Aldosterone Antagonism Is More Effective at Reducing Blood Pressure and Excessive Renal ENaC Activity in AngII-Infused Female Rats Than in Males.

BACKGROUND: AngII (angiotensin II)-dependent hypertension causes comparable elevations of blood pressure (BP), aldosterone levels, and renal ENaC (epithelial Na+ channel) activity in male and female rodents. Mineralocorticoid receptor (MR) antagonism has a limited antihypertensive effect associated with insufficient suppression of renal ENaC in male rodents with AngII-hypertension. While MR blockade effectively reduces BP in female mice with salt-sensitive and leptin-induced hypertension, MR antagonism has not been studied in female rodents with AngII-hypertension. We hypothesize that overstimulation of renal MR signaling drives redundant ENaC-mediated Na+ reabsorption and BP increase in female rats with AngII-hypertension. METHODS: We employ a combination of physiological, pharmacological, biochemical, and biophysical approaches to compare the effect of MR inhibitors on BP and ENaC activity in AngII-infused male and female Sprague Dawley rats. RESULTS: MR blockade markedly attenuates AngII-hypertension in female rats but has only a marginal effect in males. Spironolactone increases urinary sodium excretion and urinary sodium-to-potassium ratio in AngII-infused female, but not male, rats. The expression of renal MR and HSD11ß2 (11ß-hydroxysteroid dehydrogenase type 2) that determines the availability of MR to aldosterone is significantly higher in AngII-infused female rats than in males. ENaC activity is ≈2× lower in spironolactone-treated AngII-infused female rats than in males. Reduced ENaC activity in AngII-infused female rats on spironolactone correlates with increased interaction with ubiquitin ligase Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2), targeting ENaC for degradation. CONCLUSIONS: MR-ENaC axis is the primary determinant of excessive renal sodium reabsorption and an attractive antihypertensive target in female rats with AngII-hypertension, but not in males.

TRPV4 functional status in cystic cells regulates cystogenesis in autosomal recessive polycystic kidney disease during variations in dietary potassium.

Mechanosensitive TRPV4 channel plays a dominant role in maintaining [Ca2+ ]i homeostasis and flow-sensitive [Ca2+ ]i signaling in the renal tubule. Polycystic kidney disease (PKD) manifests as progressive cyst growth due to cAMP-dependent fluid secretion along with deficient mechanosensitivity and impaired TRPV4 activity. Here, we tested how regulation of renal TRPV4 function by dietary K+ intake modulates the rate of cystogenesis and mechanosensitive [Ca2+ ]i signaling in cystic cells of PCK453 rats, a homologous model of human autosomal recessive PKD (ARPKD). One month treatment with both high KCl (5% K+ ) and KB/C (5% K+ with bicarbonate/citrate) diets significantly increased TRPV4 levels when compared to control (0.9% K+ ). High KCl diet caused an increased TRPV4-dependent Ca2+ influx, and partial restoration of mechanosensitivity in freshly isolated monolayers of cystic cells. Unexpectedly, high KB/C diet induced an opposite effect by reducing TRPV4 activity and worsening [Ca2+ ]i homeostasis. Importantly, high KCl diet decreased cAMP, whereas high KB/C diet further increased cAMP levels in cystic cells (assessed as AQP2 distribution). At the systemic level, high KCl diet fed PCK453 rats had significantly lower kidney-to-bodyweight ratio and reduced cystic area. These beneficial effects were negated by a concomitant administration of an orally active TRPV4 antagonist, GSK2193874, resulting in greater kidney weight, accelerated cystogenesis, and augmented renal injury. High KB/C diet also exacerbated renal manifestations of ARPKD, consistent with deficient TRPV4 activity in cystic cells. Overall, we demonstrate that TRPV4 channel activity negatively regulates cAMP levels in cystic cells thus attenuating (high activity) or accelerating (low activity) ARPKD progression.

Practical notes on popular statistical tests in renal physiology.

