Learning Physiology From Inherited Kidney Disorders.

The identification of genes causing inherited kidney diseases yielded crucial insights in the molecular basis of disease and improved our understanding of physiological processes that operate in the kidney. Monogenic kidney disorders are caused by mutations in genes coding for a large variety of proteins including receptors, channels and transporters, enzymes, transcription factors, and structural components, operating in specialized cell types that perform highly regulated homeostatic functions. Common variants in some of these genes are also associated with complex traits, as evidenced by genome-wide association studies in the general population. In this review, we discuss how the molecular genetics of inherited disorders affecting different tubular segments of the nephron improved our understanding of various transport processes and of their involvement in homeostasis, while providing novel therapeutic targets. These include inherited disorders causing a dysfunction of the proximal tubule (renal Fanconi syndrome), with emphasis on epithelial differentiation and receptor-mediated endocytosis, or affecting the reabsorption of glucose, the handling of uric acid, and the reabsorption of sodium, calcium, and magnesium along the kidney tubule.

Parathyroid hormone and phosphate homeostasis in patients with Bartter and Gitelman syndrome: an international cross-sectional study.

BACKGROUND: Small cohort studies have reported high parathyroid hormone (PTH) levels in patients with Bartter syndrome and lower serum phosphate levels have anecdotally been reported in patients with Gitelman syndrome. In this cross-sectional study, we assessed PTH and phosphate homeostasis in a large cohort of patients with salt-losing tubulopathies. METHODS: Clinical and laboratory data of 589 patients with Bartter and Gitelman syndrome were provided by members of the European Rare Kidney Diseases Reference Network (ERKNet) and the European Society for Paediatric Nephrology (ESPN). RESULTS: A total of 285 patients with Bartter syndrome and 304 patients with Gitelman syndrome were included for analysis. Patients with Bartter syndrome type I and II had the highest median PTH level (7.5 pmol/L) and 56% had hyperparathyroidism (PTH >7.0 pmol/L). Serum calcium was slightly lower in Bartter syndrome type I and II patients with hyperparathyroidism (2.42 versus 2.49 mmol/L; P = .038) compared to those with normal PTH levels and correlated inversely with PTH (rs -0.253; P = .009). Serum phosphate and urinary phosphate excretion did not correlate with PTH. Overall, 22% of patients had low serum phosphate levels (phosphate-standard deviation score < -2), with the highest prevalence in patients with Bartter syndrome type III (32%). Serum phosphate correlated with tubular maximum reabsorption of phosphate/glomerular filtration rate (TmP/GFR) (rs 0.699; P < .001), suggesting renal phosphate wasting. CONCLUSIONS: Hyperparathyroidism is frequent in patients with Bartter syndrome type I and II. Low serum phosphate is observed in a significant number of patients with Bartter and Gitelman syndrome and appears associated with renal phosphate wasting.

Clinical and genetic spectra of autosomal dominant tubulointerstitial kidney disease due to mutations in UMOD and MUC1.

Autosomal dominant tubulointerstitial kidney disease (ADTKD) is an increasingly recognized cause of end-stage kidney disease, primarily due to mutations in UMOD and MUC1. The lack of clinical recognition and the small size of cohorts have slowed the understanding of disease ontology and development of diagnostic algorithms. We analyzed two registries from Europe and the United States to define genetic and clinical characteristics of ADTKD-UMOD and ADTKD-MUC1 and develop a practical score to guide genetic testing. Our study encompassed 726 patients from 585 families with a presumptive diagnosis of ADTKD along with clinical, biochemical, genetic and radiologic data. Collectively, 106 different UMOD mutations were detected in 216/562 (38.4%) of families with ADTKD (303 patients), and 4 different MUC1 mutations in 72/205 (35.1%) of the families that are UMOD-negative (83 patients). The median kidney survival was significantly shorter in patients with ADTKD-MUC1 compared to ADTKD-UMOD (46 vs. 54 years, respectively), whereas the median gout-free survival was dramatically reduced in patients with ADTKD-UMOD compared to ADTKD-MUC1 (30 vs. 67 years, respectively). In contrast to patients with ADTKD-UMOD, patients with ADTKD-MUC1 had normal urinary excretion of uromodulin and distribution of uromodulin in tubular cells. A diagnostic algorithm based on a simple score coupled with urinary uromodulin measurements separated patients with ADTKD-UMOD from those with ADTKD-MUC1 with a sensitivity of 94.1%, a specificity of 74.3% and a positive predictive value of 84.2% for a UMOD mutation. Thus, ADTKD-UMOD is more frequently diagnosed than ADTKD-MUC1, ADTKD subtypes present with distinct clinical features, and a simple score coupled with urine uromodulin measurements may help prioritizing genetic testing.

