New Genetic Variants Involved in the Pathogenesis of Autosomal Dominant Alport Syndrome: A Familial Case Report

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Sandra La Rosa

DOI: 10.69097/42-02-2025-07

Sandra La Rosa1, Stefania Pesce1, Simona Lombardo1, Marilena C. Fiorino2, Antonio Granata3

1 U.O.S.D. Nefrologia e Dialisi, ASP Agrigento P.O. Sciacca (AG), Italy
2 U.O.C. Anatomia Patologica ASP Agrigento P.O. Sciacca (AG), Italy
3 U.O.C. Nefrologia e Dialisi Azienda Ospedaliera Cannizzaro, Catania (CT), Italy

Corresponding author:
Sandra La Rosa
Unità Operativa di Nefrologia e Dialisi, “Ospedale Giovanni Paolo II”
Via Pompei, Sciacca (AG), Italia
Numero di telefono/Fax 0925/962481
E-mail address: slarosa76@yahoo.it

 

Abstract

Alport syndrome is a hereditary disorder characterized by hematuria, proteinuria and progressive renal failure, frequently associated with extrarenal manifestations. The pathogenic variants of the COL4A5 gene are associated with X-linked Alport syndrome while those of the COL4A3 and COL4A4 genes are associated with the autosomal recessive (AR) or dominant (AD) form. The disease is characterized by considerable phenotypic variability linked to the different genes involved and the different mutations present, so the symptoms manifest themselves in different frequencies depending on the case. The existence of an autosomal dominant form of Alport syndrome has been identified in recent years thanks to next generation gene sequencing (NGS) techniques which have made it possible to highlight unknown genetic variants of Alport syndrome. The family studied by us presents concomitant heterozygous alterations of the COL4A3 genes (c.1029+5G>A with MAF 0 and c.3211-7A>G with MAF 1:100000), heterozygous alterations of the MTHFR gene (both C677T and A1298C) and homozygous alteration of the PAI-1 gene. While the variant c.3211-7A>G, as shown by genetic databases (ClinVar), appears to be benign, the intronic variant c.1029+5G>A (caused by exon skipping) can be classified as pathogenic due to its characteristics and the fact that it co-segregates with the phenotype within the family. The histological data, in one of the sisters, highlighted the presence of a discrete global glomerular sclerosis and the ultrastructural investigation a thinning of the glomerular basement membrane. New mutational variants of the COL4A3 gene may play a role as risk variants for the development of chronic kidney disease.

Keywords: new genetic variants of COL4A3, pannel of thrombophilia, hyperhomocysteinemia, glomerulosclerosis, next generation sequencing, exon skipping

Sorry, this entry is only available in Italiano.

Introduzione

La Sindrome di Alport (AS) è una patologia glomerulare ereditaria caratterizzata da ematuria, proteinuria e progressiva compromissione della funzione renale con una prevalenza di 1 su 2000 individui per la forma X-linked ad 1 su 100 individui per la forma autosomica dominante [1]. Le prevalenze effettive della patologia sarebbero ancora maggiori se dovessimo considerare le varianti non ancora esaminate. L’alta frequenza delle varianti patogene dei geni legati alla Sindrome di Alport suggerisce che altri fattori genetici e ambientali mitigano la corrispondente manifestazione clinica della malattia [2]. Geneticamente, la Sindrome di Alport è complessa. Sono note tre tipologie di ereditarietà e un ampio numero di mutazioni sono state già identificate nei geni che codificano per il collagene di tipo IV. In genere, ogni famiglia presenta un’unica mutazione. La forma X-Linked (XL-AS; approssimativamente l’85%) causata da mutazioni nel gene COL4A5 (Xq22-25), predomina ed è seguita dalla forma Autosomica Recessiva (AR-AS) causata da mutazioni nei geni COL4A3 o COL4A4 (2q35-37). Negli ultimi anni la forma Autosomica Dominante (AD-AS) legata a mutazioni in una sola delle copie del gene COL4A3 o COL4A4 (stato di eterozigosi) è stata diagnosticata sempre con una maggiore frequenza [3]. Nel 1997 venne descritta la prima famiglia che presentava un albero genealogico suggestivo di ereditarietà autosomica dominante. La caratterizzazione di questa famiglia a livello molecolare ha messo in evidenza una mutazione di splicing a carico del gene COL4A3 con perdita dell’esone 21. Tale mutazione è presente in eterozigosi in tutti i membri affetti, rappresentando il primo riscontro molecolare della forma autosomica dominante della sindrome di Alport [4]. L’utilizzo delle nuove tecniche di sequenziamento genico (Next Generation Sequencing) che consentono di studiare contemporaneamente i tre geni responsabili della Sindrome di Alport ha consentito di dimostrare che le mutazioni dei geni COL4A3 e/o COL4A4 sono in realtà più frequenti di quanto si riteneva in passato (sono state individuate più di 1000 mutazioni diverse nei geni COL4A5, COL4A3 e COL4A4) e che la forma dominante di SA rappresenta il 20-30% dei casi [5].

Per l’individuazione delle nuove varianti sono stati condotti test di splicing sia in vivo che in vitro, i quali hanno dimostrato lo skipping dell’esone nelle varianti identificate [6]. Sono stati eseguiti test di splicing in vitro, utilizzati minigeni e analisi di mRNA di campioni, per determinare la patogenicità di alcune varianti sinonime o silenti rilevate in alcuni pazienti con sospetta sindrome di Alport Autosomica Dominante. Nel 50% di queste varianti identificate vi era lo skipping dell’esone che potrebbe essere implicato nella perdita dei siti di legame per i fattori di trascrizione dello splicing ad esempio nei siti accettori e negli enhancers dello splicing esonico. Possiamo affermare che alcune varianti possono non alterare la sequenza amminoacidica della proteina codificata ma influenzano notevolmente lo splicing pre mRNA [7].

Sono stati presi in esame diversi individui provenienti da diversi ceppi familiari, diverse razze ed etnie che sottoposti ad analisi mutazionale mediante sequenziamento dell’esone e amplificazione hanno rilevato oltre 47 mutazioni che influenzano il corretto splicing dell’mRNA. Questo risultato amplia lo spettro genotipico delle mutazioni note nella sindrome di Alport che permetterà di effettuare le giuste correlazioni genotipo-fenotipo negli individui presi in esame [8].

Il nostro obiettivo è quello di fare conoscere le nuove varianti geniche riscontrate nella famiglia oggetto di studio, in eterozigosi; queste nuove varianti determinano una ampia variabilità fenotipica e si associano, in alcuni casi, a una difficile diagnosi di Sindrome di Alport.

 

Caso clinico familiare

Descriviamo il caso clinico di una famiglia siciliana in cui sia gli uomini che le donne presentano insufficienza renale cronica avanzata (alcuni in trattamento emodialitico) e in alcuni componenti è presente solo ematuria. Abbiamo seguito due sorelle di età diversa, una di 58 anni e l’altra di 70 anni, entrate nello stesso anno in dialisi. La sorella più giovane ha una storia clinica di dermatite atopica, poliabortività, tromboflebiti ricorrenti, artralgie diffuse migranti, tumore renale (per cui è stata sottoposta a nefrectomia), microematuria sin da piccola, trait talassemico, proteinuria dapprima nefritica poi nefrosica associata a insufficienza renale cronica da qualche anno in rapida progressione; all’esame ecografico presenta inversione dell’ecogenicità cortico-midollare con associate cisti renali e con esami immunologici tutti negativi. Altresì non presenta lesioni oculari e l’esame audiometrico risulta essere nella norma.

La sorella più anziana ha una storia di microematuria, proteinuria in range nefritico (e non nefrosico come la sorella più giovane), normoacusia, dolori articolari diffusi, cisti renali, meningioma cerebrale, fibrosi epatica, tiroidite, ANA positività (1:60), trait talassemico, insufficienza renale cronica da qualche anno e perdita della memoria. Il padre e lo zio paterno erano in dialisi (il padre aveva effettuato nel ’78 biopsia renale con diagnosi di verosimile Sindrome di Alport e lo zio paterno non ha effettuato biopsia renale ma aveva proteinuria in range nefritico e all’ecografia presentava cisti renali); il nonno aveva insufficienza renale ma non era in dialisi. Al cugino (figlio dello zio paterno) per trombosi dell’oliva portale, del ramo portale di destra e della vena splenica veniva riscontrata una mutazione in omozigosi del gene MTHFR (Figura1).