Competent statistical analysis is essential to maintain rigor and reproducibility in physiological research. Unfortunately, the benefits offered by statistics are often negated by misuse or inadequate reporting of statistical methods. To address the need for improved quality of statistical analysis in papers, the American Physiological Society released guidelines for reporting statistics in journals published by the society. The guidelines reinforce high standards for the presentation of statistical data in physiology but focus on the conceptual challenges and, thus, may be of limited use to an unprepared reader. Experimental scientists working in the renal field may benefit from putting the existing guidelines in a practical context. This paper discusses the application of widespread hypothesis tests in a confirmatory study. We simulated pharmacological experiments assessing intracellular calcium in cultured renal cells and kidney function at the systemic level to review best practices for data analysis, graphical presentation, and reporting. Such experiments are ubiquitously used in renal physiology and could be easily translated to other practical applications to fit the reader's specific needs. We provide step-by-step guidelines for using the most common types of t tests and ANOVA and discuss typical mistakes associated with them. We also briefly consider normality tests, exclusion criteria, and identification of technical and experimental replicates. This review is supposed to help the reader analyze, illustrate, and report the findings correctly and will hopefully serve as a gauge for a level of design complexity when it might be time to consult a biostatistician.

Electrolyte and transporter responses to angiotensin II induced hypertension in female and male rats and mice.

AIM: Sexual dimorphisms are evident along the nephron: Females (F) exhibit higher ratios of renal distal to proximal Na+ transporters' abundance, greater lithium clearance (CLi ) more rapid natriuresis in response to saline infusion and lower plasma [K+ ] vs. males (M). During angiotensin II infusion hypertension (AngII-HTN) M exhibit distal Na+ transporter activation, lower proximal and medullary loop transporters, blunted natriuresis in response to saline load, and reduced plasma [K+ ]. This study aimed to determine whether responses of F to AngII-HTN mimicked those in M or were impacted by sexual dimorphisms evident at baseline. METHODS: Sprague Dawley rats and C57BL/6 mice were AngII infused via osmotic minipumps 2 and 3 weeks, respectively, and assessed by metabolic cage collections, tail-cuff sphygmomanometer, semi-quantitative immunoblotting of kidney and patch-clamp electrophysiology. RESULTS: In F rats, AngII-infusion increased BP to 190 mm Hg, increased phosphorylation of cortical NKCC2, NCC and cleavage of ENaC two to threefold, increased ENaC channel activity threefold and aldosterone 10-fold. K+ excretion increased and plasma [K+ ] decreased. Evidence of natriuresis in F included increased urine Na+ excretion and CLi , and decreased medullary NHE3, NKCC2 and Na,K-ATPase abundance. In C57BL/6 mice, AngII-HTN increased abundance of distal Na+ transporters, suppressed proximal-medullary transporters and reduced plasma [K+ ] in both F and M. CONCLUSION: Despite baseline sexual dimorphisms, AngII-HTN provokes similar increases in BP, aldosterone, distal transporters, ENaC channel activation and K+ loss accompanied by similar suppression of proximal and loop Na+ transporters, natriuresis and diuresis in females and males.

TRPC3 determines osmosensitive [Ca2+]i signaling in the collecting duct and contributes to urinary concentration.

It is well-established that the kidney collecting duct (CD) plays a central role in regulation of systemic water homeostasis. Aquaporin 2 (AQP2)-dependent water reabsorption in the CD critically depends on the arginine vasopressin (AVP) antidiuretic input and the presence of a favorable osmotic gradient at the apical plasma membrane with tubular lumen being hypotonic compared to the cytosol. This osmotic difference creates a mechanical force leading to an increase in [Ca2+]i in CD cells. The significance of the osmosensitive [Ca2+]i signaling for renal water transport and urinary concentration remain unknown. To examine molecular mechanism and physiological relevance of osmosensitivity in the CD, we implemented simultaneous direct measurements of [Ca2+]i dynamics and the rate of cell swelling as a readout of the AQP2-dependent water reabsorption in freshly isolated split-opened CDs of wild type and genetically manipulated animals and combined this with immunofluorescent detection of AVP-induced AQP2 trafficking and assessment of systemic water balance. We identified the critical role of the Ca2+-permeable TRPC3 channel in osmosensitivity and water permeability in the CD. We further demonstrated that TRPC3 -/- mice exhibit impaired urinary concentration, larger urinary volume and a greater weight loss in response to water deprivation despite increased AVP levels and AQP2 abundance. TRPC3 deletion interfered with AQP2 translocation to the plasma membrane in response to water deprivation. In summary, we provide compelling multicomponent evidence in support of a critical contribution of TRPC3 in the CD for osmosensitivity and renal water handling.

TRPV4 deletion protects against hypokalemia during systemic K+ deficiency.

Tight regulation of K+ balance is fundamental for normal physiology. Reduced dietary K+ intake, which is common in Western diets, often leads to hypokalemia and associated cardiovascular- and kidney-related pathologies. The distal nephron, and, specifically, the collecting duct (CD), is the major site of controlled K+ reabsorption via H+-K+-ATPase in the state of dietary K+ deficiency. We (Mamenko MV, Boukelmoune N, Tomilin VN, Zaika OL, Jensen VB, O'Neil RG, Pochynyuk OM. Kidney Int 91: 1398-1409, 2017) have previously demonstrated that the transient receptor potential vanilloid type 4 (TRPV4) Ca2+ channel, abundantly expressed in the CD, contributes to renal K+ handling by promoting flow-induced K+ secretion. Here, we investigated a potential role of TRPV4 in controlling H+-K+-ATPase-dependent K+ reabsorption in the CD. Treatment with a K+-deficient diet (<0.01% K+) for 7 days reduced serum K+ levels in wild-type (WT) mice from 4.3 ± 0.2 to 3.3 ± 0.2 mM but not in TRPV4-/- mice (4.3 ± 0.1 and 4.2 ± 0.3 mM, respectively). Furthermore, we detected a significant reduction in 24-h urinary K+ levels in TRPV4-/- compared with WT mice upon switching to K+-deficient diet. TRPV4-/- animals also had significantly more acidic urine on a low-K+ diet, but not on a regular (0.9% K+) or high-K+ (5% K+) diet, which is consistent with increased H+-K+-ATPase activity. Moreover, we detected a greatly accelerated H+-K+-ATPase-dependent intracellular pH extrusion in freshly isolated CDs from TRPV4-/- compared with WT mice fed a K+-deficient diet. Overall, our results demonstrate a novel kaliuretic role of TRPV4 by inhibiting H+-K+-ATPase-dependent K+ reabsorption in the CD. We propose that TRPV4 inhibition could be a novel strategy to manage certain hypokalemic states in clinical settings.

Formyl peptide receptor-1 activation exerts a critical role for the dynamic plasticity of arteries via actin polymerization.

Several human diseases, include cancer and stroke are characterized by changes in immune system activation and vascular contractility. However, the mechanistic foundation of a vascular immuno-physiology network is still largely unknown. Formyl peptide receptor-1 (FPR-1), which plays a vital role in the function of the innate immune system, is widely expressed in arteries, but its role in vascular plasticity is unclear. We questioned why a receptor that is crucial for immune defense, and cell motility in leukocytes, would be expressed in vascular smooth muscle cells (VSMCs). We hypothesized that activation of FPR-1 in arteries is important for the temporal reorganization of actin filaments, and consequently, changes in vascular function, similar to what is observed in neutrophils. To address our hypothesis, we used FPR-1 knockout and VSMCs lacking FPR-1. We observed that FPR-1 activation induces actin polymerization in wild type VSMCs. Absence of FPR-1 in the vasculature significantly decreased vascular contraction and induced loss of myogenic tone to elevated intraluminal pressures via disruption of actin polymerization. Actin polymerization activator ameliorated these responses. In conclusion, we have established a novel role for FPR-1 in VSMC contractility and motility, similar to the one observed in sentinel cells of the innate immune system. This discovery is fundamental for vascular immuno-pathophysiology, given that FPR-1 in VSMCs not only functions as an immune system receptor, but it also has an important role for the dynamic plasticity of arteries.

Urinary concentrating defect in mice lacking Epac1 or Epac2.

cAMP is a universal second messenger regulating a plethora of processes in the kidney. Two downstream effectors of cAMP are PKA and exchange protein directly activated by cAMP (Epac), which, unlike PKA, is often linked to elevation of [Ca2+]i. While both Epac isoforms (Epac1 and Epac2) are expressed along the nephron, their relevance in the kidney remains obscure. We combined ratiometric calcium imaging with quantitative immunoblotting, immunofluorescent confocal microscopy, and balance studies in mice lacking Epac1 or Epac2 to determine the role of Epac in renal water-solute handling. Epac1-/- and Epac2-/- mice developed polyuria despite elevated arginine vasopressin levels. We did not detect major deficiencies in arginine vasopressin [Ca2+]i signaling in split-opened collecting ducts or decreases in aquaporin water channel type 2 levels. Instead, sodium-hydrogen exchanger type 3 levels in the proximal tubule were dramatically reduced in Epac1-/- and Epac2-/- mice. Water deprivation revealed persisting polyuria, impaired urinary concentration ability, and augmented urinary excretion of Na+ and urea in both mutant mice. In summary, we report a nonredundant contribution of Epac isoforms to renal function. Deletion of Epac1 and Epac2 decreases sodium-hydrogen exchanger type 3 expression in the proximal tubule, leading to polyuria and osmotic diuresis.-Cherezova, A., Tomilin, V., Buncha, V., Zaika, O., Ortiz, P. A., Mei, F., Cheng, X., Mamenko, M., Pochynyuk, O. Urinary concentrating defect in mice lacking Epac1 or Epac2.

Compromised regulation of the collecting duct ENaC activity in mice lacking AT1a receptor.

ENaC-mediated sodium reabsorption in the collecting duct (CD) is a critical determinant of urinary sodium excretion. Existing evidence suggest direct stimulatory actions of Angiotensin II (Ang II) on ENaC in the CD, independently of the aldosterone-mineralocorticoid receptor (MR) signaling. Deletion of the major renal AT1 receptor isoform, AT1a R, decreases blood pressure and reduces ENaC abundance despite elevated aldosterone levels. The mechanism of this insufficient compensation is not known. Here, we used patch clamp electrophysiology in freshly isolated split-opened CDs to investigate how AT1a R dysfunction compromises functional ENaC activity and its regulation by dietary salt intake. Ang II had no effect on ENaC activity in CDs from AT1a R -/- mice suggesting no complementary contribution of AT2 receptors. We next found that AT1a R deficient mice had lower ENaC activity when fed with low (<0.01% Na+ ) and regular (0.32% Na+ ) but not with high (∼2% Na+ ) salt diet, when compared to the respective values obtained in Wild type (WT) animals. Inhibition of AT1 R with losartan in wild-type animals reproduces the effects of genetic ablation of AT1a R on ENaC activity arguing against contribution of developmental factors. Interestingly, manipulation with aldosterone-MR signaling via deoxycosterone acetate (DOCA) and spironolactone had much reduced influence on ENaC activity upon AT1a R deletion. Consistently, AT1a R -/- mice have a markedly diminished MR abundance in cytosol. Overall, we conclude that AT1a R deficiency elicits a complex inhibitory effect on ENaC activity by attenuating ENaC Po and precluding adequate compensation via aldosterone cascade due to decreased MR availability.

Collecting duct prorenin receptor knockout reduces renal function, increases sodium excretion, and mitigates renal responses in ANG II-induced hypertensive mice.

Augmented intratubular angiotensin (ANG) II is a key determinant of enhanced distal Na+ reabsorption via activation of epithelial Na+ channels (ENaC) and other transporters, which leads to the development of high blood pressure (BP). In ANG II-induced hypertension, there is increased expression of the prorenin receptor (PRR) in the collecting duct (CD), which has been implicated in the stimulation of the sodium transporters and resultant hypertension. The impact of PRR deletion along the nephron on BP regulation and Na+ handling remains controversial. In the present study, we investigate the role of PRR in the regulation of renal function and BP by using a mouse model with specific deletion of PRR in the CD (CDPRR-KO). At basal conditions, CDPRR-KO mice had decreased renal function and lower systolic BP associated with higher fractional Na+ excretion and lower ANG II levels in urine. After 14 days of ANG II infusion (400 ng·kg-1·min-1), the increases in systolic BP and diastolic BP were mitigated in CDPRR-KO mice. CDPRR-KO mice had lower abundance of cleaved αENaC and γENaC, as well as lower ANG II and renin content in urine compared with wild-type mice. In isolated CD from CDPRR-KO mice, patch-clamp studies demonstrated that ANG II-dependent stimulation of ENaC activity was reduced because of fewer active channels and lower open probability. These data indicate that CD PRR contributes to renal function and BP responses during chronic ANG II infusion by enhancing renin activity, increasing ANG II, and activating ENaC in the distal nephron segments.

The renal TRPV4 channel is essential for adaptation to increased dietary potassium.

To maintain potassium homeostasis, kidneys exert flow-dependent potassium secretion to facilitate kaliuresis in response to elevated dietary potassium intake. This process involves stimulation of calcium-activated large conductance maxi-K (BK) channels in the distal nephron, namely the connecting tubule and the collecting duct. Recent evidence suggests that the TRPV4 channel is a critical determinant of flow-dependent intracellular calcium elevations in these segments of the renal tubule. Here, we demonstrate that elevated dietary potassium intake (five percent potassium) increases renal TRPV4 mRNA and protein levels in an aldosterone-dependent manner and causes redistribution of the channel to the apical plasma membrane in native collecting duct cells. This, in turn, leads to augmented TRPV4-mediated flow-dependent calcium ion responses in freshly isolated split-opened collecting ducts from mice fed the high potassium diet. Genetic TRPV4 ablation greatly diminished BK channel activity in collecting duct cells pointing to a reduced capacity to excrete potassium. Consistently, elevated potassium intake induced hyperkalemia in TRPV4 knockout mice due to deficient renal potassium excretion. Thus, regulation of TRPV4 activity in the distal nephron by dietary potassium is an indispensable component of whole body potassium balance.

Sustained Elevated Adenosine via ADORA2B Promotes Chronic Pain through Neuro-immune Interaction.

The molecular mechanisms of chronic pain are poorly understood and effective mechanism-based treatments are lacking. Here, we report that mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, displayed unexpected chronic mechanical and thermal hypersensitivity due to sustained elevated circulating adenosine. Extending from Ada(-/-) mice, we further discovered that prolonged elevated adenosine contributed to chronic pain behaviors in two additional independent animal models: sickle cell disease mice, a model of severe pain with limited treatment, and complete Freund's adjuvant paw-injected mice, a well-accepted inflammatory model of chronic pain. Mechanistically, we revealed that activation of adenosine A2B receptors on myeloid cells caused nociceptor hyperexcitability and promoted chronic pain via soluble IL-6 receptor trans-signaling, and our findings determined that prolonged accumulated circulating adenosine contributes to chronic pain by promoting immune-neuronal interaction and revealed multiple therapeutic targets.

Correction: Emerging Role of the Calcium-Activated, Small Conductance, SK3 K+ Channel in Distal Tubule Function: Regulation by TRPV4.

[This corrects the article DOI: 10.1371/journal.pone.0095149.].

New perspective of ClC-Kb/2 Cl- channel physiology in the distal renal tubule.

Since its identification as the underlying molecular cause of Bartter's syndrome type 3, ClC-Kb (ClC-K2 in rodents, henceforth it will be referred as ClC-Kb/2) is proposed to play an important role in systemic electrolyte balance and blood pressure regulation by controlling basolateral Cl(-) exit in the distal renal tubular segments from the cortical thick ascending limb to the outer medullary collecting duct. Considerable experimental and clinical effort has been devoted to the identification and characterization of disease-causing mutations as well as control of the channel by its cofactor, barttin. However, we have only begun to unravel the role of ClC-Kb/2 in different tubular segments and to reveal the regulators of its expression and function, e.g., insulin and IGF-1. In this review we discuss recent experimental evidence in this regard and highlight unexplored questions critical to understanding ClC-Kb/2 physiology in the kidney.

Defective Store-Operated Calcium Entry Causes Partial Nephrogenic Diabetes Insipidus.

Store-operated calcium entry (SOCE) is the mechanism by which extracellular signals elicit prolonged intracellular calcium elevation to drive changes in fundamental cellular processes. Here, we investigated the role of SOCE in the regulation of renal water reabsorption, using the inbred rat strain SHR-A3 as an animal model with disrupted SOCE. We found that SHR-A3, but not SHR-B2, have a novel truncating mutation in the gene encoding stromal interaction molecule 1 (STIM1), the endoplasmic reticulum calcium (Ca(2+)) sensor that triggers SOCE. Balance studies revealed increased urine volume, hypertonic plasma, polydipsia, and impaired urinary concentrating ability accompanied by elevated circulating arginine vasopressin (AVP) levels in SHR-A3 compared with SHR-B2. Isolated, split-open collecting ducts (CD) from SHR-A3 displayed decreased basal intracellular Ca(2+) levels and a major defect in SOCE. Consequently, AVP failed to induce the sustained intracellular Ca(2+) mobilization that requires SOCE in CD cells from SHR-A3. This effect decreased the abundance of aquaporin 2 and enhanced its intracellular retention, suggesting impaired sensitivity of the CD to AVP in SHR-A3. Stim1 knockdown in cultured mpkCCDc14 cells reduced SOCE and basal intracellular Ca(2+) levels and prevented AVP-induced translocation of aquaporin 2, further suggesting the effects in SHR-A3 result from the expression of truncated STIM1. Overall, these results identify a novel mechanism of nephrogenic diabetes insipidus and uncover a role of SOCE in renal water handling.

Role of renal TRP channels in physiology and pathology.

Kidneys critically contribute to the maintenance of whole-body homeostasis by governing water and electrolyte balance, controlling extracellular fluid volume, plasma osmolality, and blood pressure. Renal function is regulated by numerous systemic endocrine and local mechanical stimuli. Kidneys possess a complex network of membrane receptors, transporters, and ion channels which allows responding to this wide array of signaling inputs in an integrative manner. Transient receptor potential (TRP) channel family members with diverse modes of activation, varied permeation properties, and capability to integrate multiple downstream signals are pivotal molecular determinants of renal function all along the nephron. This review summarizes experimental data on the role of TRP channels in a healthy mammalian kidney and discusses their involvement in renal pathologies.

Insulin and IGF-1 activate Kir4.1/5.1 channels in cortical collecting duct principal cells to control basolateral membrane voltage.

Potassium Kir4.1/5.1 channels are abundantly expressed at the basolateral membrane of principal cells in the cortical collecting duct (CCD), where they are thought to modulate transport rates by controlling transepithelial voltage. Insulin and insulin-like growth factor-1 (IGF-1) stimulate apically localized epithelial sodium channels (ENaC) to augment sodium reabsorption in the CCD. However, little is known about their actions on potassium channels localized at the basolateral membrane. In this study, we implemented patch-clamp analysis in freshly isolated murine CCD to assess the effect of these hormones on Kir4.1/5.1 at both single channel and cellular levels. We demonstrated that K(+)-selective conductance via Kir4.1/5.1 is the major contributor to the macroscopic current recorded from the basolateral side in principal cells. Acute treatment with 10 µM amiloride (ENaC blocker), 100 nM tertiapin-Q (TPNQ; ROMK inhibitor), and 100 µM ouabain (Na(+)-K(+)-ATPase blocker) failed to produce a measurable effect on the macroscopic current. In contrast, Kir4.1 inhibitor nortriptyline (100 µM), but not fluoxetine (100 µM), virtually abolished whole cell K(+)-selective conductance. Insulin (100 nM) markedly increased the open probability of Kir4.1/5.1 and nortriptyline-sensitive whole cell current, leading to significant hyperpolarization of the basolateral membrane. Inhibition of the phosphatidylinositol 3-kinase cascade with LY294002 (20 µM) abolished action of insulin on Kir4.1/5.1. IGF-1 had similar stimulatory actions on Kir4.1/5.1-mediated conductance only when applied at a higher (500 nM) concentration and was ineffective at 100 nM. We concluded that both insulin and, to a lesser extent, IGF-1 activate Kir4.1/5.1 channel activity and open probability to hyperpolarize the basolateral membrane, thereby facilitating Na(+) reabsorption in the CCD.

Control of ENaC-mediated sodium reabsorption in the distal nephron by Bradykinin.

Kinins, such as Bradykinin (BK), are peptide hormones of the kallikrein-kinin system. Apart from being a vasodilator, BK also increases urinary sodium excretion to reduce systemic blood pressure. It is becoming appreciated that BK modulates function of the epithelial Na(+) channel in the distal part of the renal nephron to affect tubular sodium reabsorption. In this chapter, we outline the molecular details, as well as discuss the physiological relevance of this regulation for the whole organism sodium homeostasis and setting chronic blood pressure.

IGF-1 and insulin exert opposite actions on ClC-K2 activity in the cortical collecting ducts.

Despite similar stimulatory actions on the epithelial sodium channel (ENaC)-mediated sodium reabsorption in the distal tubule, insulin promotes kaliuresis, whereas insulin-like growth factor-1 (IGF-1) causes a reduction in urinary potassium levels. The factors contributing to this phenomenon remain elusive. Electrogenic distal nephron ENaC-mediated Na(+) transport establishes driving force for Cl(-) reabsorption and K(+) secretion. Using patch-clamp electrophysiology, we document that a Cl(-) channel is highly abundant on the basolateral plasma membrane of intercalