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The discovery of new genetic mutations that cause hypertension has illuminated previously unrecognized physiological pathways. One such regulatory pathway was identified when mutations in with no lysine kinase (WNK)4, Kelch-like 3 ( KLHL3), and cullin 3 ( CUL3) were shown to cause the disease familial hyperkalemic hypertension (FHHt). Mutations in all three genes upregulate the NaCl cotransporter (NCC) due to an impaired ability to degrade WNK protein through the cullin-RING-ligase (CRL) ubiquitin-proteasome system. The CUL3 FHHt mutations cause the most severe phenotype, yet the precise mechanism by which these mutations cause the disease has not been established and current proposed models are controversial. New data have identified a possible novel mechanism involving dysregulation of CUL3 activity by the COP9 signalosome (CSN). The CSN interaction with mutant CUL3 is diminished, causing hyperneddylation of the CRL. Recent work has shown that direct renal CSN impairment mimics some aspects of the CUL3 mutation, including lower KLHL3 abundance and activation of the WNK-NCC pathway. Furthermore, in vitro and in vivo studies of CSN inhibition have shown selective degradation of CRL substrate adaptors via auto-ubiquitination, allowing substrate accumulation. In this review, we will focus on recent research that highlights the role of the CSN role in CUL3 mutations that cause FHHt. We will also highlight how these results inform other recent studies of CSN dysfunction.

The cortical collecting duct (CCD) forms part of the aldosterone-sensitive distal nephron and plays an essential role in maintaining the NaCl balance and acid-base status. The CCD epithelium comprises principal cells as well as different types of intercalated cells. Until recently, transcellular Na+transport was thought to be restricted to principal cells, whereas (acid-secreting) type A and (bicarbonate-secreting) type B intercalated cells were associated with the regulation of acid-base homeostasis. This review describes how this traditional view has been upended by several discoveries in the past decade. A series of studies has shown that type B intercalated cells can mediate electroneutral NaCl reabsorption by a mechanism involving Na+-dependent and Na+-independent Cl−/[Formula: see text] exchange, and that is energetically driven by basolateral vacuolar H+-ATPase pumps. Other research indicates that type A intercalated cells can mediate NaCl secretion, through a bumetanide-sensitive pathway that is energized by apical H+,K+-ATPase type 2 pumps operating as Na+/K+exchangers. We also review recent findings on the contribution of the paracellular route to NaCl transport in the CCD. Last, we describe cross-talk processes, by which one CCD cell type impacts Na+/Cl−transport in another cell type. The mechanisms that have been identified to date demonstrate clearly the interdependence of NaCl and acid-base transport systems in the CCD. They also highlight the remarkable versatility of this nephron segment.

The electrogenic Na+-HCO3− cotransporter 2 (NBCe2) is a newly discovered protein in the distal nephron. Our understanding is minimal regarding its physiological role in renal electrolyte transport. In this mini-review, we summarize the potential function of NBCe2 in the regulation of blood pressure, acid-base, and K+ and Ca2+ transport in the distal nephron.

Sgk1 is a relatively unstable kinase that regulates epithelial Na+transport in the distal nephron of the kidney. We identified a 5′ variant alternate transcript of human Sgk1 (Sgk1_v3) that is expressed in the connecting tubule and collecting duct, is regulated by aldosterone and insulin, and is predicted to encode an NH2-terminal variant Sgk1 isoform, Sgk1_i3. Sgk1_i3 contains a polybasic motif, KKR, in its NH2terminus that regulates ubiquitination and stability of the expressed protein in HEK293 cells. In Fisher rat thyroid, and mpkCCDc14cells, Sgk1_i3 had a significantly greater effect on Na+transport compared with Sgk1 and its stimulatory effect was dependent on the kinase domain. Sgk1_i3 increased the abundance of cleaved epithelial Na+channel (ENaC) subunits at the cell surface, which was inhibited by coexpression of Nedd4–2. Together, the data demonstrate that a renally expressed Sgk1 isoform, Sgk1_i3, shows improved stability, is regulated by insulin and aldosterone, and stimulates ENaC activity when heterologously expressed in collecting duct cells.

Aldosterone is thought to be the main hormone to stimulate the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) comprising the late distal convoluted tubule (DCT2), the connecting tubule (CNT) and the entire collecting duct (CD). There is immunohistochemical evidence for an axial gradient of ENaC expression along the ASDN with highest expression in the DCT2 and CNT. However, most of our knowledge about renal ENaC function stems from studies in the cortical collecting duct (CCD). Here we investigated ENaC function in the transition zone of DCT2/CNT or CNT/CCD microdissected from mice maintained on different sodium diets to vary plasma aldosterone levels. Single-channel recordings demonstrated amiloride-sensitive Na+channels in DCT2/CNT with biophysical properties typical for ENaC previously described in CNT/CCD. In animals maintained on a standard salt diet, the average ENaC-mediated whole cell current (Δ Iami) was higher in DCT2/CNT than in CNT/CCD. A low salt diet increased Δ Iamiin CNT/CCD but had little effect on Δ Iamiin DCT2/CNT. To investigate whether aldosterone is necessary for ENaC activity in the DCT2/CNT, we used aldosterone synthase knockout (AS−/−) mice that lack aldosterone. In CNT/CCD of AS−/−mice, Δ Iamiwas lower than that in wild-type (WT) animals and was not stimulated by a low salt diet. In contrast, in DCT2/CNT of AS−/−mice, Δ Iamiwas similar to that in DCT2/CNT of WT animals both on a standard and on a low salt diet. We conclude that ENaC function in the DCT2/CNT is largely independent of aldosterone which is in contrast to its known aldosterone sensitivity in CNT/CCD.

PGE2 plays an important role in the regulation of fluid metabolism chiefly via antagonizing vasopressin-induced osmotic permeability in the distal nephron, but its enzymatic sources remain uncertain. The present study was undertaken to investigate the potential role of microsomal PGE synthase (mPGES)-1 in the regulation of urine concentrating ability after water deprivation (WD). Following 24-h WD, wild-type (WT) mice exhibited a significant reduction in urine volume, accompanied by a significant elevation in urine osmolality compared with control groups. In contrast, in response to WD, mPGES-1 knockout (KO) mice had much less urine volume and higher urine osmolality. Analysis of plasma volume by measurement of hematocrit and by using a nanoparticle-based method consistently demonstrated that dehydrated WT mice were volume depleted, which was significantly improved in the KO mice. WD induced a twofold increase in urinary PGE2 output in WT mice, which was completely blocked by mPGES-1 deletion. At baseline, the KO mice had a 20% increase in V2 receptor mRNA expression in the renal medulla but not the cortex compared with WT controls; the expression was unaffected by WD irrespective of the genotype. In response to WD, renal medullary aquaporin-2 (AQP2) mRNA exhibited a 60% increase in WT mice, and this increase was greater in the KO mice. Immunoblotting demonstrated increased renal medullary AQP2 protein abundance in both genotypes following WD, with a greater increase in the KO mice. Similar results were obtained by using immunohistochemistry. Paradoxically, plasma AVP response to WD seen in WT mice was absent in the KO mice. Taken together, these results suggest that mPGES-1-derived PGE2 reduces urine concentrating ability through suppression of renal medullary expression of V2 receptors and AQP2 but may enhance it by mediating the central AVP response.

The renal late distal convoluted tubules and connecting tubules are sites for the fine regulation of Na+ and Ca2+ reabsorption. The role of these segments in Na+ and K+ homeostasis is possibly underestimated, as the tubules are technically difficult to isolate in sufficient numbers and purity for functional analysis. To overcome these difficulties, we have developed a transgenic mouse model expressing enhanced green fluorescent protein in late distal convoluted tubules and connecting tubules. Enhanced green fluorescent protein expression was driven by the promoter for the transient receptor potential subfamily V, member 5. Confocal fluorescence microscopy allowed detection of enhanced green fluorescent protein in living, isolated late distal convoluted tubules and connecting tubules and in the initial cortical collecting ducts. Enhanced green fluorescent protein expression was validated by double- and triple-fluorescence immunolabeling with specific tubule markers. Freshly isolated late distal convoluted tubules and connecting tubules increased their intracellular Ca2+ levels in response to the V2 receptor-specific agonist deamino-Cys,d-Arg8-vasopressin (2 × 10−10 M) after 1 min of superfusion. In addition, both late distal convoluted tubules and connecting tubules displayed a concentration-dependent intracellular Ca2+ response to 1α,25-dihydroxyvitamin D3 (range 10−10 to 10−8 M). This suggests that 1α,25-dihydroxyvitamin D3 can act through a nongenomic signaling pathway in these tubules. In conclusion, the transgenic mouse model, expressing enhanced green fluorescent protein, is suitable for rapid isolation of viable late distal convoluted tubules, connecting tubules, and initial cortical collecting ducts and provides an ideal tool for a more exhaustive functional characterization of these segments.

Vectorial Na+absorption across the aldosterone-sensitive distal nephron plays a key role in the regulation of extracellular fluid volume and blood pressure. Within this nephron segment, Na+diffuses from the urinary fluid into principal cells through an apical, amiloride-sensitive, epithelial Na+channel (ENaC), which is considered to be the rate-limiting step for Na+absorption. We have reported that increases in tubular flow rate in microperfused rabbit cortical collecting ducts (CCDs) lead to increases in net Na+absorption and that increases in laminar shear stress activate ENaC expressed in oocytes by increasing channel open probability. We therefore examined whether flow stimulates net Na+absorption ( JNa) in CCDs by increasing channel open probability or by increasing the number of channels at the apical membrane. Both baseline and flow-stimulated JNain CCDs were mediated by ENaC, as JNawas inhibited by benzamil. Flow-dependent increases in JNawere observed following treatment of tubules with reagents that altered membrane trafficking by disrupting microtubules (colchicine) or Golgi (brefeldin A). Furthermore, reducing luminal Ca2+concentration ([Ca2+]) or chelating intracellular [Ca2+] with BAPTA did not prevent the flow-dependent increase in JNa. Extracellular trypsin has been shown to activate ENaC by increasing channel open probability, and we observed that trypsin significantly enhanced JNawhen tubules were perfused at a slow flow rate. However, trypsin did not further enhance JNain CCDs perfused at fast flow rates. Similarly, the shear-induced increase in benzamil-sensitive JNain oocytes expressing protease resistance ENaC mutants was similar to that of controls. Our results suggest the rise in JNaaccompanying increases in luminal flow rates reflects an increase in channel open probability.

In assessing disorders of potassium excretion, urine composition is used to calculate the transtubular gradient (TTKG), as an estimate of tubule fluid concentration, at a point when the fluid was last isotonic to plasma, namely, within the cortical collecting duct (CCD). A mathematical model of the CCD has been developed, consisting of principal cells and α- and β-intercalated cells, and which includes Na+, K+, Cl−, HCO[Formula: see text], CO2, H2CO3, phosphate, ammonia, and urea. Parameters have been selected to achieve fluxes and permeabilities compatible with data obtained from perfusion studies of rat CCD under the influence of both antidiuretic hormone and mineralocorticoid. Both epithelial (flat sheet) and tubule models have been configured, and model calculations have focused on the determinants of the TTKG. Using the epithelial model, luminal K+concentrations can be computed at which K+secretion ceases (0-flux equilibrium), and this luminal concentration derives from the magnitude of principal cell peritubular uptake of K+via the Na-K-ATPase, relative to principal cell peritubular membrane K+permeability. When the model is configured as a tubule and examined in the context of conditions in vivo, osmotic equilibration of luminal fluid produces a doubling of the initial K+concentration, which, depending on delivered load, may be substantially greater than the zero-flux equilibrium value. Under such circumstances, the CCD will be a site for K+reabsorption, although the relatively low permeability ensures that this reabsorptive flux is likely to be small. Osmotic equilibration may also raise luminal NH3concentrations well above those in cortical blood. In this situation, diffusive reabsorption of NH3provides a mechanism for base reclamation without the metabolic cost of active proton secretion.