Iohexolo è un metodo possibile per stimare la velocità di filtrazione glomerulare?

Pubblicato il Scarica PDF

Fabrizio Cristiano

DOI: 10.69097/42-02-2025-03

Fabrizio Cristiano1,2, Guillermo Rosa Diez4, Carlos Guido Musso3,4, Jenny Cristiano5

1 UOSD Nefrologia e Dialisi Ospedale di Ortona, Asl 2 Lanciano Vasto Chieti
2 Department of Neuroscience, Imaging and Clinical Science, University "G. d'Annunzio" Chieti - Pescara, 66100 Chieti, Italy
3 Servicio de Nefrología, Hospital Italiano, Buenos Aires, Argentina
4 Research Department, Hospital Italiano de Buenos Aires. Buenos Aires, Argentina
5 UOC Farmacia Ospedaliera – Ospedale Clinicizzato di Chieti – Asl 2 Lanciano Vasto Chieti

Corrispondenza a:
Fabrizio Cristiano
UOSD Nefrologia e Dialisi Ospedale di Ortona, Asl 2 Lanciano Vasto Chieti
Department of Neuroscience, Imaging and Clinical Science, University "G. d'Annunzio" Chieti - Pescara, 66100 Chieti, Italy
E-mail: fabrizio.cristiano@asl2abruzzo.it

 

Abstract

La misurazione della velocità di filtrazione glomerulare (GFR) è essenziale nella diagnosi e gestione della malattia renale cronica (CKD) e della malattia policistica renale autosomica dominante (ADPKD), entrambe condizioni che richiedono una valutazione precisa della funzione renale. Tradizionalmente, il GFR è stato stimato usando marcatori endogeni come creatinina e cistatina C, sebbene questi possano risultare inaccurati a causa di fattori non correlati alla funzione renale, come massa muscolare e dieta. Il metodo di clearance di iohexolo, un mezzo di contrasto non ionico e idrosolubile, rappresenta un’alternativa più accurata e meno invasiva rispetto ai marcatori tradizionali come inulina o marcatori radioattivi. Iohexolo viene eliminato esclusivamente tramite filtrazione glomerulare, rendendolo altamente adatto per una stima diretta del GFR. Questo articolo descrive le procedure per la clearance di iohexolo, che prevedono prelievi di sangue a intervalli definiti dopo somministrazione endovenosa. Nei pazienti con funzione renale normale, gli intervalli di campionamento sono più frequenti, mentre nei pazienti con CKD avanzata, inclusi quelli con ADPKD, l’eliminazione dell’iohexol è più lenta e richiede intervalli più ampi per garantire un’analisi accurata della clearance. Iohexolo ha dimostrato alta precisione e riproducibilità, anche rispetto ad altri marcatori. Vi sono numerose evidenze di come l’uso di iohexolo possa monitorare efficacemente la progressione di CKD e ADPKD. In particolare, nell’ADPKD, iohexol rileva variazioni sottili ma clinicamente significative del GFR, anche nelle fasi iniziali della malattia, rendendolo utile per valutare terapie mirate. Tuttavia, l’uso di iohexolo è limitato a centri specializzati a causa dei costi elevati e dei protocolli rigorosi. Purtroppo il suo utilizzo a tutt’oggi è ancora abbastanza limitato e non esportabile in tutte le realtà europee.

Parole chiave: velocità di filtrazione glomerulare, iohexolo, malattia renale cronica

Ci spiace, ma questo articolo è disponibile soltanto in inglese.

Introduction

Glomerular filtration rate (GFR) assessment is a parameter in the diagnosis and management of chronic kidney disease (CKD), allowing an accurate estimation of renal function. Several methods have been proposed to measure GFR, including endogenous markers such as serum creatinine and cystatin C, as well as methods based on exogenous markers that are administered and subsequently quantified in plasma or urine [1]. Among exogenous markers, iohexol has recently gained attention due to its reliability, safety and accuracy in determining GFR. Iohexol is a nonionic, water-soluble, low-osmolal contrast agent, which has ideal characteristics for the assessment of GFR. Its elimination exclusively by the kidney, through glomerular filtration, makes it particularly suitable for this purpose [2].

