TECT ha comparato vadadustat con darbepoetina alfa in pazienti con MRC non in dialisi in due gruppi diversi: quelli con valori di emoglobina inferiori a 10 g/dl, non in terapia con EPO, e quelli con valori di emoglobina compresi tra 8 g/dl e 11 g /dl, in terapia con EPO.

In questi due gruppi i rispettivi 1751 e 1725 pazienti sono stati randomizzati a ricevere vadadustat o darbepoetina alfa. Dopo 52 settimane di follow-up i risultati ottenuti mostravano come vadadustat, comparato a darbepoietina alfa, raggiungeva il pre-specificato criterio di non inferiorità per l’efficacia sull’incremento dell’emoglobina, ma non su quello della sicurezza cardiovascolare, mostrando cioè un numero maggiore di eventi cardiovascolari nel gruppo di trattati con vadadustat [71].

Il trial INNO2VATE è stato eseguito su 3923 pazienti con MRC dipendenti da dialisi, randomizzati a ricevere vadadustat o darbepoetina alfa. In entrambi i gruppi sono stati valutati oltre all’efficacia di controllare i valori di emoglobina, anche gli eventi avversi cardiovascolari. Gli autori concludevano che il vadadustat non risultava essere inferiore alla darbepoietina alfa nel correggere e mantenere la concentrazione emoglobinica, con stesso profilo di sicurezza cardiovascolare [72].

Nel 2022 sono stati pubblicati i risultati di una metanalisi che ha incluso 4 trial randomizzati riguardanti la comparazione tra vadadustat e placebo e 6 trial randomizzati di confronto con eritropoietine per un totale di 8438 partecipanti. In questo lavoro si evidenziava come vadadustat rispetto ai gruppi placebo determinava un incremento dell’emoglobina, comparato con placebo e darbepoietina alfa determinava una riduzione dell’epcidina e della ferritina con aumentata capacità ferro legante. L’uso del vadadustat era invece correlato ad un maggior tasso di effetti avversi lievi-moderati come diarrea e nausea, ma non incrementava il rischio di eventi cardiovascolari maggiori e mortalità per tutte le cause [73].

Molidustat

Questo farmaco è stato testato come gli altri appartenenti alla classe in pazienti con MRC avanzata sia in trattamento dialitico che conservativo, sia in pazienti già in trattamento con eritropoietine, che naïve.

Il trial DIALOGUE comprendeva delle sottosezioni in cui molidustat veniva confrontato con placebo (in pazienti naïve per ESAs), oppure dopo sospensione della precedente terapia con epoietina alfa o darbepoietina alfa (DIALOGUE 1, 2, 4 rispettivamente) [74].

Il MIYABI program ha comparato l’efficacia del molidustat rispetto alla darbepoietina alfa in pazienti in trattamento emodialitico e già in terapia con EPO con un follow-up di 52 settimane [75].

In questi trial si dimostrava la non inferiorità di molidustat rispetto alla terapia standard eritropoietinica, la capacità di correggere l’anemia in pazienti naïve, con profili di tossicità non dissimili.

Desidustat

I due trial principali di investigazione sono stati il DRAEM-D [76] in pazienti con MRC in trattamento emodialitico ed il DREAM-ND [77], in pazienti MRC non in dialisi. Nel primo, sia pazienti in trattamento con EPO che naïve con livelli di emoglobina tra 8 ed 11 g/dl venivano randomizzati a ricevere desidustat o epoietina alfa. Nel secondo invece, pazienti con MRC in trattamento conservativo, con valori di emoglobina tra 7 e 10 g/dl, venivano randomizzati a ricevere desidustat o darbepoietina alfa.

In entrambi i trial si osservava una non inferiorità del desidustat rispetto alla terapia con EPO nel mantenere costanti i valori di emoglobina durante il follow-up, con un maggior tasso di responder nei gruppi desidustat. Nel secondo trial, inoltre si osservavano valori significativamente più bassi di epcidina e LDL nei pazienti trattati con desidustat.

Enarodustat

I trial SYMPHONY-ND [30] e SYMPHONY-HD [78] hanno valutato l’efficacia e la tollerabilità di enarodustat in due corti di popolazioni giapponesi rispettivamente in MRC conservativa ed in trattamento emodialitico.

Il SYMPHONY-ND ha randomizzato una popolazione di pazienti naïve per ESAs 1:1 ad assumere enarodustat o darbepoietina alfa con follow-up di 24 settimane. Il SYMPHONY-HD condotto su pazienti in emodialisi periodica ha randomizzato 1:1 a ricevere enarodustat o darbepoietina alfa, con follow-up di 24 settimane. In entrambi gli studi si dimostrava la non inferiorità di enarodustat a correggere e mantenere i livelli di emoglobina quando confrontato a darbepoietina alfa, con un profilo di tossicità non differente, con l’aggiunta capacità di migliorare l’assetto marziale [79, 80].

