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Mice on a low Na, high K diet (LNaHK) are able to excrete excess K and maintain K homeostasis by increasing tubular fluid flow in the distal nephron of the kidney. High tubular fluid flow increases...

The renal distal nephron [distal convoluted tubule 2 (DCT2) and connecting tubule (CNT)] is crucial for sodium and blood pressure regulation, and is tightly controlled by aldosterone. Due to diffic...

Recent studies point to direct actions of Ang II on ENaC-mediated Na+ reabsorption in the distal nephron via stimulation of AT1R that occurs independently of aldosterone-MR signaling. We used split...

The epithelial sodium channel (ENaC) is a major determinant of sodium (Na+) and water balance in the kidney. We previously reported that aldosterone (aldo) modifies expression of microRNAs (miRs) t...

It becomes recognized that Ca2+-permeable TRPV4 channel is predominantly expressed in the distal nephron and its activity is essential for [Ca2+]i elevations in response to increased tubular flow. Here, we probe whether TRPV4 function in the kidney is regulated by dietary intake of major electrolytes. We found that renal TRPV4 expression was increased by high potassium diet (5% K+) and decreased by dietary potassium restriction (0.003 % K+). Saturation of systemic mineralocorticoid status with DOCA had little effect on TRPV4 protein abundance pointing to an aldosterone-independent nature of this regulation. Increased TRPV4 expression by high potassium intake was associated with significantly augmented [Ca2+]i responses to elevated flow as was detected using Fura-2 Ca2+ imaging in freshly isolated split-opened distal nephrons. Furthermore, dietary sodium load (2% Na+) similarly increased renal TRPV4 expression and enhanced flow-induced [Ca2+]i responses in distal nephron cells. Interestingly, protein level...

The inward rectifying potassium channels of the ROMK family are present in the distal nephron of the kidney. These channels have two membrane spanning portions, between which lies a hydrophobic domain thought to confer the majority of the conductive properties of the channel. The N- and C-termini are both intracellular. In this paper we have examined the contribution of the N- and C-termini to the pore by examining the interaction of Cs+ with the channels. ROMK1 has an additional 19 amino acids on its N-terminus in comparison to ROMK2. The C-terminus of ROMK2 was extended by addition of a streptavidin tag (sfROMK2). Currents were measured following expression in Xenopus oocytes using two-electrode voltage clamp. ROMK1, ROMK2 and sfROMK2 exhibited concentration- and voltage-dependent block of inward currents by extracellular Cs+. The Hill coefficients were not significantly different from one. The mean Kd values at 0 mV were 100.6 ± 10.6, 63.1 ± 3.9 and 40.6 ± 9.4, respectively (p < 0.05). The electric distances (δ) were 0.94 ± 0.06, 1.0 ± 0.05 and 1.37 ± 0.06 respectively. The δ of sfROMK2 was greater than either ROMK1 or ROMK2 (p < 0.001). ROMK1, ROMK2 and sfROMK2 are sensitive to extracellular Cs+. Block was both concentration- and voltage-dependent. sfROMK2 is most Cs+-sensitive. ROMK1 contains an additional N-terminal 19 amino acids. Thus the pore properties of these two isoforms are subtly different, and influenced by the N-terminus. The lower Kd in sfROMK2 suggests that the streptavidin tag, and perhaps the C-terminus, also affect the pore.

The mechanism by which dietary K+ restriction induces distal–nephron Li+ reabsorption was investigated by administration of bendroflumethiazide (BFTZ) or vehicle in conscious Wistar rats. Changes in fractional excretion of Li+ following administration of amiloride (ΔFELi) were used as an index of distal tubular Li+ reabsorption. The results revealed an absence of distal tubular Li+ reabsorption in K+–replete rats (ΔFELi = 3.6±2.4%), in contrast to K+ restriction in which ΔFELi was 24.0±2.7%. The distal tubular Li+ reabsorption in K+–depleted rats was significantly reduced by preadministration of BFTZ (ΔFELi = 9.2±0.9%). The fractions of Li+ and Na+ reabsorbed in the amiloride–sensitive segment were different in K+–replete rats (9±6 vs. 60±6%), but similar in K+–depleted rats (61±5 vs. 73±4%). BFTZ administration to K+–depleted rats resulted in a proportional decrease in these fractions, suggesting competition between Na+ and Li+ for reabsorption in the distal–nephron segment during K+ depletion. These results are compatible with the hypothesis that during K+ depletion the reabsorption of Li+ in the distal–nephron segment is competitively inhibited by Na+.

The epithelial Na+channel (ENaC) controls the rate-limiting step in the process of transepithelial Na+reabsorption in the distal nephron, the distal colon, and the airways. Hereditary salt-losing syndromes have been ascribed to loss of function mutations in the α-, β-, or γ-ENaC subunit genes, whereas gain of function mutations (located in the COOH terminus of the β- or γ-subunit) result in hypertension due to Na+retention (Liddle’s syndrome). In mice, gene-targeting experiments have shown that, in addition to the kidney salt-wasting phenotype, ENaC was essential for lung fluid clearance in newborn mice. Disruption of the α-subunit resulted in a complete abolition of ENaC-mediated Na+transport, whereas knockout of the β- or γ-subunit had only minor effects on fluid clearance in lung. Disruption of each of the three subunits resulted in a salt-wasting syndrome similar to that observed in humans.

The renal mechanisms underlying sodium retention during liver cirrhosis have been difficult to elucidate. Kim and associates describe a biphasic pattern of regulation of the renal epithelial sodium channel in the common bile duct ligation model, shedding some light on this issue.

Renal tubular disorders in childhood are rare, but, when present, may produce profound electrolyte and volume disturbances. Children with hereditary tubular dysfunction commonly present in the first year of life with failure to thrive and other non-specific symptoms. Biochemical analysis of both serum and urine, together with clinical history and examination, remain fundamental for their diagnosis, whilst localization of the renal tubular defect to the proximal or distal nephron is necessary for understanding of the underlying pathophysiology and appropriate management. During recent years, important new information concerning diagnosis and treatment of selective tubular disorders has been acquired.

Primary cilia are hair-like organelles singly distributed along the apical surface of proximal and distal nephron tubules as mechanosensors. The goal of this study was to use electron microscopy to systemically evaluate cilia changes in acute tubular injury (ATI) from both transplant and native renal biopsies. Three groups of cases were included: control group 1-native biopsies without major changes in renal tubules; study group 2-native biopsies with prominent ATI; and study group 3-renal transplant biopsies with prominent ATI (delayed renal function group). Extensive search for ciliary structures along renal tubules was conducted in each case, focused on proximal tubular areas with injured (diminished) apical microvilli. Singly located cilia were found in 3/19 specimens in control group 1, 4/18 in group 2 (native ATI), and 6/24 in group 3 (transplant ATI). Importantly, there were clusters of cilia in proximal tubules with markedly diminished apical microvilli in 3/24 biopsies from 2 patients in group 3, but none from groups 1 and 2. The clusters of cilia ranged from 6 to 15 individual cilia along the apical surface with diminished apical microvilli. Under high magnifications, the cilia demonstrated 9 pairs of peripheral microtubules without a central pair of microtubules, consistent with primary cilia (9 + 0) rather than motile cilia (9 + 2). In summary, the authors found clusters of cilia in proximal tubules with remarkable apical microvillar injury in 3 renal transplant biopsies with ATI, implying a reactive, or repairing, process following tubular injury, thus they name this finding "cilia metaplasia".

We studied the characteristics of the basal and antidiuretic hormone (arginine vasotocin, AVT)-activated whole cell currents of an aldosterone-treated distal nephron cell line (A6) at two different cytosolic Ca2+ concentrations ([Ca2+] c , 2 and 30 nm). A6 cells were cultured on a permeable support filter for 10 ∼ 14 days in media with supplemental aldosterone (1 μm). At 30 nm [Ca2+] c , basal conductances mainly consisted of Cl− conductances, which were sensitive to 5-nitro-2-(3-phenylpropylamino)-benzoate. Reduction of [Ca2+] c to 2 nm abolished the basal Cl− conductance. AVT evoked Cl− conductances at 2 as well as 30 nm [Ca2+] c . In addition to Cl− conductances, AVT induced benzamil-insensitive nonselective cation (NSC) conductances. This action on NSC conductances was observed at 30 nm [Ca2+] c but not at 2 nm [Ca2+] c . Thus, cytosolic Ca2+ regulates NSC and Cl− conductances in a distal nephron cell line (A6) in response to AVT. Keeping [Ca2+] c at an adequate level seems likely to be an important requirement for AVT regulation of ion conductances in aldosterone-treated A6 cells.