 

GFR Analysis Methodology

The iohexol clearance method is a widely accepted approach to estimate glomerular filtration rate (GFR) due to its high accuracy and reproducibility. This method involves the administration of an exogenous tracer (iohexol) followed by serial blood sampling to monitor its plasma clearance. Below is a step-by-step description of the method. Iohexol is intravenously injected as a 5% solution (50 mg/mL), with the dose ranging from 5 to 10 mL (approximately 250 mg of iohexol). The precise dose is adjusted according to the patient’s body weight and renal function. Blood samples are collected at predetermined intervals based on the patient’s renal function. In patients with normal or mildly impaired renal function, blood samples are collected at shorter intervals, typically at 2, 4, and 6 hours. Patients with moderately to severely impaired renal function require extended sampling intervals, such as 2, 4, 6, 12, and 24 hours, to account for slower iohexol elimination. Plasma iohexol concentrations are measured using spectrophotometry or high-performance liquid chromatography (HPLC). The clearance rate of iohexol, directly proportional to GFR, is calculated from the plasma concentration decay curve. GFR is estimated using models such as the Brøchner-Mortensen formula, which considers the volume of distribution and elimination kinetics of iohexol. Protocol Adaptations: Specific protocols (e.g., 2-point or multi-point sampling) are tailored to optimize accuracy while reducing patient burden. Adjustments are made for variables like age, body mass, and comorbidities. This structured approach ensures a reliable estimation of GFR while accommodating physiological and pathological variations among patients. The iohexol clearance method has proven effective in clinical settings, particularly for patients with chronic kidney disease (CKD) and autosomal dominant polycystic kidney disease (ADPKD), where precise GFR monitoring is crucial.

This is necessary to capture the slower elimination and obtain adequate data to correctly calculate the clearance [3]. Each patient may present physiological variations that affect the pharmacokinetics of iohexol, so protocols may be adapted based on factors such as age and body mass (e.g., elderly or underweight patients eliminate the tracer more slowly) and comorbid conditions (e.g., liver disease).   These adjustments are aimed at obtaining a detailed and accurate profile of GFR, especially in patients with impaired renal function or significant physiological variability [4]. The concentration of iohexol is determined in the laboratory by spectrophotometry or chromatography, and the data obtained are used to calculate the plasma clearance of iohexol. The clearance of iohexol (i.e. the rate at which it is eliminated from the plasma) is proportional to the GFR. In practice, the GFR can be calculated with formulas that consider the volume of distribution and the elimination time of iohexol [5].

Renal Function Level Tracer Dose Sampling Times Additional Notes
Normal renal function 5-10 mL (250 mg) 2, 4, 6 hours Frequent sampling captures rapid elimination
Mildly impaired function 5-10 mL (250 mg) 2, 4, 6 hours Protocols remain similar to normal function
Moderate impairment 5-10 mL (250 mg) 2, 4, 6, 12 hours Sampling extended to account for slower elimination
Severe impairment 5-10 mL (250 mg) 2, 4, 6, 12, 24 hours Comprehensive sampling ensures accurate calculation over prolonged clearance
Special populations Adjusted by weight Variable intervals Tailored protocols based on age, comorbidities (e.g., liver disease), and body mass
Table 1. Detailed description of the iohexol Method for GFR Estimation. Iohexol Sampling protocols based on renal function.

 

GFR Measurement: The Standard in Renal Functionality Assessment

Unlike other previously used contrast agents, such as inulin or 125I-iothalamate, iohexol has fewer side effects, is easy to handle, and does not require a specialized center for radioactivity management, as is the case with radioactive markers. Numerous studies have shown that iohexol offers high accuracy in estimating GFR. For example, comparison of iohexol clearance with inulin confirmed the accuracy of iohexol as a valid and less invasive alternative. GFR measurement with iohexol is based on blood sampling after marker injection and calculations that take into account the concentration of the marker in the plasma at defined intervals. Iohexol clearance has been shown to be useful in patients for whom creatinine is not always accurate in estimating renal function, particularly in conditions such as CKD, acute renal failure, and in pediatric patients or those with significant comorbidities, as this method is less influenced by variables such as muscle mass, age, and diet [6].

The ability to standardize a formula for accurate estimation of GFR and to use specific formulas for interpretation of clearance values ​​has reduced errors related to GFR measurement. However, the criticality of measuring GFR with iohexol is represented by rigorous protocols for blood sampling and laboratory analysis. Errors in sampling times and measurement techniques can influence the precision of the results. Many laboratories have adopted standardized protocols to minimize these fluctuations, making the use of iohexol more common and widespread in order to consolidate its role in clinical practice and in nephrology research, especially for those particulars that may present variability in estimating GFR [7].