 

Roxadustat: attuali linee guida in Italia

Attualmente, in Italia, l’unica molecola disponibile è il roxadustat. In commercio sono presenti compresse da 20, 50, 70, 100 e 150 mg. La somministrazione deve essere fatta tre volte a settimana, in giorni non consecutivi, indipendentemente dall’assunzione di cibo. Per i pazienti che non assumono eritropoietine, la dose iniziale è di 70 mg 3 volte a settimana per pazienti con peso inferiore ai 100 kg, 100 mg per pazienti con peso maggiore di 100 kg. La dose può essere incrementata fino a 400 mg 3 volte a settimana (in pazienti in trattamento emodialitico) o 300 mg 3 volte a settimana (in pazienti non in dialisi), con controllo dell’emocromo ogni 2 settimane fino al raggiungimento dei valori target e successivamente ogni 4 settimane.

Nei pazienti in trattamento con eritropoietine è disponibile una tabella (Tabella 1) di conversione che permette di evitare un periodo di wash out per la terapia eritropoietinica, facendo assumere la prima compressa di roxadustat al posto della successiva dose di eritropoietina. Per i pazienti in trattamento emodialitico non è necessario un aggiustamento della dose del farmaco [81].

In attesa di nuove evidenze, come avviene per la ormai consolidata terapia con eritropoietine, per valori di emoglobina al di sotto di 9 g/dl si dovrebbe avviare terapia con HIF-PHIs, con raggiungimento di valori di mantenimento tra 11-12 g/dl.

È da considerare inoltre che, in caso di compromissione epatica è consigliato di dimezzare la dose in caso di compromissione moderata (Child Pugh-B) e di evitare la somministrazione in caso di compromissione severa (Child Pugh-C).

Dose di darbepoietina alfa endovenosa o sottocutanea (microgrammi/settimana) Dose di epoietina endovenosa o sottocutanea (UI/settimana) Dose di metossipoietilenglicole-epoietina beta endovenosa o sottocutanea (microgrammi/mese) Dose di roxadustat (milligrammi tre volte a settimana)
Meno di 25 Meno di 5000 Meno di 80 70
Da 25 a meno di 40 Da 5000 fino ad 8000 Da 80 fino a 120 inclusi 100
Da 40 fino ad 80 inclusi Da più di 8000 fino a 16000 incluse Da più di 120 fino a 200 inclusi 150
Più di 80 Più di 16000 Più di 200 200
In questa tabella sono riportati i dosaggi e lo schema terapeutico nel passaggio da ESA a roxadustat
Tabella 1. Dosi iniziali di roxadustat da assumere tre volte alla settimana nei pazienti che passano da un ESA a roxadustat.

Dagli studi effettuati si è visto come alcuni chelanti del fosforo quali acetato di calcio e sevelamer carbonato riducono la biodisponibilità della molecola, per tanto roxadustat deve essere assunto almeno un’ora dopo l’assunzione di tali chelanti. Per il lantanio carbonato invece non si sono osservate interazioni significative. Inoltre, roxadustat può determinare un incremento delle concentrazioni sieriche delle statine per cui, in caso di co-somministrazione è necessario valutare la possibilità di ridurre il dosaggio della stessa.

 

Conclusioni

Questa nuova classe di farmaci, utili al trattamento dell’anemia correlata a malattia renale cronica, dai numerosi studi eseguiti si è dimostrata non inferiore rispetto alla terapia standard con eritropoietine nel correggere e mantenere i valori di emoglobina in pazienti con malattia renale cronica, e potrebbe garantire, nel prossimo futuro, un’alternativa terapeutica. Un punto di forza di queste nuove molecole è rappresentato dagli effetti pleiotropici sul metabolismo lipidico e marziale. Com’è noto i pazienti con malattia renale cronica, a causa di uno stato infiammatorio cronico possono presentare alterazioni del normale metabolismo marziale con difficoltà al trattamento dell’anemia con l’eritropoietina. I farmaci HIF-PHIs grazie alla modulazione genica, determinando una soppressione dell’epcidina, un’aumentata espressione di ceruloplasmina, transferrina e recettori della transferrina, facilitano l’assorbimento e la biodisponibilità del ferro. Tale meccanismo potrebbe rendere tali molecole una valida opzione terapeutica alle anemie difficili da trattare a causa della flogosi [82, 83].