Figura 1.  Albero genealogico della famiglia.
Figura 1.  Albero genealogico della famiglia.

In considerazione della storia familiare di nefropatia e della sintomatologia clinica la sorella più giovane è stata sottoposta a biopsia renale (Figura 2). La sorella più anziana non ha effettuato la biopsia renale perché al momento della nostra osservazione i suoi reni erano già di piccole dimensioni.

Figura 2. Biopsia renale della paziente più giovane
Figura 2. Biopsia renale della paziente più giovane. A: Glomerulo con sclerosi totale; B: Glomerulo con sclerosi dell’80%; C: Altro glomerulo sclerotico all’80%; D: Due glomeruli entrambi con sclerosi totale; E: Atrofia tubulare; F: Glomerulo normale.

L’esame istologico della sorella più giovane metteva in evidenza una discreta (11 glomeruli su 14 isolati) sclerosi glomerulare globale o superiore all’80% del flocculo, fibrosi interstiziale, atrofia tubulare e lieve danno vascolare arterio-arteriosclerotico. L’indagine immunoistochimica ha messo in evidenza una positività discreta per IgM (++); altresì è stata eseguita indagine immunoistochimica per le catene alfa 3 e alfa 5 del COL IV che sono risultate entrambe normalmente espresse. L’indagine ultrastrutturale evidenziava un assottigliamento della membrana basale glomerulare (valore medio di 110-120 nanometri) con spiccata evidenza della lamina densa e in alcuni tratti con lieve ampliamento dello spazio sotto-endoteliale. La lamina densa in limitati segmenti appare reduplicata.

La sorella più anziana ha una figlia di 41 anni anch’essa con storia di microematuria fin da piccola e difficoltà nel concepimento; di seguito si riporta ultimo suo esame delle urine (Tabella 1).

Esame Urine Chimico-fisico (met. Riflettometrico)
pH 5,0 *↓ Unità di misura [5,5 – 7,5]
Glucosio 0 mg/dL [< 10]
Corpi chetonici 0 mg/dL [< 5]
Proteine < 15 mg/dL [< 15]
Rapporto proteine/creatinina – screening < 150 mg/g [< 150]
Rapporto albumina/creatinina – screening < 30 mg/g [< 30]
Emoglobina > 0.75 *↑ mg/dL [< 0,03]
Esterasi Leucocitaria                                   Vedasi dato Leucociti [Assente]
Nitriti Assenti [Assenti]
Densità Relativa
(met. rifrattometrico)		 1,016 [1,005 – 1,030]
Conduttività
(met. Impedenziometrico)		 19,5 mS/cm [3,0 – 38,0]
Esame citofluorimetrico e morfologico
(met. Citometria a flusso in fluorescenza)
Eritrociti 195 cell/µL [< 15]
Leucociti < 20 cell/µL [< 20]
Cellule Ep. Squamose 55 *↑ cell/µL [< 20]
Batteri Assenti [Assenti]
Tabella 1. Esame delle urine della figlia della paziente più anziana.

Per la storia clinica delle due sorelle si è proceduto a effettuare in entrambe uno studio genetico con analisi mutazionale del pannello dei geni associati a sindrome di Alport con il riscontro di varianti genomiche non note in letteratura. Si tratta di varianti presenti in eterozigosi del gene COL4A3; sono varianti a penetranza completa con trasmissione agli individui della stessa famiglia che differiscono tra loro per clinica, sintomatologia e comorbidità presenti al momento della rilevazione (NM_000091.4: c.1029+5G>A con MAF 0 e NM_000091.4: c.3211-7A>G con MAF 1:100000) (Figura 3).

Figura 3. Analisi genetica delle due pazienti.
Figura 3. Analisi genetica delle due pazienti.

La variante NM_000091.4: c.3211-7>G, nel database di genetica ClinVar, è risultata essere verosimilmente benigna, mentre diverso è il ruolo della variante genetica intronica c.1029+5G>A presente in eterozigosi. Quest’ultima essendo una variante genetica nuova è assente sui database di popolazione e non è descritta in letteratura scientifica, ma è predetta alterare lo splicing del gene. Quest’ultima variante sembrerebbe determinare la perdita di un introne e può avere un ruolo come variante di rischio per lo sviluppo di malattia renale cronica contribuendo a una maggiore complessità nella manifestazione clinica della Sindrome di Alport.

Tale famiglia è stata sottoposta a screening patogenetico al fine di studiarne la trasmissione delle diverse varianti mutazionali.

Per questo motivo anche la figlia della sorella più anziana è stata sottoposta a screening genetico che ha documentato la stessa variante genetica (Figura 4).

Figura 4. Analisi genetica della figlia della paziente più anziana.
Figura 4. Analisi genetica della figlia della paziente più anziana.

Le indagini hanno identificato nel gene COL4A3 in eterozigosi la nuova variante genetica intronica causata dallo skipping dell’esone: NM_000091 (COL4A3): c.1029+5G>A classificabile come patogenetica per le sue caratteristiche e per il fatto che essa co-segrega con il fenotipo all’interno della famiglia.

Per spiegare la progressione della nefropatia, si è proceduto a richiedere (in considerazione anche della storia familiare ovvero della presenza della mutazione in omozigosi del gene MTHFR nel cugino paterno) lo studio del pattern trombofilico (Tabella 2).

Biomolecolare

(La ricerca delle mutazioni è stata eseguita mediante PCR ed ibridazione con sonde allele specifiche)
Mutazione G1691A Fattore V Leiden

Assente

 Genotipo

Omozigote Normale-Wild Type
Mutazione C677T MTHFR Presente Genotipo Eterozigote
Mutazione A 1298C MTHFR Presente Genotipo Eterozigote
Fattore V° Leiden (Mutaz. H 1299 R) Assente Genotipo

Omozigote Normale-Wild Type
Mutazione G 20210 A FATT. SECONDO Assente Genotipo

Omozigote Normale-Wild Type
PAI-1 Presente Genotipo Omozigote 5G
Tabella 2. Pattern trombofilico di entrambe le pazienti.

In entrambe le nostre pazienti è stata riscontrata una mutazione del gene MTHFR C677T e A1298C in eterozigosi e quella di PAI-1 in omozigosi. La mutazione MTHFR è un difetto genetico che provoca la riduzione o la perdita di attività dell’enzima metilen-tetraidrofolato reduttasi. La conseguenza di tale fenomeno è l’aumento dei valori di omocisteina nel sangue che si associa da una diminuzione dei livelli ematici di acido folico.

Le mutazioni dell’MTHFR, presenti nelle nostre pazienti, si associano quindi a iperomocisteinemia che già vent’anni fa è stata riconosciuta caratteristica comune nei pazienti con insufficienza renale.

Nelle due sorelle infatti, vi sono livelli elevati di omocisteina (Tabella 3).

Tabella 3. Dosaggio della omocisteina di entrambe le pazienti.
Tabella 3. Dosaggio della omocisteina di entrambe le pazienti.

 

Discussione

La Sindrome di Alport è dovuta a mutazione nei geni codificanti per le catene del collagene di tipo IV che risulta essere il principale componente della membrana basale, a sua volta è interposta tra epitelio ed endotelio a livello glomerulare. Le catene del collagene di tipo IV sono dette catene alfa. Sono state identificate sei catene per il collagene di tipo IV codificate da sei geni. I geni codificanti per le catene alfa 3 ed alfa 4 (COL4A3 e COL4A4) sono localizzati sul cromosoma 2, quelli codificanti per le catene alfa 5 ed alfa 6 (COL4A5 e COL4A6) sono localizzati sul cromosoma X e i geni che codificano per le catene alfa 1 e alfa 2 (COL4A1 e COL4A2) sono localizzati sul cromosoma 13 [9]. Le catene di collagene sono ampiamente distribuite nei vari distretti corporei e per tale motivo la sintomatologia della Sindrome di Alport è molto varia e va da sintomi tipicamente associati a un danno renale ad alterazioni sensoriali (ipoacusia, lenticono e macchie retiniche) [10], a manifestazioni rare come leiomiomi soprattutto a livello bronchiale ed esofageo [11] ad aneurismi dell’aorta toraco-addominale in particolar modo nei soggetti di sesso maschile [12].