Glomerular filtration rate (GFR) is the gold standard measure of kidney function and is critical to the diagnosis and management of kidney disease. An adequate estimation of GFR requires the measurement of renal clearance of an exogenous marker with the characteristic of being filtered by the kidney and that is not subject to reabsorption, metabolism or secretion. Although inulin represents an ideal marker of glomerular filtration, it cannot be used in clinical practice to estimate glomerular filtration. 125I-iothalamate and 99mTc-diethylenetriaminepentaacetic acid (DTPA) can represent an alternative, however, being difficult to handle and with safety limits, they cannot also be used in clinical practice. A possible alternative for estimating glomerular filtration could be represented by the use of non-radioactive contrast agents such as iothalamate (ionic) [8], which in terms of estimation and precision are comparable to inulin. However, they have limitations mainly represented by the collection method of urine and potential errors affected by delayed bladder emptying, such as obstructive causes in male patients or an excessive water load. The use of an appropriate exogenous marker (51Cr-EDTA, 125I-iothalamate, iohexol) has the advantage of estimating glomerular filtration precisely by evaluating the rate of elimination of the tracer after an intravenous infusion [9] and with blood samples repeated at intervals over time, however the procedure is complicated to implement. Thanks to the Bröchner-Mortensen formula it was possible to correlate iohexol with inulin clearance with data analysis with a simplified model with analysis of six blood samples (Figure 1).  This method is currently used to measure GFR in multicenter clinical trials [10]. The Bröchner-Mortensen formula is used to estimate creatinine clearance (or glomerular filtration rate, GFR) using iohexol, a contrast agent used in nuclear medicine and radiology to assess renal function. This method is useful for calculating GFR on a blood sample collected after iohexol administration [11].

GFR correction with iohexol using Bröchner-Mortensen formula.
Figure 1. GFR correction with iohexol using Bröchner-Mortensen formula.

Iohexol for CKD Patients

In order to give an accurate estimate of GFR, iohexol is considered a valid alternative to inulin but presents practical difficulties in the estimation and accuracy of the results. The accuracy of GFR estimation with iohexol was evaluated by administering the marker on three different occasions to 24 patients and measuring its plasma clearance. The results show a low intraindividual variability (5.59%) and a high reproducibility (6.28%), demonstrating that iohexol is reliable even in patients with moderate or severe renal insufficiency (GFR < 40 mL/min/1.73 m²) [12].

The accuracy of iohexol clearance is high and is not affected by gender and stage of chronic kidney disease, making the method applicable to different types of patients. Simplified iohexol clearance measurement methods exist to measure GFR in patients with CKD, comparing their accuracy with that of the standard 10-hour two-compartment method. The study evaluates the performance of several simplified models, including a population pharmacokinetic (popPK) model and 5-, 6-, and 7-hour single-compartment models, to reduce the complexity and cost of measurements [13].

The results indicate that compared to the 8-hour reference method, the abbreviated models tend to overestimate GFR, especially in patients with an eGFR less than 40 mL/min/1.73 m². Furthermore, the popPK model is less precise and less reliable in patients with advanced CKD (stage III-IV), while the 6- and 7-hour monocompartmental models provide a more accurate estimate but show limitations compared to the standard method [8].

Iohexol represents a valid alternative to inulin for the estimation of GFR, without the need for continuous infusion or urine collection required for inulin. The iohexol plasma clearance method initially requires multiple blood samples to accurately estimate GFR. However, abbreviated methods using a single plasma sample have also been developed, which certainly simplifies the procedure but may reduce accuracy for some patients, particularly those with advanced renal failure [14].

The reliability of the single-sample method has been evaluated. Their study demonstrated that, despite a strong correlation between the multiple and single clearance methods, the accuracy of the single sample method varies significantly according to the patient’s GFR, with acceptable results for approximately 75% of patients and more significant deviations for the remaining 25% [15].

Iohexol for ADPKD patients

Due to its unique characteristics, iohexol has been studied as a marker of GFR in patients with ADPKD. Iohexol clearance, measured by plasma sampling at specific times after contrast injection, represents a valid alternative to inulin and other traditional markers and has allowed to examine the progression of the disease and the effect of potential therapies in reducing the rate of GFR decline. ADPKD is characterized by a gradual replacement of the renal parenchyma with cystic formations resulting in a progressive decrease in GFR; accurate monitoring of this decline with iohexol allows a reliable estimate of residual renal function [16]. Iohexol is particularly useful in patients with ADPKD because it allows repeatable and reliable measurements with an accurate estimate of GFR over time, essential to monitor the evolution of the disease. It also detects changes in GFR even in the early stages of the disease, when creatinine values ​​are not yet significant [17]. Iohexol is an accurate marker for measuring GFR in patients with CKD and ADPKD. Due to its high accuracy, reliability and ease of use compared to traditional markers such as inulin, iohexol allows for accurate monitoring of the progression of chronic kidney disease. Iohexol clearance-based methods, including simplified protocols, reduce the need for extensive sampling, making the process less invasive and more suitable for frequent clinical use [18].

