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Nephronophthisis (NPH) is an autosomal-recessive ciliopathy representing one of the most frequent causes of kidney failure in childhood characterized by a broad clinical and genetic heterogeneity. Applied to one of the worldwide largest cohorts of patients with NPH, genetic analysis encompassing targeted and whole exome sequencing identified disease-causing variants in 600 patients from 496 families with a detection rate of 71%. Of 788 pathogenic variants, 40 known ciliopathy genes were identified. However, the majority of patients (53%) bore biallelic pathogenic variants in NPHP1. NPH-causing gene alterations affected all ciliary modules defined by structural and/or functional subdomains. Seventy six percent of these patients had progressed to kidney failure, of which 18% had an infantile form (under five years) and harbored variants affecting the Inversin compartment or intraflagellar transport complex A. Forty eight percent of patients showed a juvenile (5-15 years) and 34% a late-onset disease (over 15 years), the latter mostly carrying variants belonging to the Transition Zone module. Furthermore, while more than 85% of patients with an infantile form presented with extra-kidney manifestations, it only concerned half of juvenile and late onset cases. Eye involvement represented a predominant feature, followed by cerebellar hypoplasia and other brain abnormalities, liver and skeletal defects. The phenotypic variability was in a large part associated with mutation types, genes and corresponding ciliary modules with hypomorphic variants in ciliary genes playing a role in early steps of ciliogenesis associated with juvenile-to-late onset NPH forms. Thus, our data confirm a considerable proportion of late-onset NPH suggesting an underdiagnosis in adult chronic kidney disease.

The overall diagnostic yield of massively parallel sequencing–based tests in patients with chronic kidney disease (CKD) is 30% for paediatric cases and 6–30% for adult cases. These figures should encourage nephrologists to frequently use genetic testing as a diagnostic means for their patients. However, in reality, several barriers appear to hinder the implementation of massively parallel sequencing–based diagnostics in routine clinical practice. In this article we aim to support the nephrologist to overcome these barriers. After a detailed discussion of the general items that are important to genetic testing in nephrology, namely genetic testing modalities and their indications, clinical information needed for high-quality interpretation of genetic tests, the clinical benefit of genetic testing and genetic counselling, we describe each of these items more specifically for the different groups of genetic kidney diseases and for CKD of unknown origin.

RNA modifications play a fundamental role in cellular function. Pseudouridylation, the most abundant RNA modification, is catalyzed by the H/ACA small ribonucleoprotein (snoRNP) complex that shares four core proteins, dyskerin (DKC1), NOP10, NHP2, and GAR1. Mutations in DKC1 , NOP10 , or NHP2 cause dyskeratosis congenita (DC), a disorder characterized by telomere attrition. Here, we report a phenotype comprising nephrotic syndrome, cataracts, sensorineural deafness, enterocolitis, and early lethality in two pedigrees: males with DKC1 p.Glu206Lys and two children with homozygous NOP10 p.Thr16Met. Females with heterozygous DKC1 p.Glu206Lys developed cataracts and sensorineural deafness, but nephrotic syndrome in only one case of skewed X-inactivation. We found telomere attrition in both pedigrees, but no mucocutaneous abnormalities suggestive of DC. Both mutations fall at the dyskerin–NOP10 binding interface in a region distinct from those implicated in DC, impair the dyskerin–NOP10 interaction, and disrupt the catalytic pseudouridylation site. Accordingly, we found reduced pseudouridine levels in the ribosomal RNA (rRNA) of the patients. Zebrafish dkc1 mutants recapitulate the human phenotype and show reduced 18S pseudouridylation, ribosomal dysregulation, and a cell-cycle defect in the absence of telomere attrition. We therefore propose that this human disorder is the consequence of defective snoRNP pseudouridylation and ribosomal dysfunction.

Bi-allelic loss of function variations in genes encoding proteins of the renin-angiotensin system (AGT, ACE, REN, AGTR1) are associated with autosomal recessive renal tubular dysgenesis, a severe disease characterized by the absence of differentiated proximal tubules leading to fetal anuria and neonatal end-stage renal disease. We identified bi-allelic loss of function mutations in ACE, the gene encoding angiotensin-converting enzyme, in 3 unrelated cases displaying progressive chronic renal failure, whose DNAs had been sent for suspicion of juvenile hyperuricemic nephropathy, nephronophthisis, and cystic renal disease, respectively. In all cases, patients were affected with anemia whose severity was unexpected regarding the level of renal failure and with important polyuro-polydipsia. Bi-allelic loss of function mutation of ACE can have atypical and sometimes late presentation with chronic renal failure, anemia (out of proportion with the level of renal failure), and polyuro-polydipsia. These data illustrate the usefulness of next generation sequencing and “agnostic” approaches to elucidate cases with chronic kidney disease of unknown etiology and to broaden the spectrum of phenotypes of monogenic renal diseases. It also raises the question of genetic modifiers involved in the variation of the phenotypes associated with these mutations.