Treatment and long-term outcome in primary nephrogenic diabetes insipidus.

BACKGROUND: Primary nephrogenic diabetes insipidus (NDI) is a rare disorder and little is known about treatment practices and long-term outcome. METHODS: Paediatric and adult nephrologists contacted through European professional organizations entered data in an online form. RESULTS: Data were collected on 315 patients (22 countries, male 84%, adults 35%). Mutation testing had been performed in 270 (86%); pathogenic variants were identified in 258 (96%). The median (range) age at diagnosis was 0.6 (0.0-60) years and at last follow-up 14.0 (0.1-70) years. In adults, height was normal with a mean (standard deviation) score of -0.39 (±1.0), yet there was increased prevalence of obesity (body mass index >30 kg/m2; 41% versus 16% European average; P < 0.001). There was also increased prevalence of chronic kidney disease (CKD) Stage ≥2 in children (32%) and adults (48%). Evidence of flow uropathy was present in 38%. A higher proportion of children than adults (85% versus 54%; P < 0.001) received medications to reduce urine output. Patients ≥25 years were less likely to have a university degree than the European average (21% versus 35%; P = 0.003) but full-time employment was similar. Mental health problems, predominantly attention-deficit hyperactivity disorder (16%), were reported in 36% of patients. CONCLUSION: This large NDI cohort shows an overall favourable outcome with normal adult height and only mild to moderate CKD in most. Yet, while full-time employment was similar to the European average, educational achievement was lower, and more than half had urological and/or mental health problems.

High-throughput sequencing contributes to the diagnosis of tubulopathies and familial hypercalcemia hypocalciuria in adults.

Hereditary tubulopathies are rare diseases with unknown prevalence in adults. Often diagnosed in childhood, hereditary tubulopathies can nevertheless be evoked in adults. Precise diagnosis can be difficult or delayed due to insidious development of symptoms, comorbidities and polypharmacy. Here we evaluated the diagnostic value of a specific panel of known genes implicated in tubulopathies in adult patients and compared to our data obtained in children. To do this we analyzed 1033 non-related adult patients of which 744 had a clinical diagnosis of tubulopathy and 289 had a diagnosis of familial hypercalcemia with hypocalciuria recruited by three European reference centers. Three-quarters of our tubulopathies cohort included individuals with clinical suspicion of Gitelman syndrome, kidney hypophosphatemia and kidney tubular acidosis. We detected pathogenic variants in 26 different genes confirming a genetic diagnosis of tubulopathy in 29% of cases. In 16 cases (2.1%) the genetic testing changed the clinical diagnosis. The diagnosis of familial hypercalcemia with hypocalciuria was confirmed in 12% of cases. Thus, our work demonstrates the genetic origin of tubulopathies in one out of three adult patients, half of the rate observed in children. Hence, establishing a precise diagnosis is crucial for patients, in order to guide care, to survey and prevent chronic complications, and for genetic counselling. At the same time, this work enhances our understanding of complex phenotypes and enriches the database with the causal variants described.

Cervical artery dissection: fibromuscular dysplasia versus vascular Ehlers-Danlos syndrome.

We report the case of a 42-year-old patient referred for suspicion of fibromuscular dysplasia in the context of a carotid artery dissection occurring after a minor trauma. Initial complaints included left hemicrania, lateral diplopia with left 6th cranial nerve palsy and pulsatile tinnitus. The work-up disclosed a large left carotid-cavernous fistula, as well as more proximal carotid lesions compatible with multifocal fibromuscular dysplasia. Personal history included colonic and uterine perforation. Family history disclosed a fatal hemorrhage due to rupture of a splenic artery aneurysm in the father and an iliac dissection in the sister. Genetic screening revealed a mutation in exon 6 of the COL3A1 gene in the index patient and her sister, confirming the diagnosis of vascular Ehlers-Danlos syndrome (vEDS). This case report shows that images suggestive of fibromuscular dysplasia may be found in patients with demonstrated vEDS. Furthermore, it reminds that in case of cervical artery dissection occurring in a young patient, all efforts should be made to diagnose the underlying condition. In particular, the existence of a family history of arterial dissection, the occurrence of a carotid-cavernous fistula and coexistence with other complications suggestive of a connective tissue disease should prompt physicians to consider the diagnosis of vEDS.

Genome-Wide Meta-Analysis Unravels Interactions between Magnesium Homeostasis and Metabolic Phenotypes.

Magnesium (Mg2+) homeostasis is critical for metabolism. However, the genetic determinants of the renal handling of Mg2+, which is crucial for Mg2+ homeostasis, and the potential influence on metabolic traits in the general population are unknown. We obtained plasma and urine parameters from 9099 individuals from seven cohorts, and conducted a genome-wide meta-analysis of Mg2+ homeostasis. We identified two loci associated with urinary magnesium (uMg), rs3824347 (P=4.4×10-13) near TRPM6, which encodes an epithelial Mg2+ channel, and rs35929 (P=2.1×10-11), a variant of ARL15, which encodes a GTP-binding protein. Together, these loci account for 2.3% of the variation in 24-hour uMg excretion. In human kidney cells, ARL15 regulated TRPM6-mediated currents. In zebrafish, dietary Mg2+ regulated the expression of the highly conserved ARL15 ortholog arl15b, and arl15b knockdown resulted in renal Mg2+ wasting and metabolic disturbances. Finally, ARL15 rs35929 modified the association of uMg with fasting insulin and fat mass in a general population. In conclusion, this combined observational and experimental approach uncovered a gene-environment interaction linking Mg2+ deficiency to insulin resistance and obesity.

Common variants in CLDN14 are associated with differential excretion of magnesium over calcium in urine.

The nature and importance of genetic factors regulating the differential handling of Ca2+ and Mg2+ by the renal tubule in the general population are poorly defined. We conducted a genome-wide meta-analysis of urinary magnesium-to-calcium ratio to identify associated common genetic variants. We included 9320 adults of European descent from four genetic isolates and three urban cohorts. Urinary magnesium and calcium concentrations were measured centrally in spot urine, and each study conducted linear regression analysis of urinary magnesium-to-calcium ratio on ~2.5 million single-nucleotide polymorphisms (SNPs) using an additive model. We investigated, in mouse, the renal expression profile of the top candidate gene and its variation upon changes in dietary magnesium. The genome-wide analysis evidenced a top locus (rs172639, p = 1.7 × 10-12), encompassing CLDN14, the gene coding for claudin-14, that was genome-wide significant when using urinary magnesium-to-calcium ratio, but not either one taken separately. In mouse, claudin-14 is expressed in the distal nephron segments specifically handling magnesium, and its expression is regulated by chronic changes in dietary magnesium content. A genome-wide approach identified common variants in the CLDN14 gene exerting a robust influence on the differential excretion of Mg2+ over Ca2+ in urine. These data highlight the power of urinary electrolyte ratios to unravel genetic determinants of renal tubular function. Coupled with mouse experiments, these results support a major role for claudin-14, a gene associated with kidney stones, in the differential paracellular handling of divalent cations by the renal tubule.

Bone marrow transplantation improves proximal tubule dysfunction in a mouse model of Dent disease.

Dent disease is a rare X-linked tubulopathy caused by mutations in the endosomal chloride-proton exchanger (ClC-5) resulting in defective receptor-mediated endocytosis and severe proximal tubule dysfunction. Bone marrow transplantation has recently been shown to preserve kidney function in cystinosis, a lysosomal storage disease causing proximal tubule dysfunction. Here we test the effects of bone marrow transplantation in Clcn5Y/- mice, a faithful model for Dent disease. Transplantation of wild-type bone marrow in Clcn5Y/- mice significantly improved proximal tubule dysfunction, with decreased low-molecular-weight proteinuria, glycosuria, calciuria, and polyuria four months after transplantation, compared to Clcn5Y/- mice transplanted with ClC-5 knockout bone marrow. Bone marrow-derived cells engrafted in the interstitium, surrounding proximal tubule cells, which showed a rescue of the apical expression of ClC-5 and megalin receptors. The improvement of proximal tubule dysfunction correlated with Clcn5 gene expression in kidneys of mice transplanted with wild-type bone marrow cells. Coculture of Clcn5Y/- proximal tubule cells with bone marrow-derived cells confirmed rescue of ClC-5 and megalin, resulting in improved endocytosis. Nanotubular extensions between the engrafted bone marrow-derived cells and proximal tubule cells were observed in vivo and in vitro. No rescue was found when the formation of the tunneling nanotubes was prevented by actin depolymerization or when cells were physically separated by transwell inserts. Thus, bone marrow transplantation may rescue the epithelial phenotype due to an inherited endosomal defect. Direct contacts between bone marrow-derived cells and diseased tubular cells play a key role in the rescue mechanism.