Altro vantaggio nell’utilizzo di queste nuove molecole risulta legato alla possibilità di una produzione più “fisiologica” dell’EPO endogena se confrontata ad una possibile disponibilità “sovrafisiologica” di eritropoietina ricombinante esogena necessaria per correggere l’anemia. Questo determinerebbe un minore feed-back negativo endocrino sugli organi eritropoietici con minori effetti collaterali, in particolar modo cardio-vascolari, rispetto alla classica terapia con eritropoietina che, in alcuni casi, per fenomeni di resistenza, necessita incrementi del dosaggio, esponendo il paziente a un maggior rischio di eventi avversi con valori di emoglobina a target [84, 85].

Presentando un buon profilo di sicurezza e limitati effetti avversi, con l’utilizzo su larga scala si potrà in futuro confermare queste iniziali evidenze, ma senza dubbio, questi farmaci andranno ad arricchire l’armamentario farmacologico del nefrologo.

 

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Correlation of Acoustic Radiation Force Impulse Imaging with Chronicity Markers in Native Renal Biopsy

Abstract

Introduction. Acoustic Radiation Force Impulse (ARFI) is an ultrasound parameter which has shown promise in assessing liver stiffness, but there are limited data on the correlation of ARFI with chronicity markers in renal biopsies.
Objectives.

  1. Determine ARFI values in ultrasound and correlate with chronicity markers in renal biopsy
  2. Determine whether ARFI can be used as a non-invasive chronicity predictor compared to renal length, Resistive Index (RI), and cortical thickness.

Patients and Methods. Two hundred and fifty patients were enrolled in the study. The ultrasound variables ARFI, renal length, RI, and cortical thickness values were assessed by the radiologist prior to renal biopsy. The biopsy slides were graded as per the Mayo Clinic consensus report scoring system by an experienced pathologist.
Results. Among 250 study participants, 167 were males and 83 were females. IgA nephropathy was the most common pathology (n=47;19%), followed by diabetic nephropathy (n=42;17%), membranous nephropathy (n=35;14%), FSGS (n=27;11%), and MCD (n=19; 8%). The mean eGFR was 55.9 ± 42.12 ml/min/1.73 m2. The average renal length was 10.086 ± 1.01 cm. The average cortical thickness was 0.707 ± 0.134 cm. Resistive index was 0.68 ± 0.09. Acoustic radiation force impulse had weak negative correlation (r=-0.286; p=0.0001) with total pathological score and weak positive correlation with eGFR (r=0.279; p=0.0001). RI was a better indicator for histologically evaluated chronicity with positive correlation coefficient (r=0.416; p=0.0005) compared to renal length, cortical thickness, and ARFI.
Conclusion. ARFI didn’t corelate with the pathological score in renal biopsies. RI had better predictive value for chronicity in native renal biopsies.

Keywords: ARFI, Resistive index, Cortical thickness, Renal length, Chronicity

Introduction

Chronic Kidney Disease (CKD) is a vexing global health issue, with its reported prevalence in India ranging from 1% to 13% [1]. Renal biopsy is considered the gold standard to assess the extent of glomerulosclerosis, interstitial fibrosis, tubular atrophy, and vascular sclerosis which influence the progression of CKD [1]. In low- to middle-income countries, biopsy assessment and the technical prowess for kidney biopsy are not easily accessible. Acoustic Radiation Force Impulse Imaging (ARFI) is a unique ultrasound technique which superimposes data involving tissue elasticity onto ultrasound-produced greyscale images. It has been found to be highly useful in assessing liver, breast, prostate, and thyroid pathologies [2, 3]. ARFI uses short-duration, brief focused acoustic pulses along the main ultrasound beam to induce tissue shear stress, which is dependent on tissue attenuation, acoustic beam intensity, and acoustic frequency [2]. These shear stresses are converted into shear waves whose speed is directly proportional to the density and elasticity of the tissue [2]. ARFI has been used previously in chronicity evaluation in kidney biopsy samples [2]. The majority of the studies [3] state that ARFI doesn’t correlate with renal histological fibrosis, but a single study [2] mentions that ARFI correlates with histological renal fibrosis in chronic kidney disease with a sensitivity of 86% and specificity of 82%. Resistive index (RI) has been previously reported to correlate well with glomerulosclerosis, vascular sclerosis, and interstitial fibrosis in renal biopsies [4]. Renal length and cortical thickness have proved to be sensitive parameters for predicting chronicity and progression in chronic kidney disease and had a good correlation with estimated glomerular filtration rate (eGFR) [5, 6]. There is a paucity of South Asian data on the correlation of ARFI with chronicity markers in renal biopsies irrespective of aetiology and hence this study was designed to mitigate this research gap.