La malattia si può presentare con una grande variabilità fenotipica, legata principalmente ai geni coinvolti e all’estensione della mutazione per cui i diversi sintomi possono appalesarsi con frequenza diversa a secondo dei casi [13]. Esiste comunque una grande difficoltà nella interpretazione delle varianti dovuta all’incompleta o conflittuale evidenza di patogenicità. Delle recenti linee guida hanno indicato che le caratteristiche cliniche, la storia familiare di insufficienza renale o persistente ematuria, insieme alla identificazione di varianti genetiche, supportano in maniera forte la diagnosi di Sindrome di Alport [14].

I familiari di I grado di una persona affetta da Sindrome di Alport in forma autosomica dominante hanno il rischio del 50% di presentare la variante patogenetica, indipendentemente dal sesso.

La forma autosomica dominante è caratterizzata da lenta progressione del danno renale, da notevole variabilità intra ed interfamiliare e da una penetranza che è del 95%. I pazienti presentano un quadro clinico caratterizzato da microematuria, proteinuria (in alcuni casi isolata) e insufficienza renale dopo la terza decade. Le manifestazioni extra-renali sono rare (non sono state descritte le anomalie oculari tipiche dell’Alport e l’ipoacusia insorge in tarda età) [15].

La progressione della malattia, a seconda dell’ereditarietà, è molto variabile e va da un andamento molto rapido che richiede una terapia renale sostitutiva in adolescenza o nella prima età adulta a uno molto lento caratterizzato da una funzione renale normale per lunghi periodi di tempo [16].

La rapida progressione della malattia renale nella sorella più giovane, che è entrata in dialisi all’età di 58 anni, probabilmente è dovuta al riscontro in quest’ultima di proteinuria in range nefrosico.

A causa delle iniziali difficoltà nell’effettuare una diagnosi corretta, abbiamo sottoposto entrambe le sorelle anche a Test di Fabry, risultato negativo (Figura 5).

Figura 5. Test di Fabry di entrambe le pazienti.
Figura 5. Test di Fabry di entrambe le pazienti.

Proprio per le difficoltà diagnostiche presenti in alcuni casi e per consentire un inquadramento affidabile, l’Alport Syndrome Classification Working Group ha proposto un nuovo schema di classificazione fondato su criteri genetici, clinici e molecolari invece che solo su tratti istologici e clinici [17] (Tabella 4).

Tabella 4. Nuovo sistema di classificazione della sindrome di Alport.
Tabella 4. Nuovo sistema di classificazione della sindrome di Alport.

L’ampia variabilità fenotipica riscontrata nella famiglia, correlata alla variante mutazionale c.1029+5G>A del gene COL4A3 (non presente in letteratura), è associata anche a mutazioni dei geni del pattern trombofilico. Entrambe le sorelle infatti presentano una mutazione combinata, in eterozigosi, sia dell’MTHFR C677T e A1298C, che è riconosciuto essere associato a un alto rischio trombotico, malattie coronariche, aborti spontanei e difetti del tubo neurale [18, 19]. La paziente più giovane è infatti andata incontro a poliabortività con conseguente difficoltà nel concepimento. Gli aborti ricorrenti in gravidanza rappresentano una condizione molto frequente nelle pazienti che presentano polimorfismi dei geni della trombofilia [20].

Altresì entrambe le sorelle presentano, in omozigosi, il polimorfismo 4G/5G del gene PAI-1, che determina, in generale, una maggiore suscettibilità al tromboembolismo venoso soprattutto se associato ad altri disordini genetici del pattern trombofilico [21] e determina anche una maggiore suscettibilità al cancro [22].

In seguito alla mutazione di MTHFR si avrà una ostacolata conversione della omocisteina in metionina e questo determinerà un aumento dell’omocisteinemia. Si parla di iperomocisteinemia quando il suo valore risulta essere superiore a 15 µmol/L.

Nelle due sorelle vi sono livelli di omocisteina pari a 79,5 µmol/L (in quella più anziana) e 22,90 µmol/L (in quella più giovane). Il valore più basso di una delle due sorelle è verosimilmente correlato all’assunzione di acido folico che la paziente assume da due anni.

Prove crescenti mostrano che un livello elevato di omocisteina (Hcy) sia associato a un aumento del rischio di malattia renale cronica (CKD) [23]. L’associazione tra omocisteina plasmatica e GFR sembra essere lineare [24]. Kai et al. hanno scoperto che l’apoptosi dei podociti indotta dall’iperomocisteinemia svolge un ruolo importante nel danno renale nei topi [25]. È stato infatti dimostrato che l’Hcy induce disfunzione endoteliale inibendo la proliferazione delle cellule endoteliali e promuovendo una risposta infiammatoria. Ciò suggerisce che esiste una relazione tra iperomocisteinemia e danno renale, che alla fine porta ad apoptosi dei podociti, glomerulosclerosi focale o globale, atrofia tubulare, fibrosi interstiziale e diminuzione dell’eGFR.

È ipotizzabile che il danno ossidativo esercitato dall’iperomocisteinemia possa essere considerato concausa di aterosclerosi e, oltre a indurre una disfunzione dell’endotelio vascolare, possa essere causa di glomerulosclerosi che porterebbe alla progressione della insufficienza renale [26].

L’ipermocisteinemia porta quindi a un danno endoteliale ed è un importante fattore di rischio per le patologie cardiovascolari [27]. Nella paziente più anziana, in cui vi sono i più alti valori di omocisteina, vi è anche un’alterazione della memoria a lungo termine. Alcuni studi hanno messo in evidenza che l’iperomocisteinemia può favorire il declino cognitivo ed è un fattore di rischio non soltanto per le patologie cerebrovascolari ma anche per la demenza degenerativa. Questo avviene perchè l’iperomocisteinemia ha un’azione vasculotossica nei confronti dei piccoli vasi cerebrali, favorisce la formazione dei ROS che promuovono l’infiammazione, inibisce il recettore GABA con conseguente riduzione dell’attività della NADPH ossidasi e quindi favorisce lo stress ossidativo, attiva il recettore NMDA che promuove l’eccitotossicità e di conseguenza la degenerazione neuronale. Tutto ciò porta al declino cognitivo [28].

Nella famiglia oggetto dello studio l’estrema variabilità fenotipica è da correlare non solo alla presenza di una variante mutazionale del gene COL4A3 ma anche a mutazioni del pattern trombofilico che, insieme, possono determinare una progressione della malattia renale e quindi potrebbero essere considerate singolarmente come varianti di rischio per lo sviluppo e la progressione dell’insufficienza renale cronica.

Nella forma autosomica dominante della sindrome di Alport è comune l’ematuria, la proteinuria (anche se può non essere presente) e l’insufficienza renale è rara anche se il suo rischio è aumentato se si aggiungono altri fattori patogenetici [29].

A un recente meeting di un Gruppo di Studio sulle varianti dell’Alport, si è stabilito di estendere lo screening genetico alla ricerca di mutazioni nei geni COL4A3, COL4A4 e COL4A5 oltre che ai pazienti con fenotipo classico (con ematuria, insufficienza renale e/o storia di ematuria o insufficienza renale familiare), anche ai pazienti con proteinuria, sindrome nefrosica steroido-resistente, pazienti con glomerulosclerosi focale e segmentale (FSGS) glomerulonefrite ad IgA e pazienti con insufficienza renale cronica senza una causa apparente [30].