 

Discussion

The use of iohexol to measure GFR has several advantages over other methods, particularly those based on creatinine. Iohexol clearance provides an accurate and direct measurement of GFR without the limitations of factors unrelated to renal function (e.g. muscle mass) that influence creatinine. This makes it particularly useful for patients with variable characteristics, such as the elderly and children, or those with impaired muscle mass [19]. Unlike traditional methods such as inulin, which require continuous infusion and multiple urine collections, iohexol measurement is less invasive and more convenient for patients, as it requires only blood samples. Iohexol has low intraindividual variability, which makes repeat GFR measurements reliable over time, allowing effective monitoring of renal function in patients with chronic or progressive renal failure. The iohexol method is used in many European centers and is integrated into guidelines for GFR monitoring. In some countries, such as Sweden, it is used as part of standard care, demonstrating the efficacy and applicability of this technique at the clinical level. These advantages make iohexol a preferable choice for measuring GFR in clinical situations where greater accuracy is needed than creatinine-based estimates [20]. The use of iohexol has been particularly effective for comparing GFR data with other parameters, such as total kidney and cyst volume, allowing a holistic assessment of disease progression. Due to the accuracy of the iohexol-based method, it has been possible to demonstrate that some experimental drug treatments were able to slow down disease progression in selected patients [21]. However, this method still has some disadvantages. Iohexol itself and the analytical processes involved (e.g., spectrophotometry, chromatography) are costly. The method requires multiple blood samples, advanced laboratory equipment, and trained personnel, which increases operational costs.

The procedure presents a considerable complexity represented by sampling rigidity, because accurate GFR estimation depends on precise timing of blood samples. Even minor deviations in sampling times can lead to errors in clearance calculation; specialized training is required, because laboratory personnel need expertise in handling iohexol and analyzing plasma concentrations, which may not be available in all healthcare settings.

The method is often restricted to tertiary care centers or research facilities due to the need for specific equipment and expertise and regions with limited healthcare infrastructure may lack the resources to implement this method. The requirement for several blood samples over time makes the method invasive and potentially uncomfortable for patients. Elderly or pediatric patients, as well as those with compromised venous access, may face difficulties with repeated sampling. It is a method that is exposed to potential errors represented by a variability in the measurement, inconsistent sampling times or variations in laboratory analysis can affect the accuracy of the results. Following a precise protocol is essential, but this may not always be feasible in high-volume clinical settings. Differences in age, body weight, comorbidities (e.g., liver disease), and body composition can affect iohexol pharmacokinetics, necessitating protocol adjustments. In patients with severely impaired renal function, prolonged clearance times require extended sampling intervals, increasing the complexity and inconvenience. Although rare, the use of iohexol may pose risks, such as allergic reactions or mild nephrotoxicity in vulnerable patients. Close monitoring is necessary to minimize adverse effects, adding to the procedural demands. Methods like creatinine- or cystatin C-based eGFR estimates, while less accurate, are more practical for routine clinical use due to lower cost and invasiveness.

Newer non-invasive or minimally invasive approaches may overshadow iohexol clearance in certain settings [22]. To reduce the complexity, abbreviated kinetic profiles have been proposed, but these tend to decrease the precision of the results, especially in patients with advanced stages of CKD, as emerged from comparative studies between standard and simplified methods (iohexol).

 

Conclusions

The iohexol clearance method is a gold standard for GFR estimation in specific clinical and research contexts, providing unmatched accuracy. However, its high costs, procedural complexity, invasiveness, and dependency on specialized resources significantly limit its applicability in routine healthcare.