 

Objectives

  1. Determine ARFI values in ultrasound and correlate with chronicity markers in renal biopsy.
  2. Determine whether ARFI can be used as a non-invasive chronicity predictor compared to renal length, resistive index, and cortical thickness.

 

Methodology

This is a prospective observational study conducted at a tertiary care hospital involving 250 participants with written informed consent and ethics approval. All eligible patients undergoing native renal biopsy above 18 years of age who consented to the study were included. The research was done in compliance with relevant national and internal regulations governing human subjects over a period of 2 years. Patients with prior renal transplant, hydronephrosis, cyst, suspected renal artery stenosis, and unwilling participants were excluded. Indications for renal biopsy included nephrotic syndrome, sub-nephrotic proteinuria, unexplained renal failure, active urinary sediments, and rapidly progressive renal failure. All clinical characteristics along with laboratory results were documented on admission. Patients’ age, gender along with lab parameters like creatinine, blood urea nitrogen, serum albumin, urine protein estimation by 24-hour urine protein were noted for study purposes. The Modification of Diet in Renal Disease (MDRD) 6 variable equation was used to estimate the estimated glomerular filtration rate (eGFR).

Ultrasound Parameter Assessment

The radiological variables like ARFI, renal length, cortical thickness, and RI were estimated 10 minutes prior to renal biopsy by a single experienced radiologist using a Philips EPIQ 7 ultrasound machine with a 3 MHz transducer. Only the left kidney was chosen for all our study participants because it was more caudal and superficially positioned, facilitating renal biopsy. Quantitative elastography (ARFI) in unit measuring scale was used to estimate shear wave velocity in the region of interest, which is the lower pole of the renal parenchyma. For measuring shear wave velocity, the renal biopsy position (prone position) was used. ARFI readings were taken by asking patients to hold their breath for 10 seconds and 5 readings were taken at lower pole of the left kidney and ARFI was estimated by software installed in the ultrasound machine as depicted in Figure 1. The average of 5 readings were used for final data using the same protocol. After the ARFI values were obtained, renal length was measured between the topmost edge of upper pole and lowermost edge of lower pole and cortical thickness was measured from cortical peri-renal fat interface and sinus pyramidal apex interface. Renal RI was calculated as peak systolic velocity-end diastolic velocity/Peak systolic velocity at the level of the arcuate arteries near the corticomedullary junction. We used 60 healthy volunteers to establish the normal control value for ARFI before the actual study. Healthy volunteers were defined as individuals without comorbidities such as diabetes mellitus or hypertension. They had structurally normal kidneys on ultrasound, bland urine analysis, and eGFR > 90 ml/min/1.73 m2 according to the MDRD 6 equation. Healthy volunteers were enrolled from the master health checkup program in our hospital after confirming the above-mentioned conditions and their ARFI values were measured by the same experienced radiologist using the same equipment which is utilised for the test volunteers. The healthy volunteers aged between 18 and 70 years.

Figure 1: ARFI reading at lower pole of the study participant.
Figure 1: ARFI reading at lower pole of the study participant. 

Renal Biopsy Procedure and Processing

Two cores of renal biopsy tissue were taken using an 18 G Bard Biopsy Gun under ultrasound guidance and subjected to light microscopy and immunofluorescence. All biopsies were stained with hematoxylin and eosin, Periodic acid Schiff, Jones methenamine silver, and Masson’s Trichrome stain. Immunofluorescent staining involved fluorescein isothiocyanate conjugated polyclonal rabbit anti-human antibodies to IgG, IgM, IgA, C1q, C3, C4, lambda and kappa light chains. All biopsies were interpreted and reported by a single experienced in-house nephropathologist. The biopsy specimen containing less than 10 glomeruli was considered inadequate and not included in this study as per guidelines for renal biopsy adequacy proposed by Agarwal et al. [7]. The pathological classification and reporting of renal biopsy specimens were based on the recommendations of Mayo clinic Consensus report on Pathological Classification, Diagnosis and reporting of glomerulonephritis proposed by Sethi et al. [8]. The detailed grading system used in this study is outlined in Table 1 and Table 2.

Compartment Score 0 Score 1 Score 2 Score 3
Interstitial fibrosis <10% 11-25% 26-50% >50%
Tubular atrophy <10% 11-25% 26-50% >50%
Glomerulosclerosis <10% 11-25% 26-50% >50%
Vascular score Intimal thickness is less than medial thickness Intimal thickness is greater than medial thickness
Table 1. Mayo Clinic Consensus Report Scoring System.
Grading Score
Severe chronic changes >8
Moderate chronic changes 5-7
Mild chronic changes 2-4
Minimal chronic changes 0-1
Table 2. Mayo Clinic Consensus Report Grading System.