Ogni catena alfa del collagene di tipo IV ha un dominio amino non collagenico (NC), un dominio intermedio collagenico e un dominio carbossi non collagenico. Il dominio intermedio collagenico ha la sequenza Glicina-Xaa-Yaa dove X e Y sono spesso Prolina e Idrossiprolina. Sono stati individuati “hotspot” mutazionali di singoli aminoacidi come ad esempio in corrispondenza della Glicina nel dominio intermedio collagenico e della Cistina nel dominio carbossi non collagenico, che vengono considerate varianti ipomorfiche con un fenotipo clinico più lieve. Non è possibile definire la soglia della MAF (minore frequenza allelica) sopra la quale queste varianti sono considerate benigne soprattutto in considerazione della differente ereditarietà dell’Alport. L’interpretazione delle varianti ipomorfiche rimane comunque una sfida [31]. L’ampio spettro fenotipico delle patologie del collagene di tipo IV e l’eterogeneità mutazionale rendono difficile la diagnosi (anche tra ematuria familiare e sindrome di Alport) soprattutto nei piccoli nuclei familiari e nei casi sporadici dove l’analisi genetica fallisce.

Le varianti di alcuni pazienti con sospetto di Sindrome di Alport non vengono rilevate né con il sequenziamento dell’esone né con i test NGS (Next Generation Sequencing) per cui sono stati analizzati gli mRNA urinari dei geni COL4A3-A5 e gli mRNA di COL4A5 dei fibroblasti cutanei che hanno mostrato le mutazioni di geni non evidenziate con altri test genetici. In particolare è stata utilizzata la PCR e il sequenziamento diretto per l’analisi degli esoni con sequenze introniche vicine, corrispondenti ad anomalie dell’mRNA in cui il salto dell’esone è stato causa di una variante intronica e la ritenzione di un frammento intronico è stata causa di ulteriori varianti. L’analisi dell’mRNA per i geni rappresentanti della Sindrome di Alport dal campione delle urine o dai fibroblasti può essere oggi usata come nuova linea di diagnosi nei pazienti con sospetto di Sindrome di Alport risultati negativi al test genetico [32].

Sono comunque necessari più studi per le patologie del collagene di tipo IV per cercare di rendere più semplice la diagnosi, la terapia e di conseguenza anche la prognosi [33].

 

Conclusioni

La Sindrome di Alport autosomica dominante è una patologia di recente riscontro, grazie all’utilizzo delle nuove tecniche di sequenziamento genico (Next Generation Sequencing), con un basso rischio di lesioni oculari e ipoacusia, ma con un significativo rischio di sviluppare insufficienza renale in età avanzata rispetto alla forma X-linked. È difficile riuscire a fare una diagnosi differenziale, specialmente nei pazienti giovani, con l’ematuria familiare benigna e con le forma X-linked della sindrome di Alport nelle famiglie dove solo le femmine sono malate. La forma autosomica dominante della Sindrome di Alport è quindi caratterizzata da una lenta progressione del danno renale, da una consistente variabilità intra ed interfamiliare e da una penetranza che sembra aggirarsi intorno al 95%. Una corretta diagnosi è possibile solo se si riesce ad effettuare una indagine clinica completa nei vari componenti della famiglia, unitamente allo studio dell’albero genealogico e all’analisi genetica; solo alla fine si riesce a conoscere la prognosi. Nel caso della famiglia oggetto di studio, oltre ad essere presenti varianti mutazionali in eterozigosi del gene COL4A3 (non presenti in letteratura), sono presenti mutazioni di alcuni geni del pattern trombofilico ed entrambe le condizioni possono giocare un ruolo importante come varianti di rischio per lo sviluppo della malattia renale cronica, contribuendo a una maggiore complessità nella manifestazione clinica della Sindrome di Alport.

 

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The link between homocysteine, folic acid and vitamin B12 in chronic kidney disease

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Andrea Angelini1, Maria Laura Cappuccilli1, Giacomo Magnoni1, Anna Laura Croci Chiocchini1, Valeria Aiello1, Angelo Napoletano1, Francesca Iacovella1, Antonella Troiano1, Raul Mancini1, Irene Capelli1, Giuseppe Cianciolo1

1 Unità Operativa di Nefrologia, Dialisi e Trapianto, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Alma Mater Studiorum Università di Bologna, Italia

Corresponding author:
Irene Capelli
Department of Nephrology, Dialysis and Transplantation
Policlinico Sant'Orsola Malpighi
Via Massarenti 9, Bologna, Italy
email: irene.capelli4@unibo.it

 

Abstract

Patients with chronic kidney disease or end-stage renal disease experience tremendous cardiovascular risk. Cardiovascular events are the leading causes of death in these patient populations, thus the interest in non-traditional risk factors such as hyperhomocysteinemia, folic acid and vitamin B12 metabolism is growing.  Hyperhomocysteinemia is commonly found in CKD patients because of impaired renal metabolism and reduced renal excretion. Folic acid, the synthetic form of vitamin B9, is critical in the conversion of homocysteine to methionine like vitamin B12. Folic acid has also been shown to improve endothelial function without lowering homocysteine, suggesting an alternative explanation for the effect of folic acid on endothelial function. Whether hyperhomocysteinemia represents a reliable marker of cardiovascular risk and cardiovascular mortality or a therapeutic target in this population remains unclear. However, it is reasonable to consider folic acid with or without methylcobalamin supplementation as appropriate adjunctive therapy in patients with CKD. The purpose of this review is to summarize the characteristics of homocysteine, folic acid, and vitamin B12 metabolism, the mechanism of vascular damage, and the outcome of vitamin supplementation on hyperhomocysteinemia in patients with CKD, ESRD, dialysis treatment, and in kidney transplant recipients.

Keywords: hyperhomocysteinemia, folic acid, vitamin B12, chronic kidney disease, end-stage renal disease, cardiovascular disease

Introduction

Chronic Kidney Disease (CKD) represents an important economic burden for health systems around the world, with an estimated global prevalence of between 11 and 13%. Rationalized measures are needed to slow the progression to end-stage kidney disease (ESRD) and to decrease cardiovascular mortality [1]. Mortality rates remain in fact above 20% per year with the use of dialysis, with more than half of all deaths related to cardiovascular disease [2]. The problem of peripheral arteries disease (PAD) is also emerging, which is more common in patients with CKD and is associated with lower limb amputations and increased mortality [3].

Traditional factors such as hypertension, dyslipidaemia and diabetes mellitus are not sufficient to explain the dramatically increased cardiovascular risk in the population with CKD/ESRD. Thus, much attention shifted to other less studied aspects of CKD such as oxidative stress, endothelial dysfunction, chronic inflammation, vascular calcification in chronic kidney disease-mineral and bone disorder (CKD-MBD) and finally hyperhomocysteinemia (HHcy) [4].

The latter, since its discovery, proved to be a plausible risk factor for the development of atherosclerotic vascular disease processes leading to cardiovascular disease (CVD) and stroke. Levels of homocysteine (Hcy) higher than 20.0 μmol/L are associated with mortality 4.5 times higher. The “homocysteine hypothesis” is supported by the fact that subjects with problems in the enzymatic pathway of homocysteine metabolism have a higher level of homocysteine than the general population and a faster progression of arteriosclerosis. Therefore, the link between cardiovascular mortality and arteriosclerosis has been the subject of debate with conflicting results [5].

The high prevalence of HHcy in patients with CKD generated interest in a potential role of HHcy as a risk factor for CKD progression and CVD [5,8,9,10].

Hcy is a non-essential, sulfur-containing, non-proteinogenic amino acid, synthetized by transmethylation of the essential, diet-derived amino acid methionine (Figure 1). Aberrant Hcy metabolism could lead to redox imbalance and oxidative stress resulting in elevated protein, nucleic acid and carbohydrate oxidation and lipoperoxidation, products known to be involved in cytotoxicity [11].

