Fiona E. Karet

Mutations in Kelch-like 3 and Cullin 3 cause hypertension and electrolyte abnormalities

Hypertension affects one billion people and is a principal reversible risk factor for cardiovascular disease. Pseudohypoaldosteronism type II (PHAII), a rare Mendelian syndrome featuring hypertension, hyperkalaemia and metabolic acidosis, has revealed previously unrecognized physiology orchestrating the balance between renal salt reabsorption and K+ and H+ excretion. Here we used exome sequencing to identify mutations in kelch-like 3 (KLHL3) or cullin 3 (CUL3) in PHAII patients from 41 unrelated families. KLHL3 mutations are either recessive or dominant, whereas CUL3 mutations are dominant and predominantly de novo. CUL3 and BTB-domain-containing kelch proteins such as KLHL3 are components of cullin–RING E3 ligase complexes that ubiquitinate substrates bound to kelch propeller domains. Dominant KLHL3 mutations are clustered in short segments within the kelch propeller and BTB domains implicated in substrate and cullin binding, respectively. Diverse CUL3 mutations all result in skipping of exon 9, producing an in-frame deletion. Because dominant KLHL3 and CUL3 mutations both phenocopy recessive loss-of-function KLHL3 mutations, they may abrogate ubiquitination of KLHL3 substrates. Disease features are reversed by thiazide diuretics, which inhibit the Na–Cl cotransporter in the distal nephron of the kidney; KLHL3 and CUL3 are expressed in this location, suggesting a mechanistic link between KLHL3 and CUL3 mutations, increased Na–Cl reabsorption, and disease pathogenesis. These findings demonstrate the utility of exome sequencing in disease gene identification despite the combined complexities of locus heterogeneity, mixed models of transmission and frequent de novo mutation, and establish a fundamental role for KLHL3 and CUL3 in blood pressure, K+ and pH homeostasis.

Mutations in VPS33B, encoding a regulator of SNARE-dependent membrane fusion, cause arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome

ARC syndrome (OMIM 208085) is an autosomal recessive multisystem disorder characterized by neurogenic arthrogryposis multiplex congenita, renal tubular dysfunction and neonatal cholestasis with bile duct hypoplasia and low gamma glutamyl transpeptidase (gGT) activity. Platelet dysfunction is common. Affected infants do not thrive and usually die in the first year of life. To elucidate the molecular basis of ARC, we mapped the disease to a 7-cM interval on 15q26.1 and then identified germline mutations in the gene VPS33B in 14 kindreds with ARC. VPS33B encodes a homolog of the class C yeast vacuolar protein sorting gene, Vps33, that contains a Sec1-like domain important in the regulation of vesicle-to-target SNARE complex formation and subsequent membrane fusion.

Inherited Distal Renal Tubular Acidosis

Renal acid-base balance may become deranged in a number of ways, some of which are the consequence of inherited disorders. Two main groups of distal renal acidopathies result either from a direct inability to secrete acid in the distal nephron (giving rise to type 1 renal tubular acidosis [RTA]) or

Non-polarized targeting of AE1 causes autosomal dominant distal renal tubular acidosis

Autosomal dominant distal renal tubular acidosis (ddRTA) is caused by mutations in SLC4A1, which encodes the polytopic chloride–bicarbonate exchanger AE1 that is normally expressed at the basolateral surface of α-intercalated cells in the distal nephron. Here we report that, in contrast with many disorders in which mutant membrane proteins are retained intracellularly and degraded, ddRTA can result from aberrant targeting of AE1 to the apical surface.