Clinical and mutational spectrum of hypoparathyroidism, deafness and renal dysplasia syndrome.

BACKGROUND: Hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome is a rare autosomal dominant disorder, secondary to mutations in the GATA-3 gene. Due to its wide range of penetrance and expressivity, the disease may not always be recognized. We herein describe clinical and genetic features of patients with HDR syndrome, highlighting diagnostic clues. METHODS: Medical records of eight patients from five unrelated families exhibiting GATA-3 mutations were reviewed retrospectively, in conjunction with all previously reported cases. RESULTS: HDR syndrome was diagnosed in eight patients between the ages of 18 and 60 years. Sensorineural deafness was consistently diagnosed, ranging from clinical hearing loss since infancy in seven patients to deafness detected only by audiometry in adulthood in one single patient. Hypoparathyroidism was present in six patients (with hypocalcaemia and inaugural seizures in two out of six). Renal abnormalities observed in six patients were diverse and of dysplastic nature. Three patients displayed nephrotic-range proteinuria and reached end-stage renal disease (ESRD) between the ages of 19 and 61 years, whilst lesions of focal and segmental glomerulosclerosis were histologically demonstrated in one of them. Interestingly, phenotype severity differed significantly between a mother and son within one family. Five new mutations of GATA-3 were identified, including three missense mutations affecting zinc finger motifs [NM_001002295.1: c.856A>G (p.N286D) and c.1017C>G (p.C339W)] or the conserved linker region [c.896G>A (p.R299G)], and two splicing mutations (c.924+4_924+19del and c.1051-2A>G). Review of 115 previously reported cases of GATA-3 mutations showed hypoparathyroidism and deafness in 95% of patients, and renal abnormalities in only 60%. Overall, 10% of patients had reached ESRD. CONCLUSIONS: We herein expand the clinical and mutational spectrum of HDR syndrome, illustrating considerable inter- and intrafamilial phenotypic variability. Diagnosis of HDR should be considered in any patient with hypoparathyroidism and deafness, whether associated with renal abnormalities or not. HDR diagnosis is established through identification of a mutation in the GATA-3 gene.

Paradoxical response to furosemide in uromodulin-associated kidney disease.

Mutations in the UMOD gene coding for uromodulin cause autosomal dominant tubulointerstitial kidney disease. Uromodulin is known to regulate transport processes in the thick ascending limb, but it remains unknown whether UMOD mutations are associated with functional tubular alterations in the early phase of the disease. The responses to furosemide and to a water deprivation test were compared in a 32-year-old female patient carrying the pathogenic UMOD mutation p.C217G and her unaffected 31-year-old sister. A single dose of furosemide induced an intense headache with exaggerated decrease in blood pressure (Δsyst: 30 versus 20 mmHg; Δdiast: 18 versus 5 mmHg) and body weight (Δ2.6 kg versus Δ0.9 kg over 3 h) in the proband versus unaffected sib. The diuretic response and the fall in urine osmolality were also more important and detected earlier in the affected sib. Water deprivation led to increased plasma osmolality and urine concentration in both siblings; however, the response to desmopressin was attenuated in the affected sib. These data reveal that mutations of uromodulin cause specific transport alterations, including exaggerated response to furosemide and a failure to maximally concentrate urine, in the early phase of the disease.

A role for Rhesus factor Rhcg in renal ammonium excretion and male fertility.

The kidney has an important role in the regulation of acid-base homeostasis. Renal ammonium production and excretion are essential for net acid excretion under basal conditions and during metabolic acidosis. Ammonium is secreted into the urine by the collecting duct, a distal nephron segment where ammonium transport is believed to occur by non-ionic NH(3) diffusion coupled to H(+) secretion. Here we show that this process is largely dependent on the Rhesus factor Rhcg. Mice lacking Rhcg have abnormal urinary acidification due to impaired ammonium excretion on acid loading-a feature of distal renal tubular acidosis. In vitro microperfused collecting ducts of Rhcg(-/-) acid-loaded mice show reduced apical permeability to NH(3) and impaired transepithelial NH(3) transport. Furthermore, Rhcg is localized in epididymal epithelial cells and is required for normal fertility and epididymal fluid pH. We anticipate a critical role for Rhcg in ammonium handling and pH homeostasis both in the kidney and the male reproductive tract.

The rhesus protein RhCG: a new perspective in ammonium transport and distal urinary acidification.