Hcy levels can be significantly reduced by supplementation with folic acid (FA), vitamin B12 and vitamin B6. However, in several randomized and controlled studies the impact of vitamin supplementation seems to be disappointing in terms of cardiovascular mortality [6,7]. The debate is still open: some studies have reported a null or harmful effect of supplementation with FA and B vitamins, including cyanocobalamin [10], while others have confirmed a link between the homeostasis of the vitamins, cardiovascular risk and CKD progression [12]. These two outcomes are ultimately considered the result of a complex interaction between the effects of HHcy, FA, enzymatic activity/gene variants, and FA fortification programs that exist in some countries [13].

 

B vitamins and homocysteine metabolism

Folic acid/Vitamin B9

The term “folate” includes several forms of vitamin B9, including tetrahydrofolic acid (the active form), methyltetrahydrofolate (the primary circulating form), methenyltetrahydrofolate, folinic acid, folacin and pteroylglutamic acid. Since the human body is not able to synthesize folate, it must be provided through the diet [14]. Folic acid comes from polyglutamates that are converted into monoglutamates in the intestine, and then transported through mucous epithelium by a specific vector [15].

Cobalamin/Vitamin B12

Vitamin B12, also known as cobalamin, is a nutrient with a key role in human health: it is essential as a cofactor for the enzyme methionine synthase and other biochemical reactions, such as beta oxidation of fatty acids or DNA synthesis, and in the production of red blood cells [17–18]. Vitamin B12 deficiency is a common cause of HHcy and a frequent feature of patients with CKD [14–16].

Cobalamin is one of the most complex coenzymes in nature. The molecule consists of a corrinic ring and a part of dimethylbenzimidazole (DMB), and the focal point of the structure is the cobalt atom, held in the center of the corrinic ring which bonds some chemical groups, the most important of which are the hydroxyl group (hydroxocobalamin, OHCbl) and group CN (cyanocobalamin, CNCbl). These are the forms most commonly used in pharmaceutical formulations for vitamin B12 supplementation.

Vitamin B12, when ingested, is complexed with salivary haptocorrin, and cobalamin is released from pancreatic proteases in the duodenum. Then, cobalamin binds to an intrinsic factor secreted by the parietal cells of the stomach: when this complex reaches the distal ileum, it is endocytosed by enterocytes through cubilin. Then, it is transported into the plasma by a plasma transport protein called transcobalamin. B12 is filtered by the glomerulus; however, urinary excretion is minimal under normal conditions, due to reabsorption in the proximal tubule [19].

Metabolism of homocysteine and folate cycle

As mentioned above, Hcy plasma levels are determined by several factors, such as genetic alterations of the methionine metabolism enzymes, and vitamin B12, vitamin B6 and folic acid deficiency. FA, playing a pivotal role in Hcy metabolism, is inert and requires to be activated in tetrahydrofolic acid, a precursor of 5-methyltetrahydrofolate (5-MTHF). Methylenetetrahydrofolate reductase (MTHFR) is a key regulatory enzyme involved in folate dependent Hcy remethylation. MTHFR catalyzes the reduction of 5,10-methyltetrahydrofolate to 5-MTHF, necessary for the normal activity of the enzyme methionine synthetase (MTS), which uses vitamin B12 as a cofactor and converts homocysteine into methionine [20]. Methionine is transformed into S-adenosylmethionine (SAM) and then converted to S-adenosylhomocysteine (SAH) through a reaction catalyzed by methionine synthase reductase (MTRR). SAM is one of the most important donors of methyl groups and is fundamental in the catabolism of various amino acids and fatty acids [21].

Hcy is the final product, derived from the hydrolysis of SAH to Hcy and adenosine, and is located at the center of two metabolic pathways: it is irreversibly degraded through the path of transsulfuration into cysteine or is remethylated to methionine (folate cycle).

  1. Transsulfuration: Firstly, Hcy combines with serine by forming cystathionine via cystathionine-beta-synthase (CBS); then, cystathionine is hydrolyzed into cysteine and alpha-ketobutyrrate from cystathionine-gamma-lyase (CTH). Human CBS is expressed in the liver, kidneys, brain and ovaries and, during the first embryogenesis, in the neural and cardiac systems.
  2. Remethylation: Hcy conversion into methionine is catalyzed by the enzyme MTS and connects the cycle of folates with Hcy metabolism. While the MTS enzyme is expressed ubiquitously, another Hcy remethylation system, betaine-Hcy methyltransferase, is expressed mainly in the liver and kidneys [1].

The main reactions of Hcy metabolism are summarized in Figure 1.

Figure 1: Schematic representation of homocysteine metabolic pathway. DHF: dihydrofolate; DMG: N,N- dimethylglycine betaine; Met: methionine; SAH: S-adenosylhomocysteine; SAM: S-adenosylmethionine; THF: tetrahydrofolate
Figure 1: Schematic representation of homocysteine metabolic pathway. DHF: dihydrofolate; DMG: N,N- dimethylglycine betaine; Met: methionine; SAH: S-adenosylhomocysteine; SAM: S-adenosylmethionine; THF: tetrahydrofolate

 

Folic acid metabolism, vitamin B12 and homocysteine in CKD

Homocysteine

Patients with CKD and ESRD have been shown to have higher blood levels of Hcy than the general population [22]. The normal plasma level is <10 μmol/L; levels of Hcy <16 μmol/L are defined as mild HHcy, while severe HHcy is diagnosed when the levels are >100 μmol/L [23]. About 80-90% of the circulating Hcy is protein-bound; 10-20% of total homocysteine (tHcy) is present as Hcy-cysteine and Hcy mixed disulfide (Hcy dimer), and <1% is present in the reduced free form [14]. In CKD, studies show that the cause of HHcy is a reduced clearance rather than an increase in production, but the exact site of altered clearance remains controversial: under physiological conditions, only non-protein related Hcy is subjected to glomerular filtration and is then mostly reabsorbed into the tubules and oxidized into carbon dioxide and sulfate in kidney cells [24]. Some data support the hypothesis that decreased Hcy removal in CKD is caused by a decreased intrarenal metabolism, through both transsulfuration and remethylation [25].

Folic acid

It has also been shown that an anionic inhibition of the membrane transport of 5-MTHF occurs in patients with CKD with a depression in the intracellular incorporation rate of folates. These results suggest that the level of folates measured in the blood of uremic individuals does not reflect its intracellular use because the uptake is altered due to anionic inhibition [26].

Vitamin B12

Mainly linked to proteins in the blood, about 20% of circulating B12 is related to holotranscobalamin (TC2). The kidney plays an important role in TC2 metabolism, as TC2 is filtered into the glomerulus and is reabsorbed into the proximal tubule. Defects in protein resorption in the proximal tubule could therefore lead to a biologically active loss of CT2 in the urine. Increased levels of TC2 were observed in patients with CKD. Despite this, there is a decrease in TC2 absorption in cells that can lead to a paradoxical increase in cell Hcy levels, despite normal total B12. Thus, a functional deficiency of B12 can occur in patients with CKD as part of an increase in TC2 leaks in the urine, lower absorption of CT2 in the proximal tubule, and lower cellular absorption of TC2.

It is also important to consider that high levels of B12 could be harmful to individuals with CKD. This is related to cyanide metabolism, which is abnormal in individuals with CKD due to the decreased glomerular filtrate. Cyanocobalamin, the most common form of B12 replacement, is metabolized into active methylcobalamin, releasing small amounts of cyanide. Under normal circumstances, methylcobalamin binds to cyanide converting it to cyanocobalamin. However, in patients with CKD, reduced cyanide clearance prevents the conversion of cyanocobalamin into the active form, and therefore integration into this form is less effective in reducing Hcy levels. In addition, the excessive amount of supplementation with cyanocobalamin can release cyanide ions that are not excreted and contribute to the onset of complications in the patient with CKD (e.g. uremic neuropathy) [27–28].