Recent developments have led to renewed interest in the mechanisms that mediate the changes in sodium excretion in response to changes in arterial pressure, a phenomenon termed pressure-natriuresis. Pressure-natriuresis occurs in the absence of changes in filtered load and thus, the changes in sodium excretion are caused by changes in net tubular sodium reabsorption. Both proximal and distal nephron segments have been implicated as the sites of altered reabsorption. The specific mechanism responsible for pressure-natriuresis remains unresolved. One proposal suggests that changes in renal interstitial fluid pressure which may be due, in part, to a less efficient autoregulation of the renal medullary circulation than of the cortex, alter sodium reabsorption. Alternatively, recent studies indicate that increases in arterial pressure increase endothelial nitric oxide formation which inhibits sodium reabsorption via direct effects on the tubules as well as hemodynamically mediated effects.

The MDCK cell has proven to be a useful model cell line for the study of properties and regulation of renal epithelial ion channels. Patch clamp studies disclosed the existence of several K+ channels and of a Cl- channel, and their regulation by hormones, cell volume, trace elements and drugs. Most hormones affect K+ channels at least in part by increasing cytosolic Ca2+. However, indirect evidence points to additional mechanisms contributing to K+ channel activation. Cell swelling activates both K+ channels and unselective anion channels. ICln, a protein cloned from MDCK cells, is either a Cl- channel or a regulator of thereof. ICln is up-regulated by cellular acidification and is crucial for rapid regulatory cell volume decrease.

The inwardly rectifying, ATP-sensitive K+ channel (ROMK) was localized by in situ hybridization in the rat kidney. Tissue in situ hybridization revealed that transcripts encoding the ROMK channel were expressed predominantly in cortical and outer medullary nephron segments. The localization of ROMK mRNA to specific nephron segments was assessed by hybridization of isolated nephron segments with an ROMK-specific probe (single segment in situ hybridization). ROMK mRNA was present in cortical and medullary thick ascending limb, distal tubule, and cortical and outer medullary collecting ducts, but not in proximal tubule. A weak hybridization was observed with inner medullary collecting ducts. To confirm these results, serial cryosections were alternatively stained by hybridization histochemistry for ROMK mRNA or by immunocytochemistry using antibodies specific for S1, S2, or S3 proximal tubular segments. Tubular cells that displayed immunoreactivity with the proximal tubular segment-specific antibodies showed little, if any, ROMK message. In addition, using an in situ hybridization and immunocytochemistry double-labeling technique, ROMK transcripts and vitamin D-dependent calcium-binding protein were shown to colocalize to the distal tubule (distal convoluted tubule and connecting tubule). The overall nephron localization of ROMK mRNA shown in these studies is consistent with the possibility that this novel channel may represent the low-conductance ATP-sensitive K+ channel that has been identified in apical membranes of thick limb and collecting duct segments and is believed to participate in K+ secretion.

Carcinoma of the collecting ducts of Bellini are rare aggressive neoplasms of distal nephron origin; it accounts for 2% of all renal cell carcinomas. Clinically, collecting duct carcinoma (CDC) is characterized by an extremely aggressive phenotype, accompanying metastatic diseases at presentation in most reported cases. Most cases of CDC have been reported with median age above 50 years. To our knowledge, this is the only second case report of CDC in a young teenager patient below 19 years with metastasis to lungs and bones at presentation.

Autosomal dominant distal renal tubular acidosis (ddRTA) is caused by mutations in SLC4A1, which encodes the polytopic chloride-bicarbonate exchanger AE1 that is normally expressed at the basolateral surface of alpha-intercalated cells in the distal nephron. Here we report that, in contrast with many disorders in which mutant membrane proteins are retained intracellularly and degraded, ddRTA can result from aberrant targeting of AE1 to the apical surface.

