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 propose a two-step approach to functionalize gold nanoparticles prepared by reducing tetrachloroauric acid by trisodium citrate in water. The chloride and citrate physisorbed on the gold nanoparticles are first displaced by thioctic acid (TA), which is then exchanged by thiols containing the desired functionality during the second step. TA bears a carboxylate group and disulfide; at high pH, the negative charge of the former stabilizes gold nanoparticles and the disulfide develops two S−Au bonds, retarding the desorption kinetics upon further functionalization. The slow kinetics of TA desorption is crucial to establishing sufficient steric stabilization for the gold core while losing electrostatic stabilization. Successful attachment of carboxylate, crown ether, cyclodextrin, pyridine, and amino functionalities is verified by surface sensitive techniques. In most cases, ca. 80% of the TA molecules are displaced. The nanoparticles are stable under solution pH where the surface group is ionized. Although at certain solution acidities, the gold sols have difficulty carrying charges and appear flocculated, the nanoparticles become dispersive after being centrifuged and redissolved in suitable buffer solutions, which indicates the successful protection of the gold cores by steric stabilization.

Summary MicroRNA (miRNA)-dependent regulation of gene expression confers robustness to cellular phenotypes and controls responses to extracellular stimuli. Although a single miRNA can regulate expression of hundreds of target genes, it is unclear whether any of its distinct biological functions can be due to the regulation of a single target. To explore in vivo the function of a single miRNA-mRNA interaction, we mutated the 3′ UTR of a major miR-155 target SOCS1 to specifically disrupt its regulation by miR-155. We found that under physiologic conditions and during autoimmune inflammation or viral infection some immunological functions of miR-155 were fully or largely attributable to the regulation of SOCS1, whereas others could be accounted only partially or not at all by this interaction. Our data suggest that the role of a single miRNA-mRNA interaction is cell type- and biological context-dependent.

Na + -K + -2Cl − cotransporters (NKCCs), including NKCC1 and renal-specific NKCC2, and the Na + -Cl − cotransporter (NCC) play pivotal roles in the regulation of blood pressure (BP) and renal NaCl reabsorption. Oxidative stress-responsive kinase-1 (OSR1) is a known upstream regulator of N(K)CCs. We generated and analyzed global and kidney tubule-specific (KSP) OSR1 KO mice to elucidate the physiological role of OSR1 in vivo, particularly on BP and kidney function. Although global OSR1 −/− mice were embryonically lethal, OSR1 +/− mice had low BP associated with reduced phosphorylated (p) STE20 (sterile 20)/SPS1-related proline/alanine-rich kinase (SPAK) and p-NKCC1 abundance in aortic tissue and attenuated p-NKCC2 abundance with increased total and p-NCC expression in the kidney. KSP-OSR1 −/− mice had normal BP and hypercalciuria and maintained significant hypokalemia on a low-K + diet. KSP-OSR1 −/− mice exhibited impaired Na + reabsorption in the thick ascending loop on a low-Na + diet accompanied by remarkably decreased expression of p-NKCC2 and a blunted response to furosemide, an NKCC2 inhibitor. The expression of total SPAK and p-SPAK was significantly increased in parallel to that of total NCC and p-NCC despite unchanged total NKCC2 expression. These results suggest that, globally, OSR1 is involved in the regulation of BP and renal tubular Na + reabsorption mainly via the activation of NKCC1 and NKCC2. In the kidneys, NKCC2 but not NCC is the main target of OSR1 and the reduced p-NKCC2 in KSP-OSR1 −/− mice may lead to a Bartter-like syndrome.Bartter syndrome | electrolytes | hormone | knockout mice | volume R ecent studies have shown that Na + -K + -2Cl − cotransporters (NKCCs) and the Na + -Cl − cotransporter (NCC) play very important roles in the regulation of blood pressure (BP) and extracellular volume. NKCCs consist of ubiquitous NKCC1 and renal-specific NKCC2. NKCC1 can modulate BP through vascular and renal effects (1-4). NKCC2 and NCC are two renal Na + cotransporters expressed in the thick ascending limbs (TALs) and distal convoluted tubules (DCTs) of the kidney, respectively, accounting for 20% and 10% of filtered Na + reabsorption (5). In human essential hypertension and salt-sensitive or spontaneously hypertensive animal models, activation of NKCC1 and NKCC2 has also been reported to play a pivotal role in the pathogenesis of hypertension (6, 7). In addition, activation of NCC by gene mutations in WNK1 and WNK4 leads to an autosomal dominant salt-sensitive hypertension known as pseudohypoaldosteronism type II (PAHII) (8). On the other hand, loss-of-function mutations in the SLC12A1 and SLC12A3 genes encoding NKCC2 and NCC can lead to renal salt-wasting hypotension with hypokalemic metabolic alkalosis, known as Bartter syndrome (BS) (9) and Gitelman syndrome (GS) (10), respectively.In vitro studies have shown that posttranscriptional phosphorylation of NKCC1/2 and NCC plays a crucial role in the regulation of normal transport activity. Oxidative stress-responsive kinase-1 (OSR1) (11) and STE20 (s...