 

Methylenetetrahydrofolate reductase polymorphisms

MTHFR plays a key role in Hcy metabolism and catalyzes the conversion of 5, 10-methylenetetrahydrofolate to 5-methyl-THF, the predominant circulating form of folate [29]. The MTHFR gene encodes the enzyme methyltetrahydrofolate reductase and is localized on chromosome 1 (1p36.3). Genetic polymorphisms involved in the homocysteine-methyonine route have been shown to result in HHcy. Although several MTHFR gene variants have been identified, the most characterized are single nucleotide polymorphisms (SNPs) in position 677 (MTHFR 677C>T), in position 1298 (MTHFR 129 8A>C), in position 1317 (MTHFR 1317T>C) and in position 1793 (MTHFR 1793G>A). It has been proposed that the two common mutations, MTHFR C677T and A1298C, may be associated with congenital abnormalities, cardiovascular diseases, strokes, cancer and clotting abnormalities [30,31].

C677T polymorphism is characterized by a point mutation at position 677 of the MTHFR gene that converts a cytosine into a thymine. It is known that when alanine replaces valine in the enzyme at the folate binding site, this polymorphism is commonly called thermolabile, because the activity of the encoded enzyme is reduced by 50-60% at 37°C and by 65% at 46°C. People who are homozygous for C677T tend to have slightly increased blood Hcy levels if their folate intake is insufficient, but normal Hcy levels if folate intake is adequate [32]. Substitution 677C>T is the most common missense variation of MTHFR, with a global prevalence of 40%. The frequency of C677T homozygosis varies depending on the ethnicity: from 1% or less among blacks in Africa and the United States, to 25.3% or more among Italians, Hispanic Americans and Colombians [30]. In contrast, the frequency of the mutant T allele in the MTHFR C677T gene in the Chinese population is 41.7%, higher than in other populations and could be an independent risk factors of early renal damage in the hypertensive Chinese population [33].

A1298C polymorphism is characterized by a point mutation in position 1298 in exon 7 of the MTHFR gene responsible for an amino acid substitution of a glutamine with an alanine in the enzyme regulatory domain. The activity of the encoded enzyme decreases, but to a lesser extent than in the case of C677T polymorphism. Subjects who are homozygotes for the A1298C allele do not appear to have increased serum Hcy levels [30,31]. According to Trovato et al., MTHFR 677C>T and A1298A>C gene polymorphisms could have a protective role on renal function as suggested by the lower frequency of both polymorphisms among a population of 630 dialysis patients in end-stage renal failure [34]. Regarding the other most common SNPs, MTHFR 1317T>C is a silent mutation, while MTHFR 1793G>A results in amino acid replacement, but with no impact on the functional activity of the enzyme [31].

The link between Hcy level and MTHFR gene polymorphisms has been investigated by Malinow et al.: homozygote subjects for the MTHFR T677 allele have shown an important reduction in the plasma levels of tHcy after FA integration. On the other hand, C677 allele homozygosity, especially subjects with higher basal folate levels, have shown a lesser tHcy reduction after FA supplementation. Finally, the carriers of the T/T genotype have shown the sharpest decrease of tHcy with FA integration [35]. This result was confirmed by Anchour et al: the simultaneous supplementation of folate and vitamin B12 was only useful in the homozygotes for the C allele and the reduction of Hcy was significantly higher in the carriers of the TT genotype than in other genotypes (CC/CT) [36]. These findings are consistent with the China Stroke Primary Prevention Trial (CSPPT), in which the largest decrease in serum Hcy was seen in the carriers of the TT genotype [37]. The relationship between MTHFR polymorphism and coronary heart disease severity showed that Hcy levels were significantly higher in patients with coronary arteries disease (CAD) than in control subjects and the genotype of MTHFR 677C>T was associated with increased CAD severity in patients at high risk for this pathology [38]. In summary, most available evidence suggests that MTHFR polymorphisms may influence folic acid and vitamin B12 treatment response in terms of Hcy lowering and cardiovascular risk reduction in patients with CKD and ESRD although indication of routine testing is matter of debate [39].

 

Endothelial damage of homocysteine and impact of CVD in ESRD patients

The pathogenic role of HHcy on the cardiovascular system in CKD and ESRD is related to the progression of atherosclerosis in the context of an already increased risk of vascular damage caused by the uremic syndrome. The mechanisms by which endothelial damage occurs are (Figure 2):

  • Oxidative stress. HHcy helps generate reactive oxygen species (ROS), reactive nitrogen species (RNS) and reactive species of thiol, thus decreasing the bioavailability of nitrogen monoxide (NO). These processes trigger latent matrix-metalloproteinase (MMP) and make the tissue inhibitor of metalloproteinase (TIMP) inactive. This leads to adverse cardiovascular remodelling, with increased collagen deposit [40]. HHcy significantly reduces the expression of the endothelial synthase nitric oxide protein (eNOS) in a dose-dependent manner and ultimately causes impaired basal production of NO, formation of radicals and subsequent endothelial damage by decreasing the bioavailability and bioactivity of NO [41].
  • Inflammation. Through the activation of the nuclear factor kappa B (NF-κB), a transcription factor known to stimulate the production of cytokines, chemokines, leukocyte adhesion molecules, HHcy induces the expression of proinflammatory chemokines MCP-1 and IL-8 in endothelial cells by enhancing transendothelial migration of monocytes, vascular inflammation and atherogenesis [42–43]. As for low-density lipoproteins (LDL), N-homocysteination produces aggregation, thus the accumulation of cholesterol, and facilitates the mediated absorption of oxidized LDL by macrophage scavenger receptors, resulting in the formation of foam cells in atherosclerosis [43–44].
  • Proliferation of smooth muscle cells. HHcy can significantly promote vascular smooth muscle cells (VSMC) proliferation, by promoting the expression of adhesion molecules, chemokines and VSMC mitogen [45]. HHcy can act directly on glomerular cells by inducing sclerosis and trigger kidney damage by reducing the plasma and tissue level of adenosine. The decrease in plasma adenosine in turn leads to a greater proliferation of VSMC, accelerating the sclerotic process in the arteries and glomeruli. In a pattern of folate-free HHcy rat, glomerular sclerosis, mesangial expansion, podocyte dysfunction, and fibrosis all occurred due to increased local oxidative stress [46].
Figure 2: Main pathogenetic pathways of endothelial damage mediated by hyperhomocysteinemia
Figure 2: Main pathogenetic pathways of endothelial damage mediated by hyperhomocysteinemia

These pathways end up amplifying the atherosclerotic process and inflammatory state present in CKD [47]. For patients with CKD and ESRD, despite the increase in Hcy levels (average level of Hcy in the general population about 10-15 μmol/L versus 25-35 μmol/L in uremic patients), the role of Hcy as a cardiovascular risk and mortality factor is still uncertain and many retrospective and interventional studies have given rise to conflicting evidence [48].

 

Folic acid supplementation in patients suffering from CKD

There is a large body of evidence indicating that folate therapy improves HHcy in the general population, but the data is less clear in CKD and ERSD patients [39,49]. The main interventional studies on the use of folic acid and vitamin B12 in CKD patients are summarized in Table 1. The benefits of folate supplementation in subjects with reduced renal function do not seem to lie entirely in the lowering of serum Hcy. Endothelial dysfunction is a key process in atherosclerosis and independently predicts cardiovascular events. High-dose FA (5 mg per day), alone or in combination with other B vitamins, appears to improve endothelial function through a largely Hcy-independent mechanism [50]. Endothelial cells can be particularly vulnerable to HHcy, as they do not express CBS, the first enzyme of the transsulfuration pathway [51]. Therefore, endothelial cells can eliminate Hcy only through remethylation, and normal activity of the enzymatic route is thus essential to prevent the increase of Hcy to a pathological level [52]. FA improves endothelial function by reducing intravascular oxidative stress; also improves intracellular superoxide generation by increasing the half-life of NO [53]. Folate therapy reduces but does not normalizes Hcy levels, frequently elevated in CKD patients. The mechanisms of this folate resistance have not been fully elucidated, yet. The entry of folate into the cell is mediated by specific folate receptors, whose expression is also modulated by the folate state, through an Hcy-dependent regulation mechanism. In peripheral mononuclear cells of hemodialysis patients, FR2 expression decreased and did not respond to changes in Hcy concentration [54].