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.

Claudins play a major role in the regulation of paracellular electrolyte reabsorption in the kidney. This review describes the recent findings of the physiological function of claudins underlying the paracellular transport mechanisms for Cl(-) reabsorption in the collecting duct.There are two parallel mechanisms for transepithelial Cl(-) reabsorption in the collecting duct that utilize the Na-driven Cl-bicarbonate exchanger (NDCBE) and the claudin-based paracellular channel. Histological studies have demonstrated the renal localization of claudin-3, claudin-4, claudin-7, and claudin-8 in the collecting duct. Molecular analyses using several collecting duct cell models have come to the conclusion that claudin-4 functions as a paracellular Cl(-) channel. The channel function of claudin-4 is conferred by a charged lysine residue (K65) in its extracellular loop. Claudin-8 is required for paracellular Cl(-) permeation through its interaction with and recruitment of claudin-4 during tight junction assembly. Claudin-7 provides the basic barrier function to the collecting duct. Genetic ablation of claudin-7 in animals results in systemic dehydration owing to the loss of extracellular ions and fluid in the kidney.The paracellular pathway in the collecting duct is an important route for transepithelial Cl(-) reabsorption that determines the extracellular NaCl content and the blood pressure. In the collecting duct cells, claudin-4 and claudin-8 interact to form a paracellular Cl(-) channel, whereas claudin-7 maintains the transepithelial resistance. Different subsets of the claudin family proteins fulfill diverse aspects of the tight junction function that will be fundamental to understanding the physiology of the paracellular pathway.

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 nephron segments beyond the proximal tubule are sometimes termed “the distal nephron”. In this chapter we will deal with the loop of Henle separately, and then consider the other components of the “distal nephron”.

These studies test whether three disease‐causing mutations in genes ( SCNN1A and SCNN1G ) encoding subunits of the epithelial Na + channel, ENaC, affect the biophysical and gating properties of this important renal ion channel. The S562P missense mutation in α ENaC and the K106_S108delinsN mutation in γ ENaC are associated with pseudohypoaldosteronism type 1 (PHA1). The N530S missense mutation in γ ENaC causes Liddle9s syndrome. Incorporation of S562P into α ENaC and K106_S108N into γ ENaC resulted in significant decreases in macroscopic ENaC currents. Conversely, incorporation of N530S into γ ENaC increased macroscopic ENaC current. The S562P substitution resulted in a nonfunctional channel. The K106_S108N mutation produced a functional channel having a normal macroscopic current–voltage relation, there was a slight but significant decrease in unitary conductance and a marked decrease in single‐channel open probability. The N530S substitution increased single‐channel open probability having no effect on the macroscopic current–voltage relation or unitary conductance of the channel. These findings are consistent with mutation of residues at 562 in α ENaC and 530 in γ ENaC, and a 3′ splice site in SCNN1G (318‐1 G→A; K106_108SdelinsN) resulting in aberrant ENaC activity due to changes in the biophysical and gating properties of the channel. Such changes likely contribute to the cellular mechanism underpinning the PHA1 and Liddle9s syndrome caused by these mutations in ENaC subunits.

Clinical findings resemble those of primary hyperaldosteronism, except that aldosterone secretion is negligible. The fault appears to lie with continuously avid sodium channels in the distal nephron, resulting in excessive salt absorption, potassium wasting, and severe hypertension. Insights gained in this disorder may help clarify more common forms of low-renin hypertension.

Low molecular weight proteins are normally, essentially, entirely catabolized by the proximal tubules. Physiological and pathological aspects of the renal interactions of three of these proteins: light chains, B-2 microglobulin, and myoglobin share similarities, but are also unique in certain ways. In pathological conditions, some may produce glomerular, proximal or distal nephron and/or vascular alterations.

The mechanisms through which aldosterone promotes apparently opposite effects like salt reabsorption and K+secretion remain poorly understood. The identification, localization, and physiological analysis of ion transport systems in distal nephron have revealed an intricate network of interactions between several players, revealing the complex mechanism behind the aldosterone paradox. We review the mechanisms involved in differential regulation of ion transport that allow the fine tuning of salt and K+balance.

The thick ascending limb of the loop of Henle reabsorbs 20–30% of filtered sodium chloride (NaCl) and generates the osmotic gradient necessary for water absorption in the distal nephron. It is second

There is now convincing evidence that in addition to the vacuolar-type H+-ATPase, a gastric-type H+/K+-ATPase participates in acidification by the distal nephron. To determine whether a similar pump exists in the turtle bladder, we examined the dependence of acid secretion on mucosal K+ and the effects of supposedly specific inhibitors of the gastric H+/K+-ATPase, omeprazole and SCH 28080. In CO2-stimulated bladders both drugs produced dose-dependent inhibition of electrogenic H+ secretion measured as the reverse short-circuit current (RSCC). At the highest concentrations tested, H+ secretion decreased 45 ± 16% with mucosal and 20 ± 7% with serosal omeprazole (p < 0.01). SCH 28080 at 400 µM produced essentially complete inhibition of H+ secretion with either mucosal or serosal application. When H+ secretion was purposefully inhibited by DIDS or an adverse mucosal pH gradient, SCH 28080 had no effect on RSCC. Removing mucosal K+ (measured K+ < 50 µM) with or without mucosal barium, had no effect on RSCC. The inhibition of RSCC by omeprazole was reversed by mercaptoethanol. Finally, HCO3 secretion, as measured by either RSCC or pH-stat titration, increased significantly in response to 400 µM SCH 28080. The results demonstrate that these compounds inhibit acid secretion by the turtle bladder but stimulate the secretion of base. In view of the total independence of acid secretion on potassium, it is unlikely that any of the bladder’s acid secretion is mediated by an H+/K+-ATPase. The most reasonable interpretation of the data is that omeprazole and SCH 28080, previously thought to be specific inhibitors of the H+/K+-ATPase, also inhibit the vacuolar H+-ATPase of the turtle bladder. The results also indicate that HCO3 secretion by the bladder employ a different mechanism of H+ transport than is used for acid secretion; there is no simple reversal of polarity in the acid- versus base-secreting cells.

The role of mechanical forces in the regulation of glomerulotubular balance in the proximal tubule (PT) and Ca2+signaling in the distal nephron was first recognized a decade ago, when it was proposed that the microvilli in the PT and the primary cilium in the cortical collecting duct (CCD) acted as sensors of local tubular flow. In this review, we present a summary of the theoretical models and experiments that have been conducted to elucidate the structure and function of these unique apical structures in the modulation of Na+, HCO3−, and water reabsorption in the PT and Ca2+signaling in the CCD. We also contrast the mechanotransduction mechanisms in renal epithelium with those in other cells in which fluid shear stresses have been recognized to play a key role in initiating intracellular signaling, most notably endothelial cells, hair cells in the inner ear, and bone cells. In each case, small hydrodynamic forces need to be greatly amplified before they can be sensed by the cell's intracellular cytoskeleton to enable the cell to regulate its membrane transporters or stretch-activated ion channels in maintaining homeostasis in response to changing flow conditions.

Background Epithelial cells are exposed to a variety of mechanical stimuli. Epithelial Na+ channels (ENaC) mediate sodium transport across apical membranes of epithelial cells that line the distal nephron, airway and alveoli, and distal colon. Early investigations into stretch sensitivity of ENaC were controversial. However, recent studies are supportive of ENaC's mechanosensitivity. This work studied whether flow-dependent activation of ENaC is modulated by changes in the state of the actin cytoskeleton and whether small GTPase RhoA is involved in flow-mediated increase of ENaC activity. Findings Pretreatment with Cytochalasin D and Latrunculin B for 20 min and 1-2 hrs to disassemble F-actin had no effect on flow-mediated increase of amiloride-sensitive current. Overexpression of ENaC with constitutively active (G14V) or dominant negative (T19N) RhoA similarly had no effect on flow-dependent activation of ENaC activity. In addition, we did not observe changes when we inhibited Rho-kinase with Y27632. Conclusions Our results suggest that the flow-dependent activation of ENaC is not influenced by small GTPase RhoA and modifications in the actin cytoskeleton.