Defects in KCNJ16 Cause a Novel Tubulopathy with Hypokalemia, Salt Wasting, Disturbed Acid-Base Homeostasis, and Sensorineural Deafness.

BACKGROUND: The transepithelial transport of electrolytes, solutes, and water in the kidney is a well-orchestrated process involving numerous membrane transport systems. Basolateral potassium channels in tubular cells not only mediate potassium recycling for proper Na+,K+-ATPase function but are also involved in potassium and pH sensing. Genetic defects in KCNJ10 cause EAST/SeSAME syndrome, characterized by renal salt wasting with hypokalemic alkalosis associated with epilepsy, ataxia, and sensorineural deafness. METHODS: A candidate gene approach and whole-exome sequencing determined the underlying genetic defect in eight patients with a novel disease phenotype comprising a hypokalemic tubulopathy with renal salt wasting, disturbed acid-base homeostasis, and sensorineural deafness. Electrophysiologic studies and surface expression experiments investigated the functional consequences of newly identified gene variants. RESULTS: We identified mutations in the KCNJ16 gene encoding KCNJ16, which along with KCNJ15 and KCNJ10, constitutes the major basolateral potassium channel of the proximal and distal tubules, respectively. Coexpression of mutant KCNJ16 together with KCNJ15 or KCNJ10 in Xenopus oocytes significantly reduced currents. CONCLUSIONS: Biallelic variants in KCNJ16 were identified in patients with a novel disease phenotype comprising a variable proximal and distal tubulopathy associated with deafness. Variants affect the function of heteromeric potassium channels, disturbing proximal tubular bicarbonate handling as well as distal tubular salt reabsorption.

Hnf4a Is Required for the Development of Cdh6-Expressing Progenitors into Proximal Tubules in the Mouse Kidney.

BACKGROUND: Hepatocyte NF 4α (Hnf4a) is a major regulator of renal proximal tubule (PT) development. In humans, a mutation in HNF4A impairs PT functions and is associated with Fanconi renotubular syndrome (FRTS). In mice, mosaic deletion of Hnf4a in the developing kidney reduces the population of PT cells, leading to FRTS-like symptoms. The molecular mechanisms underlying the role of Hnf4a in PT development remain unclear. METHODS: The gene deletion tool Osr2Cre removed Hnf4a in developing nephrons in mice, generating a novel model for FRTS. Immunofluorescence analysis characterized the mutant phenotype, and lineage analysis tested whether Cadherin-6 (Cdh6)-expressing cells are PT progenitors. Genome-wide mapping of Hnf4a binding sites and differential gene analysis of Hnf4a mutant kidneys identified direct target genes of Hnf4a. RESULTS: Deletion of Hnf4a with Osr2Cre led to the complete loss of mature PT cells, lethal to the Hnf4a mutant mice. Cdh6high, lotus tetragonolobus lectin-low (LTLlow) cells serve as PT progenitors and demonstrate higher proliferation than Cdh6low, LTLhigh differentiated PT cells. Additionally, Hnf4a is required for PT progenitors to differentiate into mature PT cells. Genomic analyses revealed that Hnf4a directly regulates the expression of genes involved in transmembrane transport and metabolism. CONCLUSIONS: Hnf4a promotes the differentiation of PT progenitors into mature PT cells by regulating the expression of genes associated with reabsorption, the major function of PT cells.

Hypomagnesemia is underestimated in children with HNF1B mutations.

BACKGROUND: Hypomagnesemia in patients with congenital anomalies of the kidneys and urinary tract or autosomal dominant tubulointerstitial kidney disease is highly suggestive of HNF1B-associated disease. Intriguingly, the frequency of low serum Mg2+ (sMg) level varies and is lower in children than in adults with HNF1B mutations that could be partially due to application of inaccurate normal limit of sMg, irrespective of age and gender. We aimed to re-assess cross-sectionally and longitudinally the frequency of hypomagnesemia in HNF1B disease by using locally derived reference values of sMg. METHODS: Fourteen children with HNF1B-associated kidney disease were included. Control group comprising 110 subjects served to generate 2.5th percentiles of sMg as the lower limits of normal. RESULTS: In both controls and patients, sMg correlated with age, gender, and fractional excretion of Mg2+. In girls, sMg concentration was higher than in boys when analyzed in the entire age spectrum (p < 0.05). In HNF1B patients, mean sMg was lower than in controls as compared with respective gender- and age-specific interval (p < 0.001). Low sMg levels (< 0.7 mmol/l) were found in 21.4% of patients at diagnosis and 36.4% at last visit, which rose to 85.7% and 72.7% respectively when using the age- and gender-adjusted reference data. Similarly, in the longitudinal observation, 23% of sMg measurements were < 0.7 mmol/l versus 79.7% when applying respective references. CONCLUSIONS: Hypomagnesemia is underdiagnosed in children with HNF1B disease. sMg levels are age- and gender-dependent; thus, the use of appropriate reference data is crucial to hypomagnesemia in children.

