Polymorphisms in the gene encoding sterile 20/SPS1-related proline/alanine-rich kinase (SPAK) associate with hypertension susceptibility in humans. SPAK interacts with WNK kinases to regulate the Na ϩ -K ϩ -2Cl Ϫ and NaMutations in WNK1/4 and N(K)CC can cause changes in BP and dyskalemia in humans, but the physiologic role of SPAK in vivo is unknown. We generated and analyzed SPAK-null mice by targeting disruption of exons 9 and 10 of SPAK. Compared with SPAK ϩ/ϩ littermates, SPAK ϩ/Ϫ mice exhibited hypotension without significant electrolyte abnormalities, and SPAK Ϫ/Ϫ mice not only exhibited hypotension but also recapitulated Gitelman syndrome with hypokalemia, hypomagnesemia, and hypocalciuria. In the kidney tissues of SPAK Ϫ/Ϫ mice, the expression of total and phosphorylated (p-)NCC was markedly decreased, but that of p-OSR1, total NKCC2, and p-NKCC2 was significantly increased. We observed a blunted response to thiazide but normal response to furosemide in SPAK Ϫ/Ϫ mice. In aortic tissues, total NKCC1 expression was increased but p-NKCC1 was decreased in SPAK-deficient mice. Both SPAK ϩ/Ϫ and SPAK Ϫ/Ϫ mice had impaired responses to the selective ␣ 1 -adrenergic agonist phenylephrine and the NKCC1 inhibitor bumetanide, suggesting that impaired aortic contractility may contribute to the hypotension of SPAKnull mice. In summary, SPAK-null mice have defects of NCC in the kidneys and NKCC1 in the blood vessels, leading to hypotension through renal salt wasting and vasodilation. SPAK may be a promising target for antihypertensive therapy. 21: 186821: -187721: , 201021: . doi: 10.1681 Sterile 20/SPS1-related proline/alanine-rich kinase (SPAK) 1,2 and oxidative stress-responsive kinase 1 (OSR1) 3 are serine/threonine kinases that share high homology in both their N-terminal catalytic and Cterminal regulatory domains and are widely distributed in the brain, pancreas, heart and kidney. Gene mutations of the NCC in the distal convoluted tubules (DCTs) and NKCC2 in the thick ascending limb of the loop of Henle (TAL) cause autosomal recessive Gitelman syndrome (GS) 12 and Bartter syndrome (BS), 13 respectively. These congenital renal tubular disorders are characterized by J Am Soc Nephrol

We have identified a novel homozygous nonsense mutation (W516X) in the kidney-type electrogenic sodium bicarbonate cotransporter 1 (NBC1) in a patient with isolated proximal renal tubular acidosis (pRTA). To specifically address the pathogenesis of this mutation, we created NBC1 W516X knock-in mice to match the patient's abnormalities. The expression of NBC1 mRNA and protein in the kidneys of NBC1(W516X/W516X) mice were virtually absent, indicating that nonsense-mediated mRNA decay (NMD) is involved in the defective transcription and translation of this mutation. These mice not only recapitulated the phenotypes of this patient with growth retardation, pRTA, and ocular abnormalities, but also showed anemia, volume depletion, prerenal azotemia, and several organ abnormalities, culminating in dehydration and renal failure with early lethality before weaning. In isolated renal proximal tubules, both NBC1 activity and the rate of bicarbonate absorption were markedly reduced. Unexpectedly, there was no compensatory increase in mRNA of distal acid/base transporters. Sodium bicarbonate but not saline administration to these mutant mice markedly prolonged their survival, decreased their protein catabolism and attenuated organ abnormalities. The prolonged survival time uncovered the development of corneal opacities due to corneal edema. Thus, NBC1(W516X/W516X) mice with pRTA represent an animal model for metabolic acidosis and may be useful for testing therapeutic inhibition of NMD in vivo.

Gitelman syndrome (GS) is characterized by salt-losing hypotension, hypomagnesemia, hypokalemic metabolic alkalosis, and hypocalciuria. To better model human GS caused by a specific mutation in the thiazide-sensitive Na(+) -Cl(-) cotransporter (NCC) gene SLC12A3, we generated a nonsense Ncc Ser707X knockin mouse corresponding to human p.Ser710X (c.2135C>A), a recurrent mutation with severe phenotypes in Chinese GS patients. Compared with wild-type or heterozygous littermates, homozygous (Hom) knockin mice fully recapitulated the phenotype of human GS. The markedly reduced Ncc mRNA and virtually absent Ncc protein expression in kidneys of Hom mice was primarily due to nonsense-mediated mRNA decay (NMD) surveillance mechanisms. Expression of epithelial Na(+) channel (Enac), Ca(2+) channels (Trpv5 and Trpv6), and K(+) channels (Romk1 and maxi-K) were significantly increased. Late distal convoluted tubules (DCT) volume was increased and DCT cell ultrastructure appeared intact. High K(+) intake could not correct hypokalemia but caused a further increase in maxi-K but not Romk1 expression. Renal tissue from a patient with GS also showed the enhanced TRPV5 and ROMK1 expression in distal tubules. We suggest that the upregulation of TRPV5/6 and of ROMK1 and Maxi-K may contribute to hypocalciuria and hypokalemia in Ncc Ser707X knockin mice and human GS, respectively.