BACKGROUND AND AIMS Mutations of NPHS2 cause autosomal recessive steroid-resistant nephrotic syndrome (SRNS). The encoded podocin, a key component of the slit diaphragm, homo-oligomerizes through two C-terminal oligomerization sites. We formerly found that specific podocin variants may exert a dominant negative effect against R229Q podocin variant secondary to an altered oligomerization [1, 2]. Recently, two families with autosomal dominant focal segmental glomerulosclerosis were found to carry a de novo heterozygous NPHS2 last-exon frameshift mutations affecting the second oligomerization site (either c.989_992delTGTC, p.L330Pfs*17 or c.988_989delCT, p.L330Vfs*15) [3]. We hypothesized that these podocin variants exert a dominant negative effect against wild type (WT) podocin. METHODS We coexpressed eGFP-tagged WT podocin with either hemagglutinin-tagged (HA) WT, p.L330Pfs*17 or p.L330Vfs*15 podocin variants in cultured podocytes. Plasma membrane was stained with CF405M-conjugated wheat germ agglutinin (WGA), and immunohistochemical staining was performed with primary anti-HA and secondary AlexaFluor568-conjugated antibodies. All analysis was done in a blinded fashion. The membrane-targeting of podocin was assessed by the ratio of the WGA-labeled perimembranous area that colocalized with podocin (Mander's overlap coefficients, MOC) and the colocalization of the coexpressed variants were estimated as Pearson correlation coefficients (PCC) by FiJi software on confocal microscopic images. MOC and PCC were compared by Holmes–Bonferroni adjusted Wilcoxon signed-rank test. RESULTS The frameshift podocin variants were not membranous in monoexpression [MOC: HA-p.L330Pfs*17 (n = 26): 0.08 (0.03–0.22) median (interquartile ranges); HA-p.L330Vfs*15 (n = 24): 0.07 (0.02–0.16)]. The GFP-tagged WT podocin strongly colocalized with the HA-tagged WT or frameshift podocin variants [PCC: WT-eGFP + HA-WT (n = 19): 0.64 (0.49–0.71); WT-eGFP + HA-L330Pfs*17 (n = 17): 0.54 (0.35–0.58); WT-eGFP + HA-L330Vfs*15 (n = 17): 0.44 (0.29–0.66)]. The GFP-WT podocin was retained in intracellular compartments in the presence of any of the two frameshift podocin variants [MOCWT(WT) = 0.58 (0.46–0.69)], MOCWT(L330Pfs*17) = 0.01 (0.002–0.029), P = 2x10-6 versus WT(WT); MOCWT(L330Vfs*15) = 0.003 (0.001–0.013), P = 2 x 10−6 versus WT(WT)]. CONCLUSIONS The dominant podocin variants retain WT podocin in intracellular compartments explaining the exceptional dominant transmission of NPHS2-associated glomerulopathy. This is in accordance with our former results showing the primary role of C-terminal hetero-oligomerization in exerting interallelic interactions of NPHS2 alleles. FUNDING MTA-SE Lendulet Research Grant (LP2015-11/2015), OTKA KH125566 and K135798, and ÚNKP-20–3-I-SE-29 from the source of the National Research, Development and Innovation Fund.

Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial nephropathy belonging to the ciliopathy disorders and known as the most common cause of hereditary end-stage renal disease in children. Yet, no curative treatment is available. The major gene, NPHP1, encodes a protein playing key functions at the primary cilium and cellular junctions. Using a medium-throughput drug-screen in NPHP1 knockdown cells, we identified 51 Food and Drug Administration-approved compounds by their ability to alleviate the cellular phenotypes associated with the loss of NPHP1; 11 compounds were further selected for their physicochemical properties. Among those compounds, prostaglandin E1 (PGE1) rescued ciliogenesis defects in immortalized patient NPHP1 urine-derived renal tubular cells, and improved ciliary and kidney phenotypes in our NPH zebrafish and Nphp1 knockout mouse models. Furthermore, Taprenepag, a nonprostanoid prostaglandin E2 receptor agonist, alleviated the severe retinopathy observed in Nphp1−/− mice. Finally, comparative transcriptomics allowed identification of key signaling pathways downstream PGE1, including cell cycle progression, extracellular matrix, adhesion, or actin cytoskeleton organization. In conclusion, using in vitro and in vivo models, we showed that prostaglandin E2 receptor agonists can ameliorate several of the pleotropic phenotypes caused by the absence of NPHP1; this opens their potential as a first therapeutic option for juvenile NPH-associated ciliopathies.