Mineralized tissues in hypophosphatemic rickets.

Hypophosphatemic rickets is caused by renal phosphate wasting that is most commonly due to X-linked dominant mutations in PHEX. PHEX mutations cause hypophosphatemia indirectly, through the increased expression of fibroblast growth factor 23 (FGF23) by osteocytes. FGF23 decreases renal phosphate reabsorption and thereby increases phosphate excretion. The lack of phosphate leads to a mineralization defect at the level of growth plates (rickets), bone tissue (osteomalacia), and teeth, where the defect facilitates the formation of abscesses. The bone tissue immediately adjacent to osteocytes often remains unmineralized ("periosteocytic lesions"), highlighting the osteocyte defect in this disorder. Common clinical features of XLH include deformities of the lower extremities, short stature, enthesopathies, dental abscesses, as well as skull abnormalities such as craniosynostosis and Chiari I malformation. For the past four decades, XLH has been treated by oral phosphate supplementation and calcitriol, which improves rickets and osteomalacia and the dental manifestations, but often does not resolve all aspects of the mineralization defects. A newer treatment approach using inactivating FGF23 antibodies leads to more stable control of serum inorganic phosphorus levels and seems to heal rickets more reliably. However, the long-term benefits of FGF23 antibody treatment remain to be elucidated.

Bartter and Gitelman syndromes: Questions of class.

Bartter and Gitelman syndromes are rare inherited tubulopathies characterized by hypokalaemic, hypochloraemic metabolic alkalosis. They are caused by mutations in at least 7 genes involved in the reabsorption of sodium in the thick ascending limb (TAL) of the loop of Henle and/or the distal convoluted tubule (DCT). Different subtypes can be distinguished and various classifications have been proposed based on clinical symptoms and/or the underlying genetic cause. Yet, the clinical phenotype can show remarkable variability, leading to potential divergences between classifications. These problems mostly relate to uncertainties over the role of the basolateral chloride exit channel CLCNKB, expressed in both TAL and DCT and to what degree the closely related paralogue CLCNKA can compensate for the loss of CLCNKB function. Here, we review what is known about the physiology of the transport proteins involved in these disorders. We also review the various proposed classifications and explain why a gene-based classification constitutes a pragmatic solution.

What does sodium-glucose co-transporter 1 inhibition add: Prospects for dual inhibition.

Epithelial glucose transport is accomplished by Na+ -glucose co-transporters, SGLT1 and SGLT2. In the intestine, uptake of dietary glucose is for its majority mediated by SGLT1, and humans with mutations in the SGLT1 gene show glucose/galactose malabsorption. In the kidney, both transporters, SGLT1 and SGLT2, are expressed and recent studies identified that SGLT2 mediates up to 97% of glucose reabsorption. Humans with mutations in the SGLT2 gene show familial renal glucosuria. In the last three decades, significant progress was made in understanding the physiology of these transporters and their potential as therapeutic targets. Based on the structure of phlorizin, a natural compound acting as a SGLT1/2 inhibitor, initially several SGLT2, and later SGLT1 and dual SGLT1/2 inhibitors have been developed. Interestingly, SGLT2 knockout or treatment with SGLT2 selective inhibitors only causes a fractional glucose excretion in the magnitude of ∼60%, an effect mediated by up-regulation of renal SGLT1. Based on these findings the hypothesis was brought forward that dual SGLT1/2 inhibition might further improve glycaemic control via targeting two distinct organs that express SGLT1: the intestine and the kidney. Of note, SGLT1/2 double knockout mice completely lack renal glucose reabsorption. This review will address the rationale for the development of SGLT1 and dual SGLT1/2 inhibitors and potential benefits compared to sole SGLT2 inhibition.

Focus on neonatal and infantile onset of nephrogenic syndrome of inappropriate antidiuresis: 12 years later.