SummaryBackground and objectives Gitelman's syndrome (GS) is an autosomal recessive renal tubular disorder caused by mutations in the SLC12A3 gene encoding the thiazide-sensitive Na ϩ -Cl Ϫ cotransporter (NCC). Despite meticulous sequencing of genomic DNA, approximately one-third of GS patients are negative or heterozygotes for the known mutations.Design, Setting, Participants, & Measurements Because blood leukocytes express NCC mRNA, we evaluate whether deep intronic mutations contribute to GS patients with uniallelic or undetectable SLC12A3 mutations. Twenty-nine patients with GS (men/women ϭ 16/13), including eight negative and 21 uniallelic SLC12A3 mutations from 19 unrelated families, and normal controls were enrolled in an academic medical center. Analysis of cDNA from blood leukocytes, sequencing of the corresponding introns of genomic DNA for abnormal transcript, and analysis of NCC protein expression from renal biopsy were performed. Results We identified nine Taiwan aboriginal patients carrying c.1670 -191C3 T mutations in intron 13and 10 nonaboriginal patients carrying c.2548ϩ253C3 T mutations in intron 21 from 14 families (14/ 19). These two mutations undetected in 100 healthy subjects created pseudoexons containing new premature termination codons. Haplotype analysis with markers flanking SLC12A3 revealed that both mutations did not have founder effects. Apical NCC expression in the DCT of renal tissue was markedly diminished in two patients carrying deep intronic mutations.Conclusions Deep intronic mutations in SLC12A3 causing defective NCC expression can be identified with the RNA-based approach in patients with GS. c.1670 -191C3 T and c.2548ϩ253C3 T are hot spot mutations that can be screened in GS patients with uniallelic or negative SLC12A3 mutations.

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We describe a 17-year-old Chinese boy who presented with progressive muscle weakness and renal failure. He was diagnosed as BS of unknown type at the age of 9 months and treated with indomethacin (2 mg/kg/day) and potassium chloride (KCl) supplementation (1.5 mEq/kg/day) for hypokalemia (2.5 mmol/l). At the age of 12 years, serum K+ was 3.0 mmol/l and creatinine reached 2.0 mg/dl. On admission, his blood pressure was normal but volume status was depleted. Urinalysis was essentially normal. Biochemical studies showed hypokalemia (K+ 2.4 mmol/l) with a high transtubular K+ gradient (TTKG) 9.6, metabolic alkalosis (HCO3- 28.4 mmol/l), normomagnesemia (2.0 mg/dl), severe renal failure (BUN 94 mg/dl, Cr 6.3 mg/dl), and hypocalciuria (urine calcium/creatinine ratio 0.02 mg/mg). Abdominal sonography revealed bilateral small size kidneys without nephrocalcinosis or renal stones. After the withdrawal of indomethacin with regular KCl and adequate fluid supplementation for 1 year, serum creatinine and K+ levels have been maintained at 4.0 mg/dl and 3.3 mmol/l, respectively. Direct sequencing of NKCC2, ROMK, ClC-Kb, and NCCT in this patient disclosed a novel homozygous missense mutation (GGG to GAG, G470E) in CLCNKB. This G470E mutation was not identified in 100 healthy Chinese subjects. Long-term therapy of non-steroidal anti-inflammatory drugs (NSAIDs), prolonged hypokalemia, chronic volume depletion, and underlying genetic variety may contribute to the deterioration of his renal function. The cautious use of NSAIDs, aggressive correction of hypokalemia, and avoidance of severe volume depletion may prevent the irreversible renal damage in patients with BS due to a Cl- channel defect.

The prevailing view is that ClC-Ka chloride channel (mouse Clc-k1) functions in thin ascending limb for urine concentration, whereas ClC-Kb (mouse Clc-k2) in thick ascending limb (TAL) for salt reabsorption, respectively. Mutations of ClC-Kb cause classic Bartter syndrome with renal salt wasting with onset from perinatal to adolescent.We study the roles of Clc-k channels in perinatal mouse kidneys using constitutive or inducible kidney-specific gene ablation and 2-D and advanced 3-D imaging of optically cleared kidneys. We show that Clc-k1 and -k2 are broadly expressed and colocalized in perinatal kidneys. Deletion of Clc-k1 and -k2 reveals that both participate in NKCC2-and NCC-mediated NaCl reabsorption in neonatal kidneys. Embryonic deletion of Clc-k2 causes tubular injury and impairs renal medulla and TAL development. Inducible deletion of Clc-k2 begins after medulla maturation produces mild salt wasting resulting from reduced NCC activity. Thus, both Clc-k1 and -k2 contribute to salt reabsorption in TAL and DCT in neonates, potentially explaining less severe phenotypes in classic Bartter. As opposed to the current understanding that salt wasting in adult Bartter patients is due to Clc-k2 deficiency in adult TAL, our results suggest that it is mainly originated from medulla and TAL defects during development. Table 2. Plasma and urine biochemistries of 8-week-old Clc-k1-null and Clc-k2-null mice WT Clc-k1 -/-WT Clc-k2 -/-Plasma BUN (mg/dL) 29.7±1.6 26.6±2.0 30.4±1.7 63.6±3.0** Creatinine (mg/dL) 0.25±0.03 0.29±0.03 0.26±0.01 0.39±0.03** Table 3 Plasma and urine biochemistries in 10-week-old inducible Clc-k2 deficient mice.

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