SummaryNephronophthisis (NPH) is an autosomal recessive tubulointerstitial nephropathy belonging to the ciliopathy disorders and known as the most common cause of hereditary end-stage renal disease in children. Yet, no curative treatment is available. The major gene, NPHP1, encodes a protein playing key functions at the primary cilium and cellular junctions. Using an in cellulo medium-throughput drug-screen, we identified 51 FDA-approved compounds and selected 11 for their physicochemical properties, including prostaglandin E1 (PGE1). PGE1 was further validated to rescue ciliogenesis in immortalized patient NPHP1-/- urine-derived renal tubular cells and corroborated by the effects of its analog PGE2. The two molecules reduced pronephric cyst occurrence in vivo in nphp4 zebrafish model, and PGE1 treatment in Nphp1-/- mice led to a significant reduction of renal tubular dilatations, partially restoring cilia length within tubules. Finally, comparative transcriptomics allowed identification of key molecules downstream PGE1. Altogether, our drug-screen strategy led to the identification of PGE1 as the first potential therapeutic molecule for NPH-associated ciliopathies.Significant statementJuvenile nephronophthisis (NPH) is a renal ciliopathy due to a dysfunction of primary cilia and a common genetic cause of end-stage renal disease in children and young adults. No curative treatment is available. This paper describes the identification of Prostaglandin E1 (PGE1) as the first potential therapeutic molecule for NPH-associated ciliopathies. We demonstrated that PGE1 rescues defective ciliogenesis and ciliary composition in NPHP1-/- patient urine-derived renal tubular cells. Furthermore, PGE1 improves ciliary and kidney phenotypes in our NPH zebrafish and Nphp1-/- mouse models. Finally, in vitro experiments as well as transcriptomic analyses pointed out several pathways downstream PGE1 as cAMP, cell-cell/cell-matrix adhesion or actin cytoskeleton. Altogether, our findings provide a new alternative for treatment of NPH.

Numerous genes for monogenic kidney diseases with classical patterns of inheritance, as well as genes for complex kidney diseases that manifest in combination with environmental factors, have been discovered. Genetic findings are increasingly used to inform clinical management of nephropathies, and have led to improved diagnostics, disease surveillance, choice of therapy, and family counseling. All of these steps rely on accurate interpretation of genetic data, which can be outpaced by current rates of data collection. In March of 2021, Kidney Diseases: Improving Global Outcomes (KDIGO) held a Controversies Conference on “Genetics in Chronic Kidney Disease (CKD)” to review the current state of understanding of monogenic and complex (polygenic) kidney diseases, processes for applying genetic findings in clinical medicine, and use of genomics for defining and stratifying CKD. Given the important contribution of genetic variants to CKD, practitioners with CKD patients are advised to “think genetic,” which specifically involves obtaining a family history, collecting detailed information on age of CKD onset, performing clinical examination for extrarenal symptoms, and considering genetic testing. To improve the use of genetics in nephrology, meeting participants advised developing an advanced training or subspecialty track for nephrologists, crafting guidelines for testing and treatment, and educating patients, students, and practitioners. Key areas of future research, including clinical interpretation of genome variation, electronic phenotyping, global representation, kidney-specific molecular data, polygenic scores, translational epidemiology, and open data resources, were also identified.

Nephropathic cystinosis is a rare disease secondary to recessive mutations of the CTNS gene encoding the lysosomal cystine transporter cystinosin, causing accumulation of cystine in multiple organs. Over the years, the disease has evolved from being a fatal condition during early childhood into a treatable condition, with patients surviving into adulthood. Data on cystinosis are limited by the rarity of the disease. Here, we have investigated factors associated with kidney and growth outcome in a very large cohort of 453 patients born between 1964 and 2016 and followed in Belgium, Germany, Austria, France, Italy, Spain, The Netherlands, Turkey and United Kingdom. From the 1970s to the 1990s, the median increase in kidney survival was 9.1 years. During these years, cysteamine, a cystine-depleting agent, was introduced for the treatment of cystinosis. Significant risk factors associated with early progression to end-stage kidney disease assessed by Cox proportional multivariable analysis included delayed initiation of cysteamine therapy and higher mean leucocyte cystine levels. No significant effect on kidney function was observed for gender, pathogenic variant of the CTNS gene, and the prescription of indomethacin or renin angiotensin system blockers. Significantly improved linear growth was associated with early use of cysteamine and lower leukocyte cystine levels. Thus, our study provides strong evidence in favor of early diagnosis and optimization of cystine depletion therapy in nephropathic cystinosis.