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD), first described in 2005, is a rare genetic X-linked disease, presenting with hyponatremia, hyposmolarity, euvolemia, inappropriately concentrated urine, increased natriuresis, and undetectable or very low arginine-vasopressine (AVP) circulating levels. It can occur in neonates, infants, or later in life. NSIAD must be early recognized and treated to prevent severe hyponatremia, which can show a dangerous impact on neonatal outcome. In fact, it potentially leads to death or, in case of survival, neurologic sequelae. This review is an update of NSIAD 12 years after the first description, focusing on reported cases of neonatal and infantile onset. The different molecular patterns affecting the AVP receptor 2 (V2R) and determining its gain of function are reported in detail; moreover, we also provide a comparison between the different triggers involved in the development of hyponatremia, the evolution of the symptoms, and modality and efficacy of the different treatments available.

Nephrolithiasis secondary to inherited defects in the thick ascending loop of henle and connecting tubules.

Twin and genealogy studies suggest a strong genetic component of nephrolithiasis. Likewise, urinary traits associated with renal stone formation were found to be highly heritable, even after adjustment for demographic, anthropometric and dietary covariates. Recent high-throughput sequencing projects of phenotypically well-defined cohorts of stone formers and large genome-wide association studies led to the discovery of many new genes associated with kidney stones. The spectrum ranges from infrequent but highly penetrant variants (mutations) causing mendelian forms of nephrolithiasis (monogenic traits) to common but phenotypically mild variants associated with nephrolithiasis (polygenic traits). About two-thirds of the genes currently known to be associated with nephrolithiasis code for membrane proteins or enzymes involved in renal tubular transport. The thick ascending limb of Henle and connecting tubules are of paramount importance for renal water and electrolyte handling, urinary concentration and maintenance of acid-base homeostasis. In most instances, pathogenic variants in genes involved in thick ascending limb of Henle and connecting tubule function result in phenotypically severe disease, frequently accompanied by nephrocalcinosis with progressive CKD and to a variable degree by nephrolithiasis. The aim of this article is to review the current knowledge on kidney stone disease associated with inherited defects in the thick ascending loop of Henle and the connecting tubules. We also highlight recent advances in the field of kidney stone genetics that have implications beyond rare disease, offering new insights into the most common type of kidney stone disease, i.e., idiopathic calcium stone disease.

Calcium-sensing receptor: evidence and hypothesis for its role in nephrolithiasis.

Calcium-sensing receptor (CaSR) is a plasma-membrane G protein-coupled receptor activated by extracellular calcium and expressed in kidney tubular cells. It inhibits calcium reabsorption in the ascending limb and distal convoluted tubule when stimulated by the increase of serum calcium levels; therefore, these tubular segments are enabled by CaSR to play a substantial role in the regulation of serum calcium levels. In addition, CaSR increases water and proton excretion in the collecting duct and promotes phosphate reabsorption and citrate excretion in the proximal tubule. These CaSR activities form a network in which they are integrated to protect the kidney against the negative effects of high calcium concentrations and calcium precipitates in urine. Therefore, the CaSR gene has been considered as a candidate to explain calcium nephrolithiasis. Epidemiological studies observed that calcium nephrolithiasis was associated with polymorphisms of the CaSR gene regulatory region, rs6776158, located within the promoter-1, rs1501899 located in the intron 1, and rs7652589 in the 5'-untranslated region. These polymorphisms were found to reduce the transcriptional activity of promoter-1. Activating rs1042636 polymorphism located in exon 7 was associated with calcium nephrolithiasis and hypercalciuria. Genetic polymorphisms decreasing CaSR expression could predispose individuals to stones because they may impair CaSR protective effects against precipitation of calcium phosphate and oxalate. Activating polymorphisms rs1042636 could predispose to calcium stones by increasing calcium excretion. These findings suggest that CaSR may play a complex role in lithogenesis through different pathways having different relevance under different clinical conditions.

MAGED2: a novel form of antenatal Bartter's syndrome.

PURPOSE OF REVIEW: Antenatal Bartter's syndrome (aBS) is the most severe form of Bartter's syndrome, requiring close follow-up, in particular during the neonatal period, primarily because of prematurity. The recent identification of a novel and very severe form of aBS merits an update on this topic. RECENT FINDING: Despite the identification of several genes involved in Bartter's syndrome, about 20% of patients clinically diagnosed with aBS remained without genetic explanation for decades. We recently identified mutations in MAGED2 as a cause of an X-linked form of aBS characterized by a very early onset of severe polyhydramnios and extreme prematurity leading to high mortality. Remarkably, all symptoms in surviving patients with MAGE-D2 mutations resolve spontaneously, within weeks after preterm birth. Interestingly, MAGE-D2 affects the expression of the sodium chloride cotransporters NKCC2 and NCC, explaining thereby the severity of the disease. Importantly, a more recent analysis of MAGED2 in a large French cohort of patients with aBS confirmed our data and showed that females can also be affected. SUMMARY: MAGE-D2 is critical for renal salt reabsorption in the fetus, amniotic fluid volume regulation, and maintenance of pregnancy. Most importantly, MAGED2 must be included in the genetic screening of every form of aBS.