Simplified protocols and further technological advancements could help mitigate these barriers, broadening its accessibility. GFR estimation with iohexol involves some secondary difficulties, both in terms of high costs and the need to procure specific materials and handle a significant number of blood samples [23]. This limits the methodology to research settings or specialized centers. Unlike the gold standard creatinine-based method, which is less expensive and practicable in all healthcare settings, the use of iohexol requires advanced equipment and specifically trained personnel. The iohexol method, if not performed correctly, tends to overestimate GFR in patients with stage III and IV chronic kidney disease (eGFR < 40 mL/min/1.73 m²). Although iohexol is currently used in several research centers and specialized clinics in Europe, it remains poorly available in many healthcare settings due to staff training requirements. This logistical limitation reduces the applicability of the method in routine clinical settings, making regular monitoring of GFR with iohexol difficult in many peripheral regions. Countries such as Sweden have integrated the method as part of standard care, demonstrating the effectiveness of this approach in an advanced healthcare setting, but its use is still limited to specific cases or large clinical studies (iohexol). In conclusion, although the iohexol method is accurate and represents a valid alternative to traditional markers such as inulin, it still has significant disadvantages that limit its large-scale adoption, especially in settings with limited resources or in the absence of specialized laboratory technical staff.

 

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Come valutare la velocità di filtrazione glomerulare e quale metodo è considerato il più affidabile?

Pubblicato il Scarica PDF

Fabrizio Cristiano

DOI: 10.69097/41-04-2024-02

Fabrizio Cristiano1,2, Cosima Posari1, Benito d’Angelo1, Alessandra Schiazza1, Anna Gigante1, Ludovico Caravelli1, Alessandra Piano1, Stefania Fulle2, Jenny Cristiano5, Ginevra di Matteo2, Guillermo Rosa Diez4, Vittore Verratti3

1 UOSD Nefrologia e Dialisi Ospedale di Ortona, Asl 2 Lanciano Vasto Chieti
2 Department of Neuroscience, Imaging and Clinical Science, University “G. d'Annunzio” Chieti - Pescara, 66100 Chieti, Italy
3 Department of Psychological, Health and Territorial Sciences, University “G. d’Annunzio” Chieti - Pescara, 66100 Chieti, Italy
4 Servicio de Nefrología, Hospital Italiano, Buenos Aires, Argentina
5 UOC Farmacia Ospedaliera – Ospedale Clinicizzato di Chieti – Asl 2 Lanciano Vasto Chieti

Corrispondenza a:
Fabrizio Cristiano
UOSD Nefrologia e Dialisi Ospedale di Ortona, Asl 2 Lanciano Vasto Chieti
Department of Neuroscience, Imaging and Clinical Science, University "G. d'Annunzio" Chieti - Pescara, 66100 Chieti, Italy;
E-mail: fabrizio.cristiano@asl2abruzzo.it

 

Abstract

La prevalenza della malattia renale cronica (CKD) continua ad aumentare a livello globale, sia per un aumento della morbilità e della mortalità associate sia per le implicazioni significative sulla qualità della vita dei pazienti e sulle economie nazionali. La malattia renale cronica spesso progredisce senza essere riconosciuta dai pazienti e dai sanitari, nonostante la diagnosi si basi su due semplici misure di laboratorio: velocità di filtrazione glomerulare stimata (eGFR) e analisi delle urine. La misura della GFR è correlata alla fisiologia renale, in particolare al concetto di clearance, con la creatinina identificata come un marcatore endogeno adatto per stimare la clearance della creatinina (CrCl). In base a questo principio sono state sviluppate varie equazioni per calcolare la CrCl o il GFR stimato (eGFR) utilizzando quattro variabili che incorporano la creatinina e alcune informazioni demografiche, come sesso ed età. Tuttavia, la misurazione della creatinina richiede la standardizzazione per ridurre al minimo la variabilità del dosaggio tra i laboratori. L’accuratezza di queste equazioni rimane controversa in alcuni sottogruppi di pazienti. Per questi motivi, sono stati ideati ulteriori modelli matematici per migliorare la stima della CrCl, in particolare quando la raccolta delle urine non è praticabile, in pazienti anziani o debilitati e in soggetti con traumi, diabete o obesità. Attualmente, l’eGFR negli adulti può essere immediatamente misurato e riportato utilizzando equazioni basate sulla creatinina tracciabili tramite spettrometria di massa con diluizione isotopica. In conclusione, sfruttando le conoscenze della fisiologia renale, l’eGFR può essere impiegato clinicamente per la diagnosi precoce e il trattamento della malattia renale cronica, nonché come strumento di sanità pubblica per stimarne la prevalenza.

Parole chiave: marcatori di filtrazione glomerulare, creatinine, cistatina C, inulina, iohexol

Ci spiace, ma questo articolo è disponibile soltanto in inglese.