Renal tubular dysgenesis (RTD) is a developmental abnormality of the nephron characterized by fetal anuria, oligohydramnios, and severe postnatal hypotension. Genetic forms have an autosomal recessive inheritance and are caused by mutations in genes encoding key components of the renin-angiotensin pathway. We report three patients from two unrelated families with RTD due to pathogenic variants of the angiotensin-converting enzyme (ACE) gene, in whom RTD was associated with microcolon. We also detail key variations of the renin-angiotensin system in one of these infants. The severe intestinal developmental abnormality culminating in microcolon and early terminal ileum perforation/necrotizing enterocolitis is a novel finding not previously associated with RTD, which points to a role of the renin-angiotensin system in gut development.

RNA modifications play a fundamental role in cellular function. Pseudouridylation, the most abundant RNA modification, is catalyzed by the H/ACA small ribonucleoprotein (snoRNP) complex that shares four core proteins, dyskerin (DKC1), NOP10, NHP2, and GAR1. Mutations in DKC1, NOP10, or NHP2 cause dyskeratosis congenita (DC), a disorder characterized by telomere attrition. Here, we report a phenotype comprising nephrotic syndrome, cataracts, sensorineural deafness, enterocolitis, and early lethality in two pedigrees: males with DKC1 p.Glu206Lys and two children with homozygous NOP10 p.Thr16Met. Females with heterozygous DKC1 p.Glu206Lys developed cataracts and sensorineural deafness, but nephrotic syndrome in only one case of skewed X-inactivation. We found telomere attrition in both pedigrees, but no mucocutaneous abnormalities suggestive of DC. Both mutations fall at the dyskerin–NOP10 binding interface in a region distinct from those implicated in DC, impair the dyskerin–NOP10 interaction, and disrupt the catalytic pseudouridylation site. Accordingly, we found reduced pseudouridine levels in the ribosomal RNA (rRNA) of the patients. Zebrafish dkc1 mutants recapitulate the human phenotype and show reduced 18S pseudouridylation, ribosomal dysregulation, and a cell-cycle defect in the absence of telomere attrition. We therefore propose that this human disorder is the consequence of defective snoRNP pseudouridylation and ribosomal dysfunction.

International audience; Steroid-resistant nephrotic syndrome (SRNS) is characterized by high-range proteinuria and most often focal and segmental glomerulosclerosis (FSGS). Identification of mutations in genes causing SRNS has improved our understanding of disease mechanisms and highlighted defects in the podocyte, a highly specialized glomerular epithelial cell, as major factors in disease pathogenesis. By exome sequencing, we identified missense mutations in TBC1D8B in two families with an X-linked early-onset SRNS with FSGS. TBC1D8B is an uncharacterized Rab-GTPase-activating protein likely involved in endocytic and recycling pathways. Immunofluorescence studies revealed TBC1D8B presence in human glomeruli, and affected individual podocytes displayed architectural changes associated with migration defects commonly found in FSGS. In zebrafish we demonstrated that both knockdown and knockout of the unique TBC1D8B ortholog-induced proteinuria and that this phenotype was rescued by human TBC1D8B mRNA injection, but not by either of the two mutated mRNAs. We also showed an interaction between TBC1D8B and Rab11b, a key protein in vesicular recycling in cells. Interestingly, both internalization and recycling processes were dramatically decreased in affected individuals' podocytes and fibroblasts, confirming the crucial role of TBC1D8B in the cellular recycling processes, probably as a Rab11b GTPase-activating protein. Altogether, these results confirmed that pathogenic variations in TBC1D8B are involved in X-linked podocytopathy and points to alterations in recycling processes as a mechanism of SRNS.

Monogenic forms of Steroid-Resistant Nephrotic Syndrome (SRNS) have been widely characterized, but genetic screening paradigms preferentially address congenital, infantile onset, and familial cases. Our aim was to characterize the distribution of disease-causing gene mutations in adults with sporadic SRNS or focal segmental glomerulosclerosis (FSGS). We selected adult patients with non-syndromic, biopsy-proven FSGS or SRNS in the absence of known family history. Strict clinical criteria included lack of response to glucocorticoids and cyclosporine, and no recurrence after kidney transplantation. Mutations in SRNS genes were detected using a targeted gene panel. Sixteen of 135 tested participants (11.8%) carried pathogenic mutations in monogenic SRNS genes, and 14 others (10.4%) carried two APOL1 high-risk alleles. Autosomal recessive disease was diagnosed in 5 participants, autosomal dominant disease in 9, and X-linked disease in 2. Four participants carried a de novo heterozygous mutation. Among the 16 participants with identified mutations in monogenic SNRS genes, 7 (43.7%) had type IV collagen mutations. Mutations in monogenic SNRS genes were identified primarily in participants with proteinuria onset before 25 years of age, while the age at disease onset was variable in those with APOL1 high-risk genotype. Mean age at diagnosis was lower and renal survival was worse in participants with identified mutations in SNRS genes than in those without mutations. We found a significant rate of pathogenic mutations in adults with SRNS, with Type IV collagen mutations being the most frequent. These findings may have immediate impact on clinical practice.

