WNK1 and WNK4 modulate CFTR activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated chloride channel. WNK kinases are widely expressed modulators of ion transport. WNK1 and WNK4, two WNK kinases that are mutated in familial hyperkalemic hypertension (FHHt), are co-expressed with CFTR in several organs, raising the possibility that WNK kinases might alter CFTR activity in vivo or that CFTR could be involved in the pathogenesis of FHHt. Here, we report that WNK1 co-localizes with CFTR protein in pulmonary epithelial cells. Co-expression of WNK1 or WNK4 with CFTR in Xenopus laevis oocytes suppresses chloride channel activity. The effect of WNK4 is dose dependent and occurs, at least in part, by reducing CFTR protein abundance at the plasma membrane. This effect is independent of WNK4 kinase activity. In contrast, the effect of WNK1 on CFTR activity requires intact WNK1 kinase activity. Moreover WNK1 and WNK4 exhibit additive CFTR inhibition. Previous reports suggest that patients with FHHt exhibit mild changes in nasal potential difference that resemble the more severe changes that occur in cystic fibrosis. We report that the FHHt-causing mutant WNK4 Q562E is a more potent inhibitor of CFTR activity than is the wild-type WNK4. Taken together, these results suggest that WNK1 and WNK4 may modulate CFTR activity; they further suggest that WNK kinases may be potential therapeutic targets for cystic fibrosis.

Key enzymes for renal prostaglandin synthesis: site-specific expression in rodent kidney (rat, mouse).

Prostanoids derived from endogenous cylooxygenase (COX)-mediated arachidonic acid metabolism play important roles in the maintenance of renal blood flow and salt and water homeostasis. The relative importance of COX-1 and COX-2 isoforms is under active investigation. We have performed a comprehensive histochemical analysis by comparing rat and mouse kidneys for cellular and subcellular localization of COX-1 and -2 and microsomal-type PGE synthase (PGES), the rate-limiting biosynthetic enzyme in PGE2 synthesis. A choice of different sera was compared, and the results were confirmed by antigen-retrieval techniques, in situ hybridization, RT-PCR, and the use of COX knockout mice. In the glomerulus, significant COX-1 expression was detected in a subset of mesangial cells. Along the renal tubule, the known COX-2 expression in cTAL and macula densa was paralleled by PGES staining. In the terminal distal convoluted tubule, connecting tubule, and cortical and medullary collecting ducts, a significant COX-1 signal was colocalized with PGES; COX-2 was not found in these sites. Intercalated cells were generally negative. Cortical fibroblasts were COX-1 and PGES positive in mice, whereas in rats only PGES could be reliably detected. Lipid-laden interstitial cells of the inner medulla were COX-1, -2, and PGES positive. Vascular smooth muscle cells were not stained. The present data support prominent functions of renal prostanoids, predominantly PGE2, by defining expression sites of the key enzymes for their biosynthesis in the rat and mouse. Results define the renal cell types involved in prostaglandin autacoid functions within spatially restricted sites such as the juxtaglomerular apparatus, mesangium, distal convolutions and collecting duct, and in compartments of the renal interstitium.

Inhibition of nNOS expression in the macula densa by COX-2-derived prostaglandin E(2).

It is well established that cyclooxygenase-2 (COX-2) and the neuronal form of nitric oxide synthase (nNOS) are coexpressed in macula densa cells and that the expression of both enzymes is stimulated in a number of high-renin states. To further explore the role of nNOS and COX-2 in renin secretion, we determined plasma renin activity in mice deficient in nNOS or COX-2. Plasma renin activity was significantly reduced in nNOS -/- mice on a mixed genetic background and in COX-2 -/- mice on either BALB/c or C57/BL6 congenic backgrounds. In additional studies, we accumulated evidence to show an inhibitory influence of PGE(2) on nNOS expression. In a cultured macula densa cell line, PGE(2) significantly reduced nNOS mRNA expression, as quantified by real-time RT-PCR. In COX-2 -/- mice, nNOS mRNA expression in the kidney, determined by real-time RT-PCR, was upregulated throughout the postnatal periods, ranging from postnatal day (PND) 3 to PND 60. The induction of nNOS protein expression and NOS activity in COX-2 -/- mice was localized to macula densa cells using immunohistochemistry and NADPH-diaphorase staining methods, respectively. Therefore, these findings reveal that the absence of either COX-2 or nNOS is associated with suppressed renin secretion. Furthermore, the inhibitory effect of PGE(2) on nNOS mRNA expression indicates a novel interaction between NO and prostaglandin-mediated pathways of renin regulation.