SaRNA-mediated activation of TRPV5 reduces renal calcium oxalate deposition in rat via decreasing urinary calcium excretion.

Hypercalciuria is a main risk factor for kidney stone  formation. TRPV5 is the gatekeeper protein for mediating calcium transport and reabsorption in the kidney. In the present study, we tested the effect of TRPV5 activation with small activating RNA (saRNA), which could induce gene expression by targeting the promoter of the gene, on ethylene glycol (EG)-induced calcium oxalate (CaOx) crystals formation in rat kidney. Five pairs of RNA activation sequences targeting the promoter of rat TRPV5 were designed and synthesized. The synthesized saRNA with the strongest activating effect was selected, and transcellular calcium transportation was tested by Fura-2 analysis. Subsequently, Sprague-Dawley rats were equally divided into three groups and fed with water, 1% EG for 28 days after injecting the negative control saRNA, 1% EG for 28 days after injecting the selected TRPV5-saRNA, respectively. The CaOx crystal formation and the 24-h urine components were assessed. In vitro study, saRNA ds-320 could significantly activate the expression of TRPV5 and transcellular calcium transportation. In vivo study, after 28 days treatment of EG, rats pre-infected with saRNA ds-320 had lower urinary calcium excretion and renal CaOx crystals formation as compared to that pre-infected with negative control saRNA. Activation of TRVP5 with saRNA ds-320 could inhibit EG-induced calcium oxalate crystals formation via promoting urine calcium reabsorption and decreasing urine calcium excretion in rats.

Salt-Losing Tubulopathies in Children: What's New, What's Controversial?

Renal tubulopathies provide insights into the inner workings of the kidney, yet also pose therapeutic challenges. Because of the central nature of sodium in tubular transport physiology, disorders of sodium handling may affect virtually all aspects of the homeostatic functions of the kidney. Yet, owing to the rarity of these disorders, little clinical evidence regarding treatment exists. Consequently, treatment can vary widely between individual physicians and centers and is based mainly on understanding of renal physiology, reported clinical observations, and individual experiences. Salt-losing tubulopathies can affect all tubular segments, from the proximal tubule to the collecting duct. But the more frequently observed disorders are Bartter and Gitelman syndrome, which affect salt transport in the thick ascending limb of Henle's loop and/or the distal convoluted tubule, and these disorders generate the greatest controversies regarding management. Here, we review clinical and molecular aspects of salt-losing tubulopathies and discuss novel insights provided mainly by genetic investigations and retrospective clinical reviews. Additionally, we discuss controversial topics in the management of these disorders to highlight areas of importance for future clinical trials. International collaboration will be required to perform clinical studies to inform the treatment of these rare disorders.

ORAI channels are critical for receptor-mediated endocytosis of albumin.

Impaired albumin reabsorption by proximal tubular epithelial cells (PTECs) has been highlighted in diabetic nephropathy (DN), but little is known about the underlying molecular mechanisms. Here we find that ORAI1-3, are preferentially expressed in PTECs and downregulated in patients with DN. Hyperglycemia or blockade of insulin signaling reduces the expression of ORAI1-3. Inhibition of ORAI channels by BTP2 and diethylstilbestrol or silencing of ORAI expression impairs albumin uptake. Transgenic mice expressing a dominant-negative Orai1 mutant (E108Q) increases albuminuria, and in vivo injection of BTP2 exacerbates albuminuria in streptozotocin-induced and Akita diabetic mice. The albumin endocytosis is Ca2+-dependent and accompanied by ORAI1 internalization. Amnionless (AMN) associates with ORAIs and forms STIM/ORAI/AMN complexes after Ca2+ store depletion. STIM1/ORAI1 colocalizes with clathrin, but not with caveolin, at the apical membrane of PTECs, which determines clathrin-mediated endocytosis. These findings provide insights into the mechanisms of protein reabsorption and potential targets for treating diabetic proteinuria.

Hyperactivation of Nrf2 in early tubular development induces nephrogenic diabetes insipidus.