Item does not contain fulltext Steroid-resistant nephrotic syndrome (SRNS) almost invariably progresses to end-stage renal disease. Although more than 50 monogenic causes of SRNS have been described, a large proportion of SRNS remains unexplained. Recently, it was discovered that mutations of NUP93 and NUP205, encoding 2 proteins of the inner ring subunit of the nuclear pore complex (NPC), cause SRNS. Here, we describe mutations in genes encoding 4 components of the outer rings of the NPC, namely NUP107, NUP85, NUP133, and NUP160, in 13 families with SRNS. Using coimmunoprecipitation experiments, we showed that certain pathogenic alleles weakened the interaction between neighboring NPC subunits. We demonstrated that morpholino knockdown of nup107, nup85, or nup133 in Xenopus disrupted glomerulogenesis. Re-expression of WT mRNA, but not of mRNA reflecting mutations from SRNS patients, mitigated this phenotype. We furthermore found that CRISPR/Cas9 knockout of NUP107, NUP85, or NUP133 in podocytes activated Cdc42, an important effector of SRNS pathogenesis. CRISPR/Cas9 knockout of nup107 or nup85 in zebrafish caused developmental anomalies and early lethality. In contrast, an in-frame mutation of nup107 did not affect survival, thus mimicking the allelic effects seen in humans. In conclusion, we discovered here that mutations in 4 genes encoding components of the outer ring subunits of the NPC cause SRNS and thereby provide further evidence that specific hypomorphic mutations in these essential genes cause a distinct, organ-specific phenotype.

The megalin/cubilin receptor complex is required for proximal tubular endocytosis and degradation of filtered albumin. An additional high-capacity retrieval pathway of intact albumin for the recovery of large amounts of filtered albumin has been proposed, possibly involving cooperation between megalin/cubilin and the neonatal Fc receptor. To clarify the potential role of such a pathway, we examined the effects of megalin/cubilin gene inactivation on tubular albumin uptake and plasma albumin levels in nephrotic, podocin knockout mice. Immunofluorescence microscopy of megalin/cubilin/podocin knockout mouse kidneys demonstrated abolishment of proximal tubule albumin uptake, in contrast to the excessive albumin accumulation observed in podocin knockout mice compared to controls. Correspondingly, urinary albumin excretion was increased 1.4 fold in megalin/cubilin/podocin compared to podocin knockout mice (albumin/creatinine: 226 vs. 157 mg/mg). However, no difference in plasma albumin levels was observed between megalin/cubilin/podocin and podocin knockout mice, as both were reduced to approximately 40% of controls. There were no differences in liver albumin synthesis by mRNA levels and protein abundance. Thus, megalin/cubilin knockout efficiently blocks proximal tubular albumin uptake in nephrotic mice but plasma albumin levels did not differ as a result of megalin/cubilin-deficiency, suggesting no significance of the megalin/cubilin-pathway for albumin homeostasis by retrieval of intact albumin.

Congenital anomalies of the kidney and urinary tract (CAKUT) occur in three to six of 1000 live births, represent about 20% of the prenatally detected anomalies, and constitute the main cause of CKD in children. These disorders are phenotypically and genetically heterogeneous. Monogenic causes of CAKUT in humans and mice have been identified. However, despite high-throughput sequencing studies, the cause of the disease remains unknown in most patients, and several studies support more complex inheritance and the role of environmental factors and/or epigenetics in the pathophysiology of CAKUT. Here, we report the targeted exome sequencing of 330 genes, including genes known to be involved in CAKUT and candidate genes, in a cohort of 204 unrelated patients with CAKUT; 45% of the patients were severe fetal cases. We identified pathogenic mutations in 36 of 204 (17.6%) patients. These mutations included five de novo heterozygous loss of function mutations/deletions in the PBX homeobox 1 gene (PBX1), a gene known to have a crucial role in kidney development. In contrast, the frequency of SOX17 and DSTYK variants recently reported as pathogenic in CAKUT did not indicate causality. These findings suggest that PBX1 is involved in monogenic CAKUT in humans and call into question the role of some gene variants recently reported as pathogenic in CAKUT. Targeted exome sequencing also proved to be an efficient and cost-effective strategy to identify pathogenic mutations and deletions in known CAKUT genes.