Statistical analysis

Continuous variables were estimated using mean, standard deviation or median. Continuous variables were evaluated using an independent sample t-test. Mann Whitney U test was used to evaluate non-normally distributed variables. Fishers’ test/Chi-square test were used to evaluate categorical variables. Pearson’s correlation coefficient was used to indicate association between different variables. Multiple comparison analysis between different groups and across the groups were done using ANOVA. ANOVA was used to determine any significant difference between the means of the groups of data. SPSS version 23.0 was used for data analysis. A p-value < 0.05 was considered significant.

 

Results

This was a prospective observational study that involved 250 random consenting individuals who underwent native renal biopsy.

Clinical and demographic characteristics

250 patients, all belonging to South Asian ethnicity, volunteered for the study. 38% (n=95) belonged to the age bracket between 46 and 60 years, 32% (n=79) belonged to the age group between 31 and 45 years, followed by 21% (n=54) of the patients in age group between 18 and 30 years. The remaining patients (n=22) were aged greater than 60 years.

Among the 250 patients in the study group, males were 65.2% (n=167) and 34.8% (n=83) were females. The study participants had IgA nephropathy (n=47; 19%) predominantly, diabetic nephropathy (n=42; 17%), Hypertensive Nephrosclerosis (HTN) (n=38, 15%), Membranous nephropathy (n=35; 14%), Minimal Change Disease (MCD) (n=19; 8%) focal segmental glomerulosclerosis (FSGS) (n=27; 11%) and others (15%).

Mean lab values of serum creatinine, albumin, blood urea nitrogen, and 24-hour urine protein of the research participants are provided in Table 3.

eGFR greater than 60 ml/min/m2 was present in 94 study participants, 36 patients had an eGFR between 46 and 60 ml/min/m2, 37 patients between 31 and 45 ml/min/m2, 38 patients between 15 and 30 ml/min/m2, and 45 patients between 0 and 14 ml/min/m2.

Lab parameters Mean values with standard deviation
Serum creatinine 2.40 ± 2.55 milligrams/deciliter
Serum albumin 3.52 ± 0.67 grams/deciliter
Serum blood urea nitrogen 62.06 ± 55.63 milligrams/deciliter
24-hour urine protein 2.62 ± 1.76 grams/day
Table 3. Lab values of study participants.

Renal Length

On ultrasound measurement, renal length greater than 10 cm was present in 128 patients (51.2%), followed by 92 patients (36.8%) with length between 9-10 cm, and finally 30 patients (12%) had length between 8-8.9 cm.

Resistive Index

176 patients (70.4%) had RI between 0.61-0.8, 47 patients (18.8%) with RI between 0 and 0.6 and 27 patients (10.8%) had RI between 0.81 and 1.1.

Cortical Thickness

Cortical thickness of 0.6-0.8 cm was present in 91 participants (76.4%), followed by 31 (12.4%) having 0.9-1 cm cortical thickness and 27 (10.8%) having cortical thickness less than or equal to 0.5 cm, and only 1 patient in the study with cortical thickness greater than 1 cm.

Acoustic Radiation Force Impulse

8 patients had ARFI value between 0 and 0.5 m/sec, 102 study participants had ARFI between 0.51 and 0.8 m/sec, 113 patients had ARFI value between 0.81-1 m/sec and 27 had values greater than 1 m/sec followed by as depicted in Figure 2. The healthy volunteers used for control values were in age bracket of 18-70 years of age. The average age of healthy volunteers was 44.236 ± 12.68 years. The control value of ARFI obtained on healthy volunteers was 1.4933 ± 0.10028 m/sec.

Figure 2. ARFI (m/sec) distribution in the study. *x axis-ARFI groups in m/sec. **y axis- Number of patients in ARFI groups
Figure 2. ARFI (m/sec) distribution in the study. *x axis-ARFI groups in m/sec. **y axis- Number of patients in ARFI groups

Depth from skin surface

The study participants were distributed as follows: 64% (n=160) had a depth between 4.1 and 6 cm, 26% (n=65) had a depth between 0 and 4 cm, and 10% (n=25) had a depth greater than 6 cm from the skin surface.

Renal Pathology Findings

The renal pathology was assessed in terms of chronicity by evaluating glomerulosclerosis, interstitial fibrosis, tubular atrophy, and vascular score on the pathology specimens and it was scored as per the recommendations of Consensus grading system [8].