NF-E2-related factor-2 (Nrf2) regulates cellular responses to oxidative and electrophilic stress. Loss of Keap1 increases Nrf2 protein levels, and Keap1-null mice die of oesophageal hyperkeratosis because of Nrf2 hyperactivation. Here we show that deletion of oesophageal Nrf2 in Keap1-null mice allows survival until adulthood, but the animals develop polyuria with low osmolality and bilateral hydronephrosis. This phenotype is caused by defects in water reabsorption that are the result of reduced aquaporin 2 levels in the kidney. Renal tubular deletion of Keap1 promotes nephrogenic diabetes insipidus features, confirming that Nrf2 activation in developing tubular cells causes a water reabsorption defect. These findings suggest that Nrf2 activity should be tightly controlled during development in order to maintain renal homeostasis. In addition, tissue-specific ablation of Nrf2 in Keap1-null mice might create useful animal models to uncover novel physiological functions of Nrf2.

Genetic causes of hypomagnesemia, a clinical overview.

Magnesium is essential to the proper functioning of numerous cellular processes. Magnesium ion (Mg2+) deficits, as reflected in hypomagnesemia, can cause neuromuscular irritability, seizures and cardiac arrhythmias. With normal Mg2+ intake, homeostasis is maintained primarily through the regulated reabsorption of Mg2+ by the thick ascending limb of Henle's loop and distal convoluted tubule of the kidney. Inadequate reabsorption results in renal Mg2+ wasting, as evidenced by an inappropriately high fractional Mg2+ excretion. Familial renal Mg2+ wasting is suggestive of a genetic cause, and subsequent studies in these hypomagnesemic families have revealed over a dozen genes directly or indirectly involved in Mg2+ transport. Those can be classified into four groups: hypercalciuric hypomagnesemias (encompassing mutations in CLDN16, CLDN19, CASR, CLCNKB), Gitelman-like hypomagnesemias (CLCNKB, SLC12A3, BSND, KCNJ10, FYXD2, HNF1B, PCBD1), mitochondrial hypomagnesemias (SARS2, MT-TI, Kearns-Sayre syndrome) and other hypomagnesemias (TRPM6, CNMM2, EGF, EGFR, KCNA1, FAM111A). Although identification of these genes has not yet changed treatment, which remains Mg2+ supplementation, it has contributed enormously to our understanding of Mg2+ transport and renal function. In this review, we discuss general mechanisms and symptoms of genetic causes of hypomagnesemia as well as the specific molecular mechanisms and clinical phenotypes associated with each syndrome.

Adenosine, type 1 receptors: role in proximal tubule Na+ reabsorption.

Adenosine type 1 receptor (A1 -AR) antagonists induce diuresis and natriuresis in experimental animals and humans. Much of this effect is due to inhibition of A1 -ARs in the proximal tubule, which is responsible for 60-70% of the reabsorption of filtered Na(+) and fluid. Intratubular application of receptor antagonists indicates that A1 -AR mediates a portion of Na(+) uptake in PT and PT cells, via multiple transport systems, including Na(+) /H(+) exchanger-3 (NHE3), Na(+) /PO4(-) co-transporter and Na(+) -dependent glucose transporter, SGLT. Renal microperfusion and recollection studies have shown that fluid reabsorption is reduced by A1 -AR antagonists and is lower in A1 -AR KO mice, compared to WT mice. Absolute proximal reabsorption (APR) measured by free-flow micropuncture is equivocal, with studies that show either lower APR or similar APR in A1 -AR KO mice, compared to WT mice. Inhibition of A1 -ARs lowers elevated blood pressure in models of salt-sensitive hypertension, partially due to their effects in the proximal tubule.

Deletion of the angiotensin II type 1 receptor-associated protein enhances renal sodium reabsorption and exacerbates angiotensin II-mediated hypertension.

Angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP) promotes AT1R internalization along with suppression of pathological activation of tissue AT1R signaling. However, the functional significance of ATRAP in renal sodium handling and blood pressure regulation under pathological stimuli is not fully resolved. Here we show the blood pressure of mice with a gene-targeted disruption of ATRAP was comparable to that of wild-type mice at baseline. However, in ATRAP-knockout mice, angiotensin II-induced hypertension was exacerbated and the extent of positive sodium balance was increased by angiotensin II. Renal expression of the sodium-proton antiporter 3, a major sodium transporter in the proximal tubules, urinary pH, renal angiotensinogen production, and angiotensin II content was unaffected. Stimulation of the renal expression and activity of the epithelial sodium channel (ENaC), a major sodium transporter in the distal tubules, was significantly enhanced by chronic angiotensin II infusion. The circulating and urinary aldosterone levels were comparable. The blood pressure response and renal ENaC expression by aldosterone were not affected. Thus, ATRAP deficiency exacerbated angiotensin II-mediated hypertension by pathological activation of renal tubular AT1R by angiotensin II. This directly stimulates ENaC in the distal tubules and enhances sodium retention in an aldosterone-independent manner.