Introduction

The prevalence of chronic kidney disease (CKD) continues to escalate globally, accompanied by an increase in morbidity, mortality, and significant implications for the quality of life of patients and the economies of nations. Any clinical condition resulting from a reduction in the number of functioning nephrons can progress into chronic renal failure, defined by the KDIGO guidelines as “abnormalities in kidney structure or function, present for 3 months, with health implications” [1].

In the real world, chronic kidney disease is a silent ailment often progressing unnoticed by patients and physicians, although the diagnosis relies on two simple laboratory measures: estimated GFR (eGFR) and urine analysis (screening for albuminuria/proteinuria). The glomerular filtration rate remains the premier comprehensive indicator of renal function as it assesses renal clearance and is directly related to the functioning renal mass, serving to classify CKD into stages, calculate medication dosages, and prepare for invasive studies with contrast medium. Early diagnosis of chronic kidney disease aids in delaying progression and reducing associated morbidity and mortality.

 

Identification of the Glomerular Filtration Process for GFR Measurement in Clinical Practice

Carl Ludwig (1816-1895), pioneered of glomerular filtration identified the glomerulus as a filter. This filtration is regulated by the hydrostatic pressure and modulated by the contraction and vasodilation of the afferent and efferent arterioles. He further hypothesized that the filtered volume decreased along the tubules due to reabsorption, thereby concentrating the end products in the urine [2]. However, to apply the concept of GFR in clinical settings, it was imperative to identify a solute removed solely by filtration, without reabsorption or secretion in the nephron. Later Paul Rehberg pinpointed creatinine as such a solute, given its endogenous production, filtration, and presumed lack of reabsorption or excretion.

Comparative summary of GFR estimation equations
Figure 1. Comparative summary of GFR estimation equations, including Cockcroft-Gault, simplified MDRD-4, CKD-EPI creatinine and cystatin C, and the FAS method. These formulas incorporate variables such as age, weight, serum creatinine, and patient demographics to determine renal function.

 

Estimation of GFR with Endogenous Markers

Creatinine-Based Glomerular Filtration Estimation

Creatinine remains the most widely utilized endogenous marker for estimating renal function in clinical practice, research, and animal models. It is a waste product of regular muscle metabolism. Creatinine, not being protein-bound, is freely filtered by the glomeruli; however, its synthesis is not constant, as it is determined by daily protein intake and muscle trophism. It is also subject to both secretory and reabsorptive mechanisms [3]. These conditions restrict the utility of creatinine as a renal function marker. Gender differences in tubular secretion have also been documented: males may secrete more creatinine than females, which could result in discrepancies in GFR estimation between male and female animals [4].

The initial method to measure creatinine, developed in 1886, was the alkaline picric acid reaction of Jaffé (a colorimetric method). This method’s interference with chromogens, such as bilirubin, glucose, or hemoglobin, led to inaccuracies in humans. In rodents, non-specific chromogens could overestimate creatinine by a factor of five. Different methods have been adapted to measure serum creatinine. The enzymatic determination, now considered the reference method in rodents, was validated in 2007 with various reactions with the aid of creatininase, creatinase, and sarcosine oxidase [5]. The measurement of creatinine in serum is prone to different types of error, interferences and imprecision. Serum creatinine certainly represents the most practical and least expensive measurement for stable glomerular filtration rate, however it presents some limitations in the interpretation of the results which may be secondary to both tubular secretion and the presence of muscle mass and protein intake. Even the absolute value of creatinine is subject to some variations such as the reference intervals of each analysis method of each laboratory with the risk of altering each glomerular filtration rate analysis equation. There are limitations in estimating creatinine secondary to muscle trophism because it is a product of muscle catabolism and results difficult in patients with extremely low or high muscular mass (e.g., anorexia, obesity or weight lifter). Creatinine is secreted by tubules and this explains why creatinine cleareance overestimates true GFR. Drugs, such as trimethoprim and cimetidine, also interfere with this tubular secretion and this explains why during their intake there is an increase in creatinine values ​​without evident alterations in GFR. The absolute value of creatinine could be altered in some pathological conditions such as liver failure and rhabdomyolysis. The absolute value of creatinine has physiological limits for an accurate estimate of the glomerular filtration rate [20].

Creatinine Clearance Over 24 Hours and Estimation of GFR Using Endogenous and Exogenous Markers

24-hour creatinine clearance has been a prevalent method for assessing GFR in animal models. Yet, it is crucial to acknowledge that the limitations of serum creatinine as a renal function marker impact the precision and accuracy of the 24-hour collection [6]. Blood samples are necessary to measure serum creatinine.