Localization and Regulation of the ATP6V0A4 (a4) Vacuolar H+-ATPase Subunit Defective in an Inherited Form of Distal Renal Tubular Acidosis

Vacuolar-type H(+)-ATPases (V-H(+)-ATPases) are the major H(+)-secreting protein in the distal portion of the nephron and are involved in net H(+) secretion (bicarbonate generation) or H(+) reabsorption (net bicarbonate secretion). In addition, V-H(+)-ATPases are involved in HCO(3)(-) reabsorption in the proximal tubule and distal tubule. V-H(+)-ATPases consist of at least 13 subunits, the functions of which have not all been elucidated. Mutations in the accessory ATP6V0A4 (a4 isoform) subunit have recently been shown to cause an inherited form of distal renal tubular acidosis in humans. Here, the localization of this subunit in human and mouse kidney was studied and the regulation of expression and localization of this subunit in mouse kidney in response to acid-base and electrolyte intake was investigated. Reverse transcription-PCR on dissected mouse nephron segments amplified a4-specific transcripts in proximal tubule, loop of Henle, distal convoluted tubule, and cortical and medullary collecting duct. a4 protein was localized by immunohistochemistry to the apical compartment of the proximal tubule (S1/S2 segment), the loop of Henle, the intercalated cells of the distal convoluted tubule, the connecting segment, and all intercalated cells of the entire collecting duct in human and mouse kidney. All types of intercalated cells expressed a4. NH(4)Cl or NaHCO(3) loading for 24 h, 48 h, or 7 d as well as K(+) depletion for 7 and 14 d had no influence on a4 protein expression levels in either cortex or medulla as determined by Western blotting. Immunohistochemistry, however, demonstrated a subcellular redistribution of a4 in response to the different stimuli. NH(4)Cl and K(+) depletion led to a pronounced apical staining in the connecting segment, cortical collecting duct, and outer medullary collecting duct, whereas NaHCO(3) loading caused a stronger bipolar staining in the cortical collecting duct. Taken together, these results demonstrate a4 expression in the proximal tubule, loop of Henle, distal tubule, and collecting duct and suggest that under conditions in which increased V-H(+)-ATPase activity is required, a4 is regulated by trafficking but not protein expression. This may allow for the rapid adaptation of V-H(+)-ATPase activity to altered acid-base intake to achieve systemic pH homeostasis. The significance of a4 expression in the proximal tubule in the context of distal renal tubular acidosis will require further clarification.

Molecular cloning and characterization of novel tissue-specific isoforms of the human vacuolar H(+)-ATPase C, G and d subunits, and their evaluation in autosomal recessive distal renal tubular acidosis.

Several of the 13 subunits comprising mammalian H(+)-ATPases have multiple isoforms, encoded by separate genes and with differing tissue expression patterns, which may play an important role in the intracellular localization and activity of H(+)-ATPases. Here we report the cloning of three previously uncharacterized human genes, ATP6V1C2, ATP6V1G3 and ATP6V0D2, encoding novel H(+)-ATPase subunit isoforms C2, G3 and d2, respectively. We demonstrate that these novel genes are expressed in kidney and few other tissues, and confirm previous reports that the C1, G1 and d1 isoforms are ubiquitously expressed, while G2 is brain-specific. Previously we have shown that mutations in two kidney-specific genes, ATP6V1B1 and ATP6V0A4, encoding the H(+)-ATPase B1 and a4 subunit isoforms, cause recessive distal renal tubular acidosis (dRTA). As the genes reported here are expressed mainly in kidney, we assessed their candidacy as causative genes for recessive dRTA in eight kindreds unlinked to either known disease locus. Although no potential disease-causing mutations were seen in this cohort, this does not rule out a role for these genes in other families. The identification of these three novel tissue-specific isoforms supports the hypothesis that subunit differences may play a key role in the structure, site and function of H(+)-ATPases within the cell.

Mechanisms in Hyperkalemic Renal Tubular Acidosis

The form of renal tubular acidosis associated with hyperkalemia is usually attributable to real or apparent hypoaldosteronism. It is therefore a common feature in diabetes and a number of other conditions associated with underproduction of renin or aldosterone. In addition, the close relationship between potassium levels and ammonia production dictates that hyperkalemia per se can lead to acidosis. Here I describe the modern relationship between molecular function of the distal portion of the nephron, pathways of ammoniagenesis, and hyperkalemia.

