Abstract. WNK1 is a member of a novel serine/threonine kinase family, With-No-K, (lysine). Intronic deletions in the encoding gene cause Gordon syndrome, an autosomal dominant, hypertensive, hyperkalemic disorder particularly responsive to thiazide diuretics, a first-line treatment in essential hypertension. To elucidate the novel WNK1 BP control pathway active in distal nephron, WNK1 expression in mouse was studied. It was found that WNK1 is highly expressed in testis Ͼ heart, lung, kidney, placenta Ͼ skeletal muscle, brain, and widely at low levels. Several WNK1 transcript classes are demonstrated, showing tissue-, developmental-, and nephron-segment-specific expression. Importantly, in kidney, the most prominent transcripts are smaller than elsewhere, having the first four exons replaced by an alternative 5'-exon, deleting the kinase domain, and showing strong distal nephron expression, whereas larger transcripts show low-level widespread distribution. Alternative splicing of exons 11 and 12 is prominent-for example, transcripts containing exon 11 are abundant in neural tissues, testis, and secondary renal transcripts but are predominantly absent in placenta. The transcriptional diversity generated by these events would produce proteins greatly differing in both structure and function. These findings help further define and clarify the role of WNK1 and the thiazideresponsive pathway relevant to essential hypertension in which it participates.

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...

To test growth effects of angiotensin II (ANG II) in porcine vascular smooth muscle cells (VSMC) and potential ANG II synergy with epidermal growth factor (EGF), we exposed subconfluent, near-quiescent porcine aortic VSMC to ANG II, EGF, or ANG II + EGF (each 10(-9) M) in Dulbecco's modified Eagle's-Ham's F-12 medium with insulin + 0.4% fetal calf serum (FCS) selected for minimal ANG II-degrading capacity. Cell number and DNA and protein synthesis (by [3H]-thymidine and [35S]methionine incorporation, respectively) were determined serially over 1-6 days. ANG II alone induced an early 20% increase and then a plateau in cell number over the 0.4% FCS control (P < 0.01; n = 8), thus without sustained increase in proliferation rate. Yet ANG II alone did not increase fractional DNA or protein synthesis (each as cpm/10(3) cells) and, by flow cytometry, reduced S phase fraction without increase in cell size. EGF alone induced brisk DNA synthesis yet minimal cell division over days 0-4, thus late-cycle arrest. ANG II + EGF, despite no increase in fractional DNA or protein synthesis rates over EGF alone, induced significant indomethacin-resistant dose-dependent (P < 0.001) increase in cell proliferation rate over EGF alone with a median effective dose of 5 x 10(-10) M ANG II, thus proliferative synergy. We propose that 1) ANG II induces a subpopulation of cells arrested in or beyond S phase to proceed through mitosis but does not influence G1 traversal or S phase entry and 2) ANG II + EGF achieve proliferative synergy by complementary actions at sequential cell cycle loci, with EGF supporting progression from G0/G1 to S phase and ANG II inducing completion of mitosis by cells already in or beyond S phase ("late-cycle completion").

To characterize growth effects of epidermal growth factor (EGF) in subconfluent quiescent porcine aortic vascular smooth muscle cells (VSMC), we measured DNA and protein synthesis by [3H]thymidine (Thd) and [35S]methionine (Met) incorporation, respectively, and cell proliferation rates over 0-6 days in Dulbecco's modified Eagle's-Ham's F-12 media containing 0.4% fetal calf serum (FCS) and insulin. EGF induced dose-dependent [3H]Thd uptake (P less than 0.001); after 10(-9) M EGF, DNA synthesis rate peaked at 24 h, averaging 77% of the response to 10% FCS, and then declined steeply with nadir at 48-60 h. Unexpectedly, EGF failed to induce cell proliferation in the first 4 days, leaving this initial burst of DNA synthesis (12-60 h) uncoupled from cell division. A second lesser but sustained phase of increased DNA synthesis, apparent by day 3-4, was associated with a small increase in cell number on day 6 (P less than 0.05). The early unsustained burst of DNA synthesis reflects EGF's potent mitogenic efficacy for DNA synthesis (G1- to S-phase traversal), probably acting on a subset of cells partially synchronized initially at an EGF-responsive G0/G1 locus; the minimal cell division despite brisk DNA synthesis documents EGF's limited efficacy for (or inhibition of) late cell-cycle events required for completion of mitosis. Late cell-cycle processes are thus rate limiting. EGF also increased protein synthetic rate over control (P less than 0.03) but to a lesser degree (P less than 0.01) than 10% FCS. Indomethacin (10(-6) M) did not alter DNA or proliferative responses to 10(-9) M EGF but transiently augmented EGF-induced protein synthesis (P less than 0.025) at 24 h only.(ABSTRACT TRUNCATED AT 250 WORDS)