GFR Estimation Using Cystatin-C

Cystatin-C (CysC) is a low molecular weight protein (13KDa) of the family of cysteine ​​protease inhibitors. It is produced by all the nucleated cells of the body, filtered by the glomerulus, and then reabsorbed and metabolised by tubular epithelial cells, excluding its use for clearance on 24 hour urine. Like cratinine, the determination of cystatin C is influenced by factors such as sex, age and chronic inflammatory state [7], but it provides a more precise estimate of glomerular filtration as it is not affected by variables such as muscle mass and activity, or dietary protein intake.

 

GFR Estimation with Exogenous Markers: Inulin Clearance

The fructose polymer inulin has always represented a specific method for medical students for measuring glomerular filtration [8] due to the intrinsic characteristics of the molecule; in fact inulin is not metabolised, does not bind to plasma proteins and is freely filtered by the glomeruli without being reabsorbed or secreted by the tubular cells. However, considering inulin as the gold standard of the glomerular filtration method presents some limitations: the high cost and cumbersome methods for developing the process such as use with radioactive markers, poor solubility in water and demanding preparation for the solution to be injected (substance dissolve, filter and heat at high temperatures for many hours to remove inulin fragments). Once prepared, inulin is administered as a single intravenous bolus or continuous infusion and plasma and/or urine are collected at different times to calculate clearance. All these steps do not make this method universal.

 

Sinistrin: The New Inulin?

The measurement of GFR can also be obtained by evaluating the kinetics of Sinistrin FITC and in particular by estimating the half-life. Sinistrin has the advantage of having a lower molecular weight (3500 Da) compared to inulin, it is hightly soluble in aqueous solvents at room temperature, it can be used and labeled with FITC fluorescein [9]. Unlike inulin, it does not require any filtration and has the advantage of being able to be used using transcutaneous devices. An instrument composed of two LEDs is required for measuring fluorescence and transcutaneous GFR. The method consists in the intravenous infusion of Sinistrin with the FITC chromophore which emits the fluorescence captured by the instrument. Transcutaneous measurement has proven to be a good method for measuring renal function in murine models and has the advantage, especially in animals, of measuring glomerular filtration in the absence of particular traumas [21].

 

Transcutaneous Methods for GFR Measurement

To determine glomerular filtration, the intravenous injection of a sinister FITC molecule was studied and then the variation in fluorescence was studied using a device positioned on the skin. The change in fluorescence is used to calculate the elimination half-life of the marker and then convert the half-life data to GFR (ml/min). The main advantage of this method is its non-invasiveness, however it has limitations as it is an indirect method for measuring GFR and therefore requires conversion factors. The main advantage is its independence from blood/urine sampling and laboratory tests with real-time GFR examination, however a limitation to be evaluated is the high cost of the device ($1000) which makes it impractical for clinical practice [10].

 

Radiolabeled Tracers

The two most commonly used radiolabeled markers are ethylenediaminetetraacetic acid with radioactive chromium-51 (51Cr-EDTA) and diethylenetriamine pentaacetic acid with radioactive technetium-99 (99mTc-DTPA), both of which are low molecular weight and freely filtered by the glomerulus.

The method consists in measuring the plasma and urinary clearance of single intravenous injections of radiolabeled substances or alternatively intraperitoneal injection [11]. Blood and urine samples are taken and processed using a gamma counter that estimates GFR. 99mTc-DTPA has been used in healthy male Wistar rats and in animals with chronic kidney disease or doxorubicin-induced nephritic syndrome [12]. The main limitation of this technique derives from the use of radioisotopes, which are not easy to find and which require special authorization and specific conservation; furthermore it presents toxicity for operators who must use specific precautions and careful waste management.

99mTc can dissociate from DTPA and up to 13% of 99mTc-DTPA can bind to plasma proteins, resulting in an underestimate of GFR [13]. These markers could be useful for verifying GFR but are not preferable in clinical practice.

 

Non-Radiolabeled Contrast Agents in GFR Assessment

Among the various possibilities for measuring GFR is iothalamate, an ionic contrast agent derived from tri-iodobenzoic acid with a molecular weight of 637 Kda. Bell proposed a rapid HPLC method to detect iothalamate and para-aminohippuric acid in rat serum and urine [14], giving an estimate of both GFR and renal blood flow. This method is not easy to apply as it involves both central venous catheterization, a method not without serious side effects, and the simultaneous collection of blood and urine.