Urinary acidification is a complex process requiring the coordinated action of enzymes and transport proteins and resulting in the removal of acid and the regeneration of bicarbonate. Proton secretion is mediated by luminal H(+)-ATPases and requires the parallel movement of NH3, and its protonation to NH4(+), to provide sufficient buffering. It has been long assumed that ammonia secretion is a passive process occurring by means of simple diffusion driven by the urinary trapping of ammonium. However, new data indicate that mammalian cells possess specific membrane proteins from the family of rhesus proteins involved in ammonia/µm permeability. Rhesus proteins were first identified in yeast and later also in plants, algae, and mammals. In rodents, RhBG and RhCG are expressed in the collecting duct, whereas in humans only RhCG was detected. Their expression increases with maturation of the kidney and accelerates after birth in parallel with other acid-base transport proteins. Deletion of RhBG in mice had no effect on renal ammonium excretion, whereas RhCG deficiency reduces renal ammonium secretion strongly, causes metabolic acidosis in acid-challenged mice, and impairs restoration of normal acid-base status. Microperfusion experiments or functional reconstitution in liposomes demonstrates that ammonia is the most likely substrate of RhCG. Similarly, crystal structures of human RhCG and the homologous bacterial AmtB protein suggest that these proteins may form gas channels.

Evaluating PVALB as a candidate gene for SLC12A3-negative cases of Gitelman's syndrome.

BACKGROUND: Loss-of-function mutations in SLC12A3 coding for the thiazide-sensitive NaCl cotransporter (NCC) cause Gitelman's syndrome (GS), a recessively inherited salt-losing tubulopathy. Most GS patients are compound heterozygous. However, up to 30% of GS patients carry only a single mutant allele, and a normal SLC12A3 screening is also observed in a small subset of patients. Locus heterogeneity could explain the lack of detection of mutant SLC12A3 alleles in GS patients. The renal phenotype of the parvalbumin knockout mice pointed to PVALB as a candidate gene for GS for SLC12A3-negative cases. METHODS: PCR and direct sequencing of PVALB was performed in 132 GS patients in whom only one or no (N = 79) mutant SLC12A3 allele was found. The possible interference of biallelic SNPs (single nucleotide polymorphisms) on normal transcription or normal splicing was investigated. Genotyping of 110 anonymous blood donors was performed to determine the allelic frequency in the normal population. RESULTS: No sequence variants resulting in amino acid substitution or truncated protein within the PVALB gene were found in the 264 chromosomes tested. Ten biallelic SNPs, including six novel polymorphisms, were identified: five in the 5' UTR, none of them affecting predicted regulatory elements; three in the coding region, without alteration of the consensus splice sites, and two in the 3' UTR. The observed allelic frequencies did not differ significantly between GS patients and controls. CONCLUSION: Our results strongly suggest that mutations in the PVALB gene are not involved in GS patients who harbour a single or no mutant SLC12A3 allele.

Renal expression of parvalbumin is critical for NaCl handling and response to diuretics.

The distal convoluted tubule (DCT) plays an essential role in the reabsorption of NaCl by the kidney, a process that can be inhibited by thiazide diuretics. Parvalbumin (PV), a Ca(2+)-binding protein that plays a role in muscle fibers and neurons, is selectively expressed in the DCT, where its role remains unknown. We therefore investigated the renal phenotype of PV knockout mice (Pvalb(-/-)) vs. wild-type (Pvalb(+/+)) littermates. PV colocalized with the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) in the early DCT. The Pvalb(-/-) mice showed increased diuresis and kaliuresis at baseline with higher aldosterone levels and lower lithium clearance. Acute furosemide administration increased diuresis and natriuresis/kaliuresis, but, surprisingly, did not increase calciuria in Pvalb(-/-) mice. NaCl supplementation of Pvalb(-/-) mice increased calciuria at baseline and after furosemide. The Pvalb(-/-) mice showed no significant diuretic response to hydrochlorothiazide, but an accentuated hypocalciuria. A decreased expression of NCC was detected in the early DCT of Pvalb(-/-) kidneys in the absence of ultrastructural and apoptotic changes. The PV-deficient mice had a positive Ca(2+) balance and increased bone mineral density. Studies in mouse DCT cells showed that endogenous NCC expression is Ca(2+)-dependent and can be modulated by the levels of PV expression. These results suggest that PV regulates the expression of NCC by modulating intracellular Ca(2+) signaling in response to ATP in DCT cells. They also provide insights into the Ca(2+)-sparing action of thiazides and the pathophysiology of distal tubulopathies.