 

Use of folate and vitamin B12 in the prevention of cardiovascular mortality and in slowing the progression of CKD

The role of folic acid and vitamin B12 supplementation in reducing mortality and preventing progression to ESRD is still to be determined. According to the meta-analysis of Heinz et al. of retrospective, prospective and observational studies on total 5123 patients, HHcy emerged as a risk factor for cardiovascular events and mortality in ESRD, especially in those subjects who do not receive additional FA (in countries without fortification programmes). Prospective studies have shown that in patients with ESRD, a 5 μmol/L increase in Hcy concentration is associated with a 7% increase in the risk of total mortality and a 9% increase in the risk of cardiovascular events. The level of Hcy in these patients seems to have decreased of 13 to 31 μmol/L due to supplementation with B vitamins in intervention studies. This was associated with a 27% reduction in the risk of cardiovascular events, although mortality had not decreased [55].

The minimum dose of folic acid to achieve a reduction of Hcy is debated: non-diabetic ESRD patients can respond to a daily dose of 5 mg FA, but diabetic patients with ESRD may need up to 15 mg to reduce the Hcy level more than 20% and have benefits on CVD risk, regardless of FA fortification. In addition, simultaneous administration with vitamin B12 is more effective in counteracting HHcy [56]. In non-diabetic patients with mild to moderate CKD a treatment strategy with pravastatin, vitamin E and Hcy reduction therapy (vitamin B12 and folate) leads to a significant reduction in the progression of carotid stenosis and a significant improvement in endothelial function and urinary excretion of albumin. However, no significant effect on the eGFR has been observed [57]. Similar results have emerged in the meta-analysis of Quin et al. including studies carried out from 1966 to 2011, for a total of 3886 patients with ESRD and CKD, where the relationship between supplementation with B12, FA and CVD had been analyzed after 24 months of treatment. FA therapy reduced the risk of CVD by 15%. Greater benefits were observed in those trials with a treatment duration >24 months, a decrease in Hcy level >20% (P = 0.007), and no or partial FA fortification (P = 0.04). The positive effect was seen when Hcy levels decreased >20%, even in the presence of FA fortification [58]. However, a reduction in Hcy secondary to high-dose FA therapy does not correspond to an increase in survival nor to a reduction of cardiovascular events according to randomized double-blind studies [59]. In the meta-analysis by Pan et al. (10 studies of patients in CKD), Hcy-lowering therapy is not associated with reduction of CVD, stroke and all-cause mortality [60]. However, the cohort of patients recruited had a high number of diabetic patients from areas with a grain fortification program.

Although HHcy is associated with increased CKD progression and albuminuria [61], the DIVINE study investigated the effects of Hcy-lowering therapy with high doses of folate (40 mg/day), vitamin B12 (1000 mg/day) and vitamin B6 (2 mg/day) in patients with diabetic nephropathy and showed that this treatment regimen does not increase survival or slow progression in ESRD, but rather leads to a higher incidence of cardiovascular events and a greater decrease in eGFR [62]. A possible explanation for these negative results can be attributed to the high load of cardiovascular comorbidity and to suboptimal therapy compliance. In addition, the study considered the CKD and ESRD population together and not separately. The above-mentioned China Stroke Primary Prevention Trial (CSPPT), a large, randomized study among adults with high blood pressure without a history of stroke or myocardial infarction, found that a therapy with ACE inhibitors and FA significantly reduced the relative risk of first stroke by 21%, more than ACE inhibitors alone. Among individuals with MTHFR 677 CC or CT genotypes, those with the lowest basal folate levels have the highest risk of stroke and benefit the most from FA therapy. In addition, individuals with the TT genotype may require a higher dosage of FA to exceed biologically insufficient levels [37]. An exploratory analysis by subgroups to assess the effect of treatment on primary outcome in various subgroups of CKD participants showed that the reduction in the risk of CKD progression was more represented in the diabetes subgroup [63]. Of note, CSPPT study selected a population without fortification of cereals with folic acid.

Several factors including age, baseline Hcy levels, FA fortification of grains, B12 status, renal function, comorbidities, and medications could modify the effects of folic acid and vitamin B12 on cardiovascular risk. The available evidence regarding the effect of Hcy lowering therapies on CKD progression is controversial and further studies are needed, with CKD progression as primary endpoint and with a more homogeneous population selection [39].

 

The role of folate and vitamin B12 therapy

ESRD patients in chronic dialysis treatment

In many cases, the literature has shown that dialysis and ESRD patients are a peculiar population whose response to certain factors is opposite to that of the general population, a condition that has been called “reverse epidemiology” [64]. A curious example is hypocholesterolemia, identified as a predictor of higher mortality in dialysis patients [65]. Similarly, data from our group have previously shown that a higher BMI protects ESRD patients from coronary artery calcifications [66], in line with a meta-analysis by Lowrie et al, based on 43,334 hemodialysis patients, indicating an improved survival associated with increased BMI values [67].

In line with this theory, very low Hcy levels appear to be associated with worse clinical outcomes, longer hospitalization, and higher mortality from all causes, and cardiovascular mortality in ESRD patients [68]. The combined effect of protein-energy malnutrition and inflammation may partly explain the apparent paradox represented by the inverse relationship between Hcy level and mortality in patients with ESRD [14].

The study of Sohoo et al. examined a cohort of 12,968 hemodialysis patients treated with vitamin B12 for 5 years, to observe the relationship between serum folate/B12 and mortality. Concentrations of B12 ≥550 pg/mL are associated with increased mortality from all causes in hemodialysis patients, regardless of sociodemographic data and laboratory variables [12]. The effectiveness of high-dose folic acid in event prevention in ESRD was evaluated in a randomized study. A total of 510 patients on chronic dialysis were randomized to 1.5 or 15 mg of FA contained in a renal multivitamin with a median follow-up of 24 months. Composite mortality rates and cardiovascular events did not differ between the FA groups. High basal Hcy was associated with lower event rates, which would confirm an inverse relationship between Hcy and events in ESRD patients. The administration of FA at high doses did not affect event rates [69]. Similar studies have come to the same conclusion: the Atherosclerosis and Folic Acid Supplementation Trial (ASFAST) recruited a total of 315 subjects with chronic kidney failure (most of them in dialysis) who were randomized to 15 mg FA per day or placebo and followed for a median of 3.6 years. Total Hcy in plasma is reduced by 19% in the FA group but this does not slow down the progression of atherosclerosis nor improve morbidity or cardiovascular mortality in patients [57].

Supplementation with B vitamins along with FA could be an alternative in reducing vascular oxidative stress. However, the randomized multicenter study conducted in double-blind by Heinz et al. on 650 patients in hemodialysis undergoing supplementation with FA, vitamin B12 and vitamin B6, showed that such therapies did not reduce total mortality and had no significant effect on the risk of cardiovascular events in patients with end-stage kidney disease [62]. Normalization of Hcy levels is difficult to achieve in dialysis patients with FA alone: according to Righetti et al., only 12% of a cohort of 81 patients in chronic dialysis has reached normal levels of Hcy. However, this condition has again shown no benefit in terms of survival [70].

The changes in the uremic patient’s metabolism described in the previous sections leave an open question regarding FA and vitamin B12 supplementation in dialysis. Another study by Righetti suggested that folate therapy to lower Hcy can reduce cardiovascular events in dialysis patients [71]. In a study by our group on a population of 341 patients in chronic dialysis, group A was treated with 50 mg i.v. of 5-MTHF, and group B was treated with 5 mg/d of oral FA. Both groups received vitamin B6 and B12. Our data showed that I.V. 5-MTHF appears to improve survival in hemodialysis patients regardless of the lowering of Hcy [72]. This latest evidence confirms that the role of FA and vitamin B12 should be better understood in this category of patients, both at the biochemical level and at the level of clinical outcomes.