Uromodulin mutations causing Familial Juvenile Hyperuricaemic Nephropathy lead to protein maturation defects and retention in the endoplasmic reticulum

Familial juvenile hyperuricaemic nephropathy (FJHN), an autosomal dominant disorder, is caused by mutations in the UMOD gene, which encodes Uromodulin, a glycosylphosphatidylinositol-anchored protein that is expressed in the thick ascending limb of the loop of Henle and excreted in the urine. Uromodulin contains three epidermal growth factor (EGF)-like domains, a cysteine-rich region which includes a domain of eight cysteines and a zona pellucida (ZP) domain. Over 90% of UMOD mutations are missense, and 62% alter a cysteine residue, implicating a role for protein misfolding in the disease. We investigated 20 northern European FJHN probands for UMOD mutations. Wild-type and mutant Uromodulins were functionally studied by expression in HeLa cells and by the use of western blot analysis and confocal microscopy. Six different UMOD missense mutations (Cys32Trp, Arg185Gly, Asp196Asn, Cys217Trp, Cys223Arg and Gly488Arg) were identified. Patients with UMOD mutations were phenotypically similar to those without UMOD mutations. The mutant Uromodulins had significantly delayed maturation, retention in the endoplasmic reticulum (ER) and reduced expression at the plasma membrane. However, Gly488Arg, which is the only mutation we identified in the ZP domain, was found to be associated with milder in vitro abnormalities and to be the only mutant Uromodulin detected in conditioned medium from transfected cells, indicating that the severity of the mutant phenotypes may depend on their location within the protein. Thus, FJHN-causing Uromodulin mutants are retained in the ER, with impaired intracellular maturation and trafficking, thereby indicating mechanisms whereby Uromodulin mutants may cause the phenotype of FJHN.

Vacuolar H+-ATPase d2 subunit: molecular characterization, developmental regulation, and localization to specialized proton pumps in kidney and bone.

The ubiquitous multisubunit vacuolar-type proton pump (H+- or V-ATPase) is essential for acidification of diverse intracellular compartments. It is also present in specialized forms at the plasma membrane of intercalated cells in the distal nephron, where it is required for urine acidification, and in osteoclasts, playing an important role in bone resorption by acid secretion across the ruffled border membrane. It was reported previously that, in human, several of the renal pumps constituent subunits are encoded by genes that are different from those that are ubiquitously expressed. These paralogous proteins may be important in differential functions, targeting or regulation of H+-ATPases. They include the d subunit, where d1 is ubiquitous whereas d2 has a limited tissue expression. This article reports on an investigation of d2. It was first confirmed that in mouse, as in human, kidney and bone are two of the main sites of d2 mRNA expression. d2 mRNA and protein appear later during nephrogenesis than does the ubiquitously expressed E1 subunit. Mouse nephron-segment reverse transcription-PCR revealed detectable mRNA in all segments except thin limb of Henles loop and distal convoluted tubule. However, with the use of a novel d2-specific antibody, high-intensity d2 staining was observed only in intercalated cells of the collecting duct in fresh-frozen human kidney, where it co-localized with the a4 subunit in the characteristic plasma membrane-enhanced pattern. In human bone, d2 co-localized with the a3 subunit in osteoclasts. This different subunit association in different tissues emphasizes the possibility of the H+-ATPase as a future therapeutic target.

Revised Nomenclature for Mammalian Vacuolar-Type H+-ATPase Subunit Genes

To date, the nomenclature of mammalian genes encoding the numerous subunits and their many isoforms that comprise the family of vacuolar H(+)-ATPases has not been systematic, resulting in confusion both in the literature and among investigators. We present the official new system for these genes, approved by both Human and Mouse Gene Nomenclature Committees.