A comprehensive analysis of gene expression profiles in distal parts of the mouse renal tubule

The distal parts of the renal tubule play a critical role in maintaining homeostasis of extracellular fluids. In this review, we present an in-depth analysis of microarray-based gene expression profiles available for microdissected mouse distal nephron segments, i.e., the distal convoluted tubule (DCT) and the connecting tubule (CNT), and for the cortical portion of the collecting duct (CCD; Zuber et al., Proc Natl Acad Sci USA 106:16523-16528, 2009). Classification of expressed transcripts in 14 major functional gene categories demonstrated that all principal proteins involved in maintaining the salt and water balance are represented by highly abundant transcripts. However, a significant number of transcripts belonging, for instance, to categories of G-protein-coupled receptors or serine/threonine kinases exhibit high expression levels but remain unassigned to a specific renal function. We also established a list of genes differentially expressed between the DCT/CNT and the CCD. This list is enriched by genes related to segment-specific transport functions and by transcription factors directing the development of the distal nephron or collecting ducts. Collectively, this in silico analysis provides comprehensive information about relative abundance and tissue specificity of the DCT/CNT and the CCD expressed transcripts and identifies new candidate genes for renal homeostasi

Renal and Brain Isoforms of WNK3 Have Opposite Effects on NCCT Expression

Mutations in the WNK kinases WNK1 and WNK4 cause a rare familial form of hypertension (Gordon syndrome) by increasing expression of the thiazide-sensitive co-transporter NCCT in the kidney. Regulation of NCCT expression involves a scaffold of proteins composed of several kinases, including the third member of the WNK kinase family, WNK3. This protein, expressed in several tissues including kidney and brain, displays splice variation around exons 18 and 22. We expressed these proteins in Xenopus oocytes and found that the renal isoform of WNK3 increased but the brain isoform decreased NCCT expression and activity. Introduction of a kinase-inactivating mutation into renal WNK3 reversed its action on NCCT, and the same mutation in the brain isoforms led to loss of function. We also studied the effect of phosphorylation of a key NCCT threonine (T58) on the effects of WNK3/4 coexpression; NCCT mutants with a T58A or T58D substitution had the same surface expression as T58 but had significantly altered transporter activity; however, both isoforms of WNK3 as well as WNK4 still modulated expression of these NCCT mutants. Finally, experiments using kinase-dead STE20/SPS1-related proline/alanine-rich kinase (SPAK), a putative downstream target for WNKs, revealed that brain WNK3 acts in tandem with SPAK, whereas renal WNK3 seems to upregulate NCCT through a SPAK-independent pathway. Taken together, these results suggest that the C-terminal motifs contributed by exons 18 and 22 play an important role in the actions of WNK3 isoforms on NCCT

Role of the WNK-activated SPAK kinase in regulating blood pressure

Mutations within the with-no-K(Lys) (WNK) kinases cause Gordon's syndrome characterized by hypertension and hyperkalaemia. WNK kinases phosphorylate and activate the STE20/SPS1-related proline/alanine-rich kinase (SPAK) protein kinase, which phosphorylates and stimulates the key Na(+):Cl(−) cotransporter (NCC) and Na(+):K(+):2Cl(−) cotransporters (NKCC2) cotransporters that control salt reabsorption in the kidney. To define the importance of this pathway in regulating blood pressure, we generated knock-in mice in which SPAK cannot be activated by WNKs. The SPAK knock-in animals are viable, but display significantly reduced blood pressure that was salt-dependent. These animals also have markedly reduced phosphorylation of NCC and NKCC2 cotransporters at the residues phosphorylated by SPAK. This was also accompanied by a reduction in the expression of NCC and NKCC2 protein without changes in messenger RNA (mRNA) levels. On a normal Na(+)-diet, the SPAK knock-in mice were normokalaemic, but developed mild hypokalaemia when the renin–angiotensin system was activated by a low Na(+)-diet. These observations establish that SPAK plays an important role in controlling blood pressure in mammals. Our results imply that SPAK inhibitors would be effective at reducing blood pressure by lowering phosphorylation as well as expression of NCC and NKCC2. See accompanying Closeup by Maria Castañeda-Bueno and Gerald Gamba (DOI 10.1002/emmm.200900059)

αENaC-Mediated Lithium Absorption Promotes Nephrogenic Diabetes Insipidus

Lithium-induced nephrogenic diabetes insipidus (NDI) is accompanied by polyuria, downregulation of aquaporin 2 (AQP2), and cellular remodeling of the collecting duct (CD). The amiloride-sensitive epithelial sodium channel (ENaC) is a likely candidate for lithium entry. Here, we subjected transgenic mice lacking αENaC specifically in the CD (knockout [KO] mice) and littermate controls to chronic lithium treatment. In contrast to control mice, KO mice did not markedly increase their water intake. Furthermore, KO mice did not demonstrate the polyuria and reduction in urine osmolality induced by lithium treatment in the control mice. Lithium treatment reduced AQP2 protein levels in the cortex/outer medulla and inner medulla (IM) of control mice but only partially reduced AQP2 levels in the IM of KO mice. Furthermore, lithium induced expression of H+-ATPase in the IM of control mice but not KO mice. In conclusion, the absence of functional ENaC in the CD protects mice from lithium-induced NDI. These data support the hypothesis that ENaC-mediated lithium entry into the CD principal cells contributes to the pathogenesis of lithium-induced NDI

Sodium and potassium balance depends on αENaC expression in connecting tubule.

Mutations in α, β, or γ subunits of the epithelial sodium channel (ENaC) can downregulate ENaC activity and cause a severe salt-losing syndrome with hyperkalemia and metabolic acidosis, designated pseudohypoaldosteronism type 1 in humans. In contrast, mice with selective inactivation of αENaC in the collecting duct (CD) maintain sodium and potassium balance, suggesting that the late distal convoluted tubule (DCT2) and/or the connecting tubule (CNT) participates in sodium homeostasis. To investigate the relative importance of ENaC-mediated sodium absorption in the CNT, we used Cre-lox technology to generate mice lacking αENaC in the aquaporin 2-expressing CNT and CD. Western blot analysis of microdissected cortical CD (CCD) and CNT revealed absence of αENaC in the CCD and weak αENaC expression in the CNT. These mice exhibited a significantly higher urinary sodium excretion, a lower urine osmolality, and an increased urine volume compared with control mice. Furthermore, serum sodium was lower and potassium levels were higher in the genetically modified mice. With dietary sodium restriction, these mice experienced significant weight loss, increased urinary sodium excretion, and hyperkalemia. Plasma aldosterone levels were significantly elevated under both standard and sodium-restricted diets. In summary, αENaC expression within the CNT/CD is crucial for sodium and potassium homeostasis and causes signs and symptoms of pseudohypoaldosteronism type 1 if missing

Meta-analysis of genome-wide association studies identifies six new Loci for serum calcium concentrations

Calcium is vital to the normal functioning of multiple organ systems and its serum concentration is tightly regulated. Apart from CASR, the genes associated with serum calcium are largely unknown. We conducted a genome-wide association meta-analysis of 39,400 individuals from 17 population-based cohorts and investigated the 14 most strongly associated loci in ≤ 21,679 additional individuals. Seven loci (six new regions) in association with serum calcium were identified and replicated. Rs1570669 near CYP24A1 (P = 9.1E-12), rs10491003 upstream of GATA3 (P = 4.8E-09) and rs7481584 in CARS (P = 1.2E-10) implicate regions involved in Mendelian calcemic disorders: Rs1550532 in DGKD (P = 8.2E-11), also associated with bone density, and rs7336933 near DGKH/KIAA0564 (P = 9.1E-10) are near genes that encode distinct isoforms of diacylglycerol kinase. Rs780094 is in GCKR. We characterized the expression of these genes in gut, kidney, and bone, and demonstrate modulation of gene expression in bone in response to dietary calcium in mice. Our results shed new light on the genetics of calcium homeostasis