Glomerulosclerosis

In the 250 participants, 41.60% (n=104) had glomerulosclerosis between 10-25%, 36.8% (n=92) had glomerulosclerosis < 10%, 18% (n=40) had glomerulosclerosis between 26-50%, and 5.6% (n=14) had glomerulosclerosis greater than 50%.

Vascular Score

88.8% (n=222) of participants had intimal thickness < medial thickness, and 11.2% (n=28) had intimal thickness greater than medial thickness.

Interstitial Fibrosis

There were 32.8% (n=82) of participants having interstitial fibrosis < 10% on biopsy, 32.4% (n=81) having interstitial fibrosis between 10-25%, 22% (n=55) having interstitial fibrosis between 26-50%, and 12.8% (n=32) having interstitial fibrosis greater than 50%.

Tubular Atrophy

In the study, 39.2% (n=98) of participants had 10-25% tubular atrophy on biopsy and 26% (n=65) had tubular atrophy less than 10%. This is followed by 22% (n=55) having tubular atrophy between 26-50% and 12.8% (n=32) having tubular atrophy greater than 50%.

Chronicity grading in renal biopsy

In the study, mild chronic changes were present in 35.60% (n=89) of patients, minimal chronic changes in 30.4% (n=76), moderate chronic changes in 61 patients (24.4%), and 9.6% (n=24) had severe chronic changes depicted in Figure 3.

Figure 3. Chronicity Grading of Renal Biopsy on basis of total score.
Figure 3. Chronicity Grading of Renal Biopsy on basis of total score.

Correlation of ARFI, resistive index, cortical thickness, and renal length with total pathological score

In the statistical analysis detailed in Table 4, ARFI was found to have weak negative correlation with total pathological score (r = -0.286). Resistive index had a better correlation among the various ultrasound parameters analysed (r = +0.416) albeit weak compared to ARFI, renal length (r = -0.312), and cortical thickness (r = -0.342).

Parameter R value p-value
ARFI -0.286 0.0001
Resistive index +0.416 0.0005
Cortical thickness -0.312 0.0001
Renal length -0.342 0.0001
Table 4. Correlation of ARFI, resistive index, cortical thickness and renal length with pathological score.

Correlation of ARFI, resistive index, cortical thickness and renal length with eGFR

ARFI was found to have weak positive correlation with eGFR, as described in Table 5. Among all the ultrasound parameters assessed, the resistive index had better weak correlation with eGFR (r = -0.412). Cortical thickness didn’t have the statistical significance and correlation with eGFR. Renal length had very weak correlation (r = +0.167) with eGFR in this study.

Parameter R value p-value
ARFI +0.279 0.0001
Resistive index -0.412 0.0005
Cortical thickness 0.131 0.038
Renal length 0.167 0.008
Table 5. Correlations of ARFI, Resistive index, cortical thickness and renal length with eGFR.

Performance of ARFI in between chronicity groups

ARFI couldn’t differentiate between minimal and mild groups of chronicity scoring and also couldn’t differentiate between moderate and severe chronicity groups on inter-group analysis as depicted in Table 6. However, ARFI values were higher in minimal chronic changes group compared to moderate chronic changes group. Mean ARFI values in different chronicity groups are mentioned in Table 7. A similar trend was noticed in minimal, moderate chronic changes compared to severe chronic changes group.

Multiple Comparisons
Dependent Variable: ARFI
Least significant difference
(I) grade (J) grade Mean Difference (I-J) Std. Error Sig.
Minimal chronic changes (0-1) Mild chronic changes (2-4) 0.01152 0.02811 0.682
Moderate chronic changes (5-7) 0.11730* 0.03094 0.000
Severe chronic changes (≥8) 0.13333* 0.04214 0.002
Mild chronic changes (2-4) Minimal chronic changes (0-1) -0.01152 0.02811 0.682
Moderate chronic changes (5-7) 0.10578* 0.02991 0.000
Severe chronic changes (≥8) 0.12182* 0.04139 0.004
Moderate chronic changes (5-7) Minimal chronic changes (0-1) -0.11730* 0.03094 0.000
Mild chronic changes (2-4) -0.10578* 0.02991 0.000
Severe chronic changes (≥8) 0.01604 0.04336 0.712
Severe chronic changes (>=8) Minimal chronic changes (0-1) -0.13333* 0.04214 0.002
Mild chronic changes (2-4) -0.12182* 0.04139 0.004
Moderate chronic changes (5-7) -0.01604 0.04336 0.712
Table 6. Performance of ARFI in between chronicity groups.
Chronicity groups Number of patients Mean ARFI (m/sec) Std. Deviation
Minimal chronic changes (0-1) 76 0.885 0.18263
Mild chronic changes (2-4) 89 0.8735 0.1624
Moderate chronic changes (5-7) 61 0.7677 0.20374
Severe chronic changes (≥8) 24 0.7517 0.16857
Total 250 0.8395 0.1872
Table 7. Mean ARFI values in different chronicity groups. 