There is a great interest in exploring the mechanisms of oedema in nephrotic syndrome (NS) as evidenced by 2 parallel studies published in Acta Physiologica (1, 2). This has generated an editorial comment (3) which we respond to by this rebuttal. The interest in the nephrotic edema reflects not only the significant negative impact this has on the quality of life, but also the difficulties associated with identifying optimal treatment due to the lack of pathophysiological insight.

International audience; BACKGROUNDProteinuria is considered an unfavorable clinical condition that accelerates renal and cardiovascular disease. However, it is not clear whether all forms of proteinuria are damaging. Mutations in CUBN cause Imerslund-Gräsbeck syndrome (IGS), which is characterized by intestinal malabsorption of vitamin B12 and in some cases proteinuria. CUBN encodes for cubilin, an intestinal and proximal tubular uptake receptor containing 27 CUB domains for ligand binding.METHODSWe used next-generation sequencing for renal disease genes to genotype cohorts of patients with suspected hereditary renal disease and chronic proteinuria. CUBN variants were analyzed using bioinformatics, structural modeling, and epidemiological methods.RESULTSWe identified 39 patients, in whom biallelic pathogenic variants in the CUBN gene were associated with chronic isolated proteinuria and early childhood onset. Since the proteinuria in these patients had a high proportion of albuminuria, glomerular diseases such as steroid-resistant nephrotic syndrome or Alport syndrome were often the primary clinical diagnosis, motivating renal biopsies and the use of proteinuria-lowering treatments. However, renal function was normal in all cases. By contrast, we did not found any biallelic CUBN variants in proteinuric patients with reduced renal function or focal segmental glomerulosclerosis. Unlike the more N-terminal IGS mutations, 37 of the 41 proteinuria-associated CUBN variants led to modifications or truncations after the vitamin B12-binding domain. Finally, we show that 4 C-terminal CUBN variants are associated with albuminuria and slightly increased GFR in meta-analyses of large population-based cohorts.CONCLUSIONCollectively, our data suggest an important role for the C-terminal half of cubilin in renal albumin reabsorption. Albuminuria due to reduced cubilin function could be an unexpectedly common benign condition in humans that may not require any proteinuria-lowering treatment or renal biopsy.FUNDINGATIP-Avenir program, Fondation Bettencourt-Schueller (Liliane Bettencourt Chair of Developmental Biology), Agence Nationale de la Recherche (ANR) Investissements d'avenir program (ANR-10-IAHU-01) and NEPHROFLY (ANR-14-ACHN-0013, to MS), Steno Collaborative Grant 2018 (NNF18OC0052457, to TSA and MS), Heisenberg Professorship of the German Research Foundation (KO 3598/5-1, to AK), Deutsche Forschungsgemeinschaft (DFG) Collaborative Research Centre (SFB) KIDGEM 1140 (project 246781735, to CB), and Federal Ministry of Education and Research (BMB) (01GM1515C, to CB).

BACKGROUND: Deletions or inactivating mutations of the cystinosin gene CTNS lead to cystine accumulation and crystals at acidic pH in patients with nephropathic cystinosis, a rare lysosomal storage disease and the main cause of hereditary renal Fanconi syndrome. Early use of oral cysteamine to prevent cystine accumulation slows progression of nephropathic cystinosis but it is a demanding treatment and not a cure. The source of cystine accumulating in kidney proximal tubular cells and cystine's role in disease progression are unknown.METHODS: To investigate whether receptor-mediated endocytosis by the megalin/LRP2 pathway of ultrafiltrated, disulfide-rich plasma proteins could be a source of cystine in proximal tubular cells, we used a mouse model of cystinosis in which conditional excision of floxed megalin/LRP2 alleles in proximal tubular cells of cystinotic mice was achieved by a Cre-LoxP strategy using Wnt4-CRE. We evaluated mice aged 6-9 months for kidney cystine levels and crystals; histopathology, with emphasis on swan-neck lesions and proximal-tubular-cell apoptosis and proliferation (turnover); and proximal-tubular-cell expression of the major apical transporters sodium-phosphate cotransporter 2A (NaPi-IIa) and sodium-glucose cotransporter-2 (SGLT-2).RESULTS: Wnt4-CRE-driven megalin/LRP2 ablation in cystinotic mice efficiently blocked kidney cystine accumulation, thereby preventing lysosomal deformations and crystal deposition in proximal tubular cells. Swan-neck lesions were largely prevented and proximal-tubular-cell turnover was normalized. Apical expression of the two cotransporters was also preserved.CONCLUSIONS: These observations support a key role of the megalin/LRP2 pathway in the progression of nephropathic cystinosis and provide a proof of concept for the pathway as a therapeutic target.