 

Iohexol/Iohexol-DBS

Iohexol (Omnipaque™, GE Healthcare) is a molecule used as a contrast agent. It is excreted unmetabolised by glomerular filtration, without reabsorption or secretion by renal tubular cells without undergoing hepatic metabolism or interference with blood cells. Its use as a reference method for measuring GFR was established almost 30 years ago in humans [15]. In recent years, the filtration of iohexol in mice has been studied by intravenous injection and subsequent blood sampling for pharmacokinetic analysis. Iohexol is measured by HPLC chromatographic analysis. Schultz et al. described the plasma clearance of iohexol in rats in 2014 using liquid chromatography-electrospray-mass spectrometry (LC-ESI-MS). They administered different doses of iohexol via the tail vein to male HsdRCCHan:WIS rats, and the animals were sacrificed at different times after infection with iohexol (15, 30, 60, and 90 minutes) to obtain blood samples. Passos et al. validated the plasma clearance of iohexol in rats [16] against the “classical” gold standard, inulin clearance, using capillary electrophoresis, observing a correlation between iohexol and inulin clearance (r = 0.792). However, the procedure required large amounts of blood. Carrara proposed the measurement of GFR through experiments on mice using the following scheme: administration of iohexol (129.4 mg) intravenously and subsequent determination on four blood samples after the infusion at times (20, 40, 120, 140 minutes) [17]. While Luis-Lima proposed a further simplified scheme with fewer side effects, always in mice; intravenous administration of 6.47 mg of iohexol and subsequent blood sampling (approximately 10 μL each) after the infusion at times (15, 35, 55 and 75 minutes) with determination of iohexol by HPLC-UV on the blood and with factor correction equal to 0.89. The advantage of both methods was represented by the fact that they were comparable in their results not only in mice with normal renal function but also in mice with CKD and with a single kidney following nephrectomy [16].

This method has the advantage of using a small quantity of blood, approximately 10 μL, offering the advantage of carrying out serial samples over time to evaluate the progress of renal function.

Rodríguez-Rodríguez AE et al. have proposed the possibility of using dried blood samples (DBS) while maintaining adequate precision in sample processing [16]. The method consisted of sampling 5 μL of blood with heparin tubes at times 15, 30, 45, 60, 75 minutes after the infusion of Iohexol and subsequent drying of the blood sample on filter paper (Whatman 903, GE Healthcare) to 24 hours and subsequent extraction with 5% perchloric acid with centrifuge [18]. The measurement of Iohexol was carried out with the HPLC method; this procedure showed high precision in the determination of GFR in mice.

Turner established a new method of determining GFR using Iohexol with two blood samples and compared it to better known methods such as inulin, creatinine and cystatin-C [19]. Intravenous infusion of 25 mg/kg of Iohexol was performed and blood samples were taken at times 2, 5, 10, 20, 30, 60, 90, 120, 180, 240, 300 minutes; the result shawed that the samples taken at the 30 and 90 minute periods represented the average of the values ​​of all eleven blood samples. Thus, Iohexol was proposed as a method to determine GFR through a single intravenous infusion of 25 mg/kg of Iohexol, with subsequent measurements taken within 30 and 90 minutes.

Iohexol represents a precise method for measuring GFR however it may have measurement errors due to sample preparation.

 

Conclusions

The study of the various methods for calculating GFR is still a topic of study today so that we can achieve a simple, rapid and reproducible measurement in every peripheral structure. The ideal method should avoid 24-hour urine collection, reduce the amount of blood, avoid radiolabeled substances and speed in sample calculation. We have listed several types, each with potential disadvantages. Creatinine and cystatin-C, despite being widely used, sometimes have limitations in determining the real GFR. Radiolabeled markers (99mTc-DTPA and 51Cr-EDTA) are cheap but unsafe and should be replaced with an alternative method. Inulin represents the most precise method but is difficult to reproduce in a clinical environment due to the costs and complexity of the procedure. Iothalamate is less precise than inulin but more convenient and easier to use. Iohexol represents a precise and safe method but to date it has been studied in mouse models. An alternative may be represented by fluorescent markers such as FITC inulin or FITC sinistrin, also used in the transcutaneous method with the advantage of instantaneous measurement and no use of optimal methodical blood sampling in animals [6]. In conclusion, the method for measuring GFR should depend on the care setting, the resources available, the experience of the researcher and the safety and well-being of the animals.

 

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