Performance of ARFI in different grades of interstitial fibrosis and tubular atrophy

ARFI scores decrease as the grades of interstitial fibrosis increase in the renal biopsy specimen, as depicted in Table 8. ARFI couldn’t differentiate between renal biopsies showing < 10% tubular atrophy and those with 10-25% tubular atrophy. Similarly, it couldn’t distinguish between 26-50% tubular atrophy and >50 % tubular atrophy, as described in Table 9.

Descriptive statistics
ARFI with interstitial fibrosis
N Mean ARFI Std. Deviation Std. Error 95% Confidence Interval for Mean
Lower Bound Upper Bound
<10% 65 0.8937 0.18363 0.02278 0.8482 0.9392
10-25% 98 0.8612 0.16891 0.01706 0.8274 0.8951
26-50% 55 0.7915 0.19645 0.02649 0.7383 0.8446
>50% 32 0.7453 0.18565 0.03282 0.6784 0.8122
Total 250 0.8395 0.18720 0.01184 0.8162 0.8628
Table 8. Trends of ARFI values with different grades of interstitial fibrosis.
Multiple Comparisons
Dependent Variable: ARFI
Tubular atrophy
(I) tubular (J) tubular Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
<10% 10-25% 0.03247 0.02899 0.264 -0.0246 0.0896
26-50% 0.10224* 0.0332 0.002 0.0368 0.1676
>50% 0.14838* 0.03913 0 0.0713 0.2255
10-25% <10% -0.03247 0.02899 0.264 -0.0896 0.0246
26-50% 0.06977* 0.03053 0.023 0.0096 0.1299
>50% 0.11591* 0.0369 0.002 0.0432 0.1886
26-50% <10% -0.10224* 0.0332 0.002 -0.1676 -0.0368
10-25% -0.06977* 0.03053 0.023 -0.1299 -0.0096
>50% 0.04614 0.04029 0.253 -0.0332 0.1255
>50% <10% -0.14838* 0.03913 0 -0.2255 -0.0713
10-25% -0.11591* 0.0369 0.002 -0.1886 -0.0432
26-50% -0.04614 0.04029 0.253 -0.1255 0.0332
Table 9. Multiple comparisons between ARFI and tubular atrophy.

 

Discussion

This a prospective observational study of 250 patients who underwent ultrasound-guided left-sided renal biopsy from September 2017 to April 2019. It represents the largest reported South Asian population study compared to previous studies conducted by Wang et al. [9] and Guo et al. [10], which had 45 and 64 patients respectively. The merit of this work lies in the South Asian ethnic diversity of the study population. We used 60 healthy volunteers to obtain the normal ARFI control value, adding scientific merit and novelty compared to previous studies [9, 10].

In our study, 65.2% (n=167) of participants were males, while 34.8% (n=83) were female, and the mean age was 43.192 + 13.7008 years. The higher proportion of males in our research aligns with the hospital outpatient statistics and is consistent with a previous study [9].

The pathological grading in our research follows a standardized protocol as per the Consensus Report on Pathologic Classification, Diagnosis, and Reporting of GN [8]. This study stands out as one of the large-scale studies that adopted a standardized protocol for grading chronicity, irrespective of the acute and chronic aetiology warranting renal biopsy. The previous studies [9, 10] didn’t employ standardized pathological guidelines for evaluating chronicity in renal biopsy, lacking a reliable scoring system – which is instead one of the merits of our study. This scoring system solely focused on the chronicity markers like glomerulosclerosis, interstitial fibrosis, tubular atrophy, and vascular sclerosis in renal biopsy, thereby obviating the need to classify the renal aetiology as acute or chronic and enabling smooth comparison with ultrasound variables.

This study comprised both acute, chronic, and proteinuric renal diseases. The predominant pathology observed on renal biopsy was IgA nephropathy (19%, n=47), followed by diabetic nephropathy (17%, n=42). The previous study [9] similarly showed the presence of IgA nephropathy (n=31), followed by membranous nephropathy (n=4). The cases of diabetic nephropathy enrolled in our study had some atypical presentations like rapid progression of azotaemia not explained by the natural clinical course of diabetes and microhaematuria which warranted renal biopsy in our study participants. The predominance of IgA nephropathy in our renal biopsies signifies the burden of IgA nephropathy as a predominant primary glomerular disease in South Asian population.