Study, year Duration, design Population Treatment Outcomes

Nanayakkara PW et al, 2007 [57]

2 yrs, double-blind RCT 93 patients with mild to moderate CKD Pravastatin, vitamin E, and homocysteine lowering therapy (daily 5 mg FA + 100 mg vitamin B6 + 1 mg vitamin B12) vs placebo

In the treatment group significant reduction in CC-IMT, increase in BA-FMD, improvement in endothelial function and urinary albumin excretion, no effect on eGFR

Jamison RL et al, 2008 [58]

7 yrs, double-blind RCT 2056 patients with CKD (n=1305) or ESRD (n=751) and HHcy (> 15 mmol/L) Daily 40 mg FA + 100 mg vitamin B6 + 2 mg vitamin B12 vs placebo

In the treatment group significant lowering of Hcy levels, no effect on secondary outcomes (MI, stroke, and amputations time to dialysis and mortality)

Zoungas S et al, 2006 [61]

3.6 yrs, double-blind RCT 315 patients with CKD Daily 15 mg FA vs placebo

In the treatment group lowering by 19% of Hcy levels, no effect on secondary outcomes (change of IMT, artery function MI, stroke, cardiovascular death and overall cardiovascular events)

Heinz J et al, 2010 [62]

6 yrs, double-blind RCT 650 ESRD patients under hemodialysis treatment 5 mg FA + 50 mg vitamin B12 + 20 mg vitamin B6 (active treatment) vs or 0.2 mg FA, 4 mg vitamin B12 + 1.0 mg vitamin B6 (placebo) 3 times/week for 2 yrs

No effect on total mortality and fatal or nonfatal cardiovascular events

Xu X et al, 2016 [63]

4.5 yrs, double-blind RCT 1671 patients with CKD Daily 10 mg enalapril + 0.8 mg FA (n=7545) vs 10 mg enalapril alone (n=7559)

In patients receiving enalapril + FA   the risk for CKD progression and the rate of eGFR decline were decreased by 56% and 44%, respectively

Wrone EM et al, 2004 [63]

2 yrs, RCT 510 ESRD patients under hemodialysis treatment Daily 1, 5, or 15 mg FA contained in a renal multivitamin

No effect of high-dose FA administration on the rates of cardiovascular events and mortality

Righetti M et al, 2003 [70]

1 yr, RCT 81 ESRD patients under hemodialysis treatment Daily 15 mg FA (n=25) vs 5 mg FA (n=26) vs untreated (n=30)

No significant improvement of HHcy, regardless of FA dose, but treated patients tended towards a decreased rate of cardiovascular events.

Righetti M et al, 2006 [71]

871 days (median follow-up, range 132-1825 days), single-center, open, randomized prospective trial 114 ESRD patients under hemodialysis treatment 5 mg daily FA, or 5 mg every other day (if serum FA levels were up the normal high limit of 16.8 ng/mL) + vitamin B complex (250 mg B1 + 250 mg B6 + 500 mg B12, if plasma vitamin B12 values were below the normal limit of 200 ng/L)

Lower rate of cardiovascular events in treated patients with low Hcy levels

Cianciolo G et al, 2008 [72]

55 months, randomized prospective study 341 ESRD patients under hemodialysis treatment Patients were randomized into two groups: group A (n=174) treated with I.V. 50 mg 5-MTHF (Prefolic) three times a week (end of each dialysis session) vs group B (n=167) treated with daily 5 mg FA. Both groups also received I.V. 300 mg vitamin + 1 g vitamin B12 at the end of the dialysis session.

Both FA acid and 5-MTHF decreased Hcy levels, and I.V. 5-MTHF improved survival in hemodialysis independent from Hcy lowering. CRP but not HHcy resulted to be the main risk factor for mortality in hemodialysis patients

Buccianti G et al, 2001 [74]

6 months, cross-sectional clinical study 55 ESRD patients under hemodialysis treatment 27 patients with macrocytosis treated the end of each dialysis session with I.V. 0.9 mg folinic acid + 0.5 mg cyanocobalamin + 1.5 mg hydroxycobalamin vs 28 untreated patients

Intermittent I.V. administration of folinic acid combined with vitamin B12 resulted in lower HHCy plasma concentration, but the effect was also related to genotype and dialysis modality

Bostom AG et al, 2011 [78]

5 yrs, multi-center

double-blind RCT

 4110 stable kidney transplant recipients Participants were randomized to receive either a high dose (n=2056) of FA (5.0 mg), vitamin B6 (pyridoxine; 50 mg) and vitamin B12 (cyanocobalamin; 1.0 mg) or a low dose (n=2054) of vitamin B6 (1.4 mg) and vitamin B12 (2.0 µg) and no FA.

In the high dose treatment arm, a significant reduction in Hcy level was achieved, but without any beneficial impact on cardiovascular outcomes, all-cause mortality, or allograft failure
Table 1: Summary of major interventional studies on folic acid / vitamin B12 administration in patients with CKD

BA-FMD: brachial artery flow-mediated dilatation; CC-IMT: carotid intima-media thickness; CKD: chronic kidney disease; eGFR: estimated glomerular filtration rate; ESRD: end-stage renal disease; FA: folic acid; HHcy: homocysteinemia; I.V.: intravenous; MI: myocardial infarction; RCT: randomized controlled trial; yr(s): year(s)

Role of FA and B12 supplementation in CKD anemia

In uremia-related anemia, unless patients with CKD and ESRD show significant folate depletion, additional FA supplementation does not appear to have a beneficial effect on erythropoiesis or response to recombinant human erythropoietin therapy (rHuEPO). However, measurements of folate circulating in the serum do not necessarily reflect folate reserves in tissues, and folate measurements in red blood cells provide a more accurate representation. The low concentrations of folate in red blood cells in these patients suggest the need for FA supplement [73]. Megaloblastic anemia, that occurs in vitamin deficiencies frequently found in uremic patients, results from inhibition of DNA synthesis during the production of red blood cells [74]. When cobalamin levels become inadequate, DNA synthesis is compromised, and the cell cycle cannot progress from the G2 growth phase to the mitosis phase. This leads to continuous cell growth without division, and then to macrocytosis [14]. In patients with CKD, folate and vitamin B12 deficiency may represent an important factor in renal anemia and hyporesponsiveness to rHuEPO therapy [75].

Kidney transplant recipients

In kidney transplants, several factors such as dialysis vintage, anemia, immunosuppression, inflammatory state, and dysmetabolic alterations can affect the cardiovascular risk [76,77]. The effect of supplementation of FA, vitamin B12 and vitamin B6 on CVD and mortality reduction has been studied by the Folic Acid for Vascular Outcome Reduction in Transplantation (FAVORIT) study. Kidney transplant recipients were randomized to a daily multivitamin drug containing high doses of folate (5.0 mg), vitamin B12 (1.0 mg) and vitamin B6 (50 mg), or placebo. Despite the actual lowering the Hcy, the incidence of CVD, mortality from all causes and the onset of kidney failure dependent on dialysis did not differ between the two treatment arms [78]. A longitudinal ancillary study of the FAVORIT trial has recently indicated that the integration of high-dose B vitamins results in a modest cognitive benefit in patients with high base values. It should be noted that almost all subjects had no shortage of folate or B12, thus the potential cognitive benefits of folate and B12 supplementation in individuals with poor vitamin B status remain controversial [79].

 

Future perspectives and conclusion

At present, the results from available trials do not provide complete support for considering alterations in FA and vitamin B12 as reliable indices of CVD risk in CKD and ESRD population. Moreover, these factors do not represent a validated therapeutic target to cardiovascular risk reduction and CKD progression.

However, there is some evidence to indicate that the incidence of stroke and CKD progression might be controlled using more targeted FA therapy (baseline FA levels may have an impact on the efficacy of the FA intervention therapy), in particular among those with the MTHFR 677TT genotype and low to moderate folate levels and in countries without a grain fortification program [37,63]. However, in both general population and CKD patients, it remains a matter of debate if beneficial effects of FA therapy are due to its direct antioxidant effect or to a reduction in HHcy.

Discordant results in terms of CKD progression and cardiovascular risk, in the analyzed studies, result from differences in patient characteristics and FA treatment schemes among trials and may be influenced by the degree of cardiovascular and renal impairment.

In conclusion FA with or without vitamin B12 supplementation is an appropriate adjunctive therapy in patients with CKD and ESRD on dialysis treatment, in these cases FA may be supplemented pharmacologically after careful evaluation of folate status.

 

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