AIM: Activation of sodium reabsorption by urinary proteases has been implicated in sodium retention associated with nephrotic syndrome. The study was designed to test the hypothesis that nephrotic proteinuria in mice after conditional deletion of podocin leads to urokinase dependent, amiloride-sensitive plasmin-mediated sodium and water retention.METHODS: Ten days after podocin knockout, urine and feces was collected for 10 days in metabolic cages and analysed for electrolytes, plasminogen, protease activity, and ability to activate γENaC by patch clamp and western blot. Mice were treated with amiloride (2.5 mg-kg-1 for 2 days and 10 mg-kg-1 for 2 days) or an anti-urokinase-type plasminogen activator (uPA) targeting antibody (120 mg-kg-1 /24h) and compared to controls.RESULTS: Twelve days after deletion, podocin-deficient mice developed significant protein-and albuminuria associated with increased body weight, ascites, sodium accumulation, and suppressed plasma renin. This was associated with increased urinary excretion of plasmin and plasminogen that correlated with albumin excretion, urine protease activity co-migrating with active plasmin, and ability of urine to induce an amiloride-sensitive inward current in M1 cells in vitro. Amiloride treatment in podocin-deficient mice resulted in weight loss, increased sodium excretion, normalization of sodium balance and prevention of the activation of plasminogen to plasmin in urine in a reversible way. Administration of uPA targeting antibody abolished urine activation of plasminogen, attenuated sodium accumulation and prevented cleavage of γENaC.CONCLUSIONS: Nephrotic range glomerular proteinuria leads to urokinase dependent intratubular plasminogen activation and γENaC cleavage which contributes to sodium accumulation. This article is protected by copyright. All rights reserved.

Apolipoprotein L1 (APOL1) risk variants are strongly associated with sporadic focal segmental glomerulosclerosis in populations with African ancestry. We determined the frequency of G1/G2 variants in patients with steroid-resistant nephrotic syndrome/focal segmental glomerulosclerosis with African or French West Indies origin in France and its relationships with other steroid-resistant nephrotic syndrome genes. In a cohort of 152 patients (139 families), the APOL1 risk variants were genotyped: the two risk allele (high risk) genotype was found in 43.1% of subjects compared to 18.9% in a control population (P0.0001). Compared to patients in the low risk group, patients in the high-risk group were more likely to originate from French West Indies than from Africa. APOL1 high-risk genotype was more frequent in young adult patients, but it was also found in children and it was associated with a bad renal prognosis. APOL1 high-risk genotype was usually not associated with other causative mutation in known monogenic steroid-resistant nephrotic syndrome genes and families in which multiple individuals have focal segmental glomerulosclerosis may have APOL1-associated disease. After renal transplantation, recipients of deceased-donor kidney transplantation from individuals with recent African ancestry possessing two APOL1 high-risk variants have slightly shorter allograft survival. Effects of APOL1 are independent of other traditional risk factors. Recently it was shown that black living kidney donors homozygous for APOL1 high-risk alleles have significantly lower glomerular filtration rate and increased risk of end-stage renal disease after donation. However, APOL1 genotype may not summarize by itself the totality of this risk. We showed that living kidney donors of African ancestry in France with low-risk APOL1 genotype have lower postdonation eGFR increase and lower baseline kidney volume compared to Caucasian donors.

Little is known about the long-term progression of adult nephropathic cystinosis patients. Our objective was to study central nervous system complications in cystinosis patients in the era of early cysteamine treatment, using advanced neuroimaging techniques. Neurological examination and multimodal brain 3 Tesla MRI were performed in 21 adult cystinosis patients, including 18 infantile cystinosis patients, 20 controls matched for age and renal function, and 12 healthy controls. Differences in gray matter volume and rest cerebral blood flow (CBF) using arterial spin labeling sequence were investigated using whole-brain voxel-based approach. Median age was 33.8 years (18.7-65.8). Seven patients (38.9%) presented with at least one central nervous system clinical abnormality: two (11.1%) with seizures, three (16.7%) with memory defects, five (27.8%) with cognitive defect, and one (5.5%) with stroke-like episode. These patients had a worse compliance to treatment (compliance score 2 vs 1, P = .03) and received a lower median cysteamine dose (0.9 g/day vs 2.1 g/day, P = .02). Among patients with infantile cystinosis, 13 (72.2%) showed cortical atrophy, which was absent in controls, but it was not correlated with symptoms. Cystinosis patients showed a significant gray matter decrease in the middle frontal gyrus compared with healthy controls and a significant negative correlation between the cystine blood level and rest CBF was observed in the right superior frontal gyrus, a region associated with executive function. Compliance to cysteamine treatment is a major concern in these adult patients and could have an impact on the development of neurological and cognitive complications.