The average renal length in our study is 10.086 ± 1.01 cm. The average eGFR was 55.9 ± 42.12 ml/min/1.73 m2 in our patient population. Resistive index in our study ranged from 0.68 ± 0.09. These findings were quite similar to earlier research [11], which had resistive index of 0.69 ± 0.10, and average renal length of 9.8 ± 1.1 cm. However, in contrast to the previous study [11], the resistive index had weak albeit significant correlation with eGFR in our study. Resistive index is an integrated ultrasound variable signifying arterial compliance, pulsatility, and peripheral resistance, which is predictive of vascular stiffness and sclerosis contributing to hypertension and CKD progression [12].

Splendiani et al. [13] demonstrated that RI > 0.70 was predictor of dismal renal prognosis. Their study showed that RI had excellent correlation with eGFR decline, with significant p-value (p < 0.0001). This contrasts with our findings, as RI and eGFR weakly correlated in our study. Previous research [14, 15] concluded that renal RI and eGFR had significant correlation, which contrasts our research findings, where we demonstrated a weak correlation of RI with eGFR.

The average cortical thickness was 0.707 ± 0.134 cm in our study. An earlier seminal study [16] evaluated the impact of ultrasound parameters with renal histology and studied the correlation of parameters like renal length, parenchymal thickness, cortical echogenicity on the chronicity observed in renal histopathology. His study [16] concluded that renal length was a good indicator for chronicity in renal biopsy. Our research showed a very weak agreement of renal length with total pathological score (r=-0.342) which contrasted with the previous study [16]. Renal length is susceptible to interobserver variation and is dependent on the pathology contributing to acute and chronic dysfunction thereby contributing to its unreliability in predicting chronicity [5, 17].

ARFI and the total pathological score had a weak negative correlation (r= -0.286; p=0.0001). It couldn’t differentiate between minimal and mild group of chronicity scoring and also couldn’t differentiate between moderate and severe chronicity group. It couldn’t differentiate between renal biopsies showing < 10% tubular atrophy and 10-25% tubular atrophy. Similarly, it couldn’t distinguish between 26-50% tubular atrophy and >50 tubular atrophy groups on intergroup analysis. Our study findings were in partial agreement with the earlier study [9] which showed no correlation between ARFI and renal histopathological changes suggestive of fibrosis, and contrasted findings of Goya et al. [18]. ARFI didn’t correlate strongly with eGFR in our study, which was dissimilar to findings of Asano K et al. [19]. This disparity in findings may be due to predominant diabetic patients in the previous studies [18, 19] thereby raising the possibility of influence of chronic pathology like diabetes mellitus on ARFI readings.

It was found that resistive index was better in predicting interstitial fibrosis and tubular atrophy and chronicity compared to cortical thickness, ARFI and renal length when chronicity score was used as a fixed variable in regression analysis. Resistive index correlated better with chronicity score among the ultrasound variables measured in our research protocol. On analysis using eGFR as a fixed variable, resistive index outperformed ARFI in correlating better with eGFR. These findings were discordant with the previous studies done by Cui G et al. [20] and Hu Q et al. [21].

ARFI is influenced by age and depth and they are considered independent variables influencing ARFI readings [22] which were not analysed in our study. But our study was an honest attempt to uniformly stratify chronicity into multiple groups irrespective of the acute or chronic aetiology emphasising that loss of kidney function, and progression to CKD is influenced by the chronicity markers like glomerulosclerosis, vascular sclerosis, interstitial fibrosis, and tubular atrophy [23].

 

Conclusion

ARFI imaging doesn’t correlate with chronicity markers and total pathological score in renal biopsies and is not beneficial in differentiating various groups of chronicity in renal biopsies. Resistive index had better predictive value for chronicity in native renal biopsies compared to ARFI, renal length, and cortical thickness.

 

Merits of this research

The merits of this study include it being the largest study done in Southern Asia comparing ARFI with chronicity in renal biopsies with a standardized pathological grading system. This study uniformly stratifies chronicity by using total pathological score independent of acute and chronic aetiology for renal biopsy which is the novelty of this research.

 

Limitations

The limitation of this research includes non-analysis of factors affecting ARFI values including depth, age, and renal blood flow. We didn’t evaluate the variation of ARFI with respect to poles and different regions of the same kidney. Our findings were restricted to the biopsied left kidney of the study participants, and we didn’t analyse variations of ARFI values in the right kidney. Our study didn’t analyse the effect of acute or chronic aetiology for renal dysfunction on ARFI values.

 

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