T he role of WNK1 and WNK4 in control of electrolyte balance and BP first became apparent with their mutation being associated with familial hyperkalemic hypertension (FHHt; also known as Gordon syndrome and pseudohypoaldosteronism type 2), a human autosomal dominant disorder that features hypertension, hyperkalemia, and acidosis that usually are hyperresponsive to thiazide diuretics (1,2). FHHt can be caused by intronic deletions in WNK1 or missense mutations in WNK4 (3). Mutations in either cause a broadly similar phenotype, suggesting that WNK1 and WNK4 function in a common pathway. Unlike most monogenic disorders that affect BP, which feature reciprocal Na ϩ and K ϩ (and/or H ϩ ) imbalances and share a relationship to the aldosterone pathway (4), FHHt features concurrent NaCl and K ϩ (and/or H ϩ ) retention (1,3,5). This unusual characteristic indicates the existence of a novel "WNK pathway" functioning in normal physiology, which may allow the "independent of aldosterone" regulation of K and Na balance (and extracellular volume) by the kidney, ultimately also maintaining BP within the normal range. The BP-regulatory role of this WNK pathway is conserved in evolution as WNK1ϩ/Ϫ mice are hypotensive (6).Previously, we and others demonstrated that a 5Ј-truncated kinase-deficient isoform (WNK1-S) predominates in kidney (7), this being conserved between human and mouse (7-9). Isoform-specific probes distinguished ubiquitous low-level expression of full-length WNK1-long (WNK1-L) from abundant WNK1-S expression in distal nephron. Recent Xenopus oocyte studies implicate WNK4 in inhibition of NaCl reabsorption by thiazide-sensitive Na ϩ Cl Ϫ co-transporter (NCC) (10,11) and/or Materials and Methods Animal TreatmentAll procedures were carried out under provisions of ethically approved licenses and involved adult, 25-to 30-g, male C57BL/6 mice (Charles River, Margate, UK). Modified electrolyte feeds for mice were obtained from Special Diet Services (Witham, UK). RNA ExtractionAt conclusion of treatments of mice, both kidneys were removed under terminal anesthetic, immediately frozen on dry ice, and stored at Ϫ80°C. Frozen kidneys were fragmented and immediately homogenized in TRIzol Reagent (Invitrogen, Paisley, UK), and total RNA was extracted following the manufacturer's guidelines. Real-Time PCRAssays used ABI PRISM 7900 relative quantification real-time methods (Applied Biosystems, Foster City, CA). PCR was performed in 384-well plates (AB Gene) and used 10-l reactions that contained 5.0 l of TaqMan Master Mix (Applied Biosystems), 200 nM of each primer, 5 nM of probe, and 4.5 l of template (1:40 dilution of cDNA synthesized as described previously [7]). PCR conditions involved 95°C for 10 min, then 40 cycles of 95°C for 15 s and 60°C for 60 s. Standard template dilution curves enabled target gene quantification and normalization to the endogenous control TATA-Box Binding Protein (TBP). All group values were calibrated to their control groups.Validation studies using mouse renal RNA established TBP as an excelle...

Naringin, a major flavonoid in citrus, has potential for preventing atherosclerosis. The presence in the colon of a large amount of naringin after oral intake might affect the gut microbiota. We investigated the role of gut microbiota remodeling in the alleviation of atherosclerosis by naringin. Naringin significantly alleviated atherosclerosis and lowered the serum and liver cholesterol levels by 24.04 and 28.37% in ApoE −/− mice fed with a high-fat diet. Nontarget metabolomics showed that naringin modulated the hepatic levels of cholesterol derivatives and bile acids. Naringin increased the excretion of bile acids and neutral sterols by 1.6-and 4.3-fold, respectively. The main potential pathway by which naringin alleviated atherosclerosis was the gut microbiota−liver− cholesterol axis. Naringin modulated the abundances of bile salt hydrolase-and 7α-dehydroxylase-producing bacteria, promoting bile acid synthesis from cholesterol by upregulating CYP7A1 via suppression of the FXR/FGF15 pathway. In addition, naringin facilitated reverse cholesterol transport by downregulating PCSK9/IDOL. The results provide insight into the atherosclerosisalleviation mechanisms of citrus flavonoids and a scientific basis for the development of functional foods containing citrus flavonoids.

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The results showed that Cd significantly induced MMP collapse and typical mitophagosomes formation, increased LC3-II/LC3-I ratio and PINK1 level, and decreased mitochondrial mass, revealing that Cd could induce mitophagy. However, NAC or ALC pretreatment markedly decreased Cd-induced ROS and simultaneously rescued MMP and mitochondrial mass, suggesting ROS played a crucial role in regulating mitophagy. NAC or ALC also dramatically lessened PINK1 level and mitochondrial accumulation of Parkin, indicating that ROS were related to PINK1/Parkin pathway. Notably, CsA compromised Cd-induced mitophagy, PINK1 accumulation and Parkin translocation while failed to block ROS increase, suggesting ROS functioned as an upstream signal for PINK1/Parkin pathway. Taken together, the results indicated that Cd induced ROS-mediated mitophagy through PINK1/Parkin pathway in kidneys of mice. The present study proposes a new perspective to evaluate the nephrotoxicity and its molecular mechanism under Cd exposure in vivo.