Recent evidence suggests that the presence of more than one pathogenic mutation in a single patient is more common than previously anticipated. One of the challenges hereby is to dissect the contribution of each gene mutation, for which animal models such as Drosophila can provide a valuable aid. Here, we identified three families with mutations in ADD3, encoding for adducin-γ, with intellectual disability, microcephaly, cataracts and skeletal defects. In one of the families with additional cardiomyopathy and steroid-resistant nephrotic syndrome (SRNS), we found a homozygous variant in KAT2B, encoding the lysine acetyltransferase 2B, with impact on KAT2B protein levels in patient fibroblasts, suggesting that this second mutation might contribute to the increased disease spectrum. In order to define the contribution of ADD3 and KAT2B mutations for the patient phenotype, we performed functional experiments in the Drosophila model. We found that both mutations were unable to fully rescue the viability of the respective null mutants of the Drosophila homologs, hts and Gcn5, suggesting that they are indeed pathogenic in flies. While the KAT2B/Gcn5 mutation additionally showed a significantly reduced ability to rescue morphological and functional defects of cardiomyocytes and nephrocytes (podocyte-like cells), this was not the case for the ADD3 mutant rescue. Yet, the simultaneous knockdown of KAT2B and ADD3 synergistically impaired kidney and heart function in flies as well as the adhesion and migration capacity of cultured human podocytes, indicating that mutations in both genes may be required for the full clinical manifestation. Altogether, our studies describe the expansion of the phenotypic spectrum in ADD3 deficiency associated with a homozygous likely pathogenic KAT2B variant and thereby identify KAT2B as a susceptibility gene for kidney and heart disease in ADD3-associated disorders.

Inflammation is involved in the pathogenesis of many disorders. However, the underlying mechanisms are often unknown. Here, we test whether cystinosin, the protein involved in cystinosis, is a critical regulator of galectin-3, a member of the β-galactosidase binding protein family, during inflammation. Cystinosis is a lysosomal storage disorder and, despite ubiquitous expression of cystinosin, the kidney is the primary organ impacted by the disease. Cystinosin was found to enhance lysosomal localization and degradation of galectin-3. In Ctns-/- mice, a mouse model of cystinosis, galectin-3 is overexpressed in the kidney. The absence of galectin-3 in cystinotic mice ameliorates pathologic renal function and structure and decreases macrophage/monocyte infiltration in the kidney of the Ctns-/-Gal3-/- mice compared to Ctns-/- mice. These data strongly suggest that galectin-3 mediates inflammation involved in kidney disease progression in cystinosis. Furthermore, galectin-3 was found to interact with the pro-inflammatory cytokine Monocyte Chemoattractant Protein-1, which stimulates the recruitment of monocytes/macrophages, and proved to be significantly increased in the serum of Ctns-/- mice and also patients with cystinosis. Thus, our findings highlight a new role for cystinosin and galectin-3 interaction in inflammation and provide an additional mechanistic explanation for the kidney disease of cystinosis. This may lead to the identification of new drug targets to delay cystinosis progression.

NPHS2, encoding podocin, is the major gene implicated in steroid-resistant nephrotic syndrome. Its c.686G>A, p.R229Q variant is the first human variant with a mutation-dependent pathogenicity; it is only pathogenic when trans-associated to specific mutations. Secondary to its high allele frequency in the European, South Asian, African, and Latino populations, its benign trans-associations can be accidentally identified in affected patients. Distinguishing pathogenic and benign p.R229Q associations can be challenging. In this paper, we present the currently known pathogenic and benign associations, and show that a rare p.R229Q association can be considered pathogenic if the variant in trans meets the following criteria; it affects the 270-351 residues and alters but does not disrupt the oligomerization, its p.R229Q association is found in a family with slowly progressing focal segmental glomerulosclerosis, but is expected to be rare in the general population ( 15% of the p.R229Q associations identified so far in patients are benign.

Nephrotic syndrome is broadly a disorder of the glomerular filtration barrier, but in practice the site of dysfunction in the great majority of pathologies is in the podocyte. Genetic causes of nephrotic syndrome provide the strongest proof of this. Almost all the genetic associations with nephrotic syndrome are podocyte proteins. Some basement membrane protein mutations associated with nephrotic syndrome may act through signalling to podocytes, or by causing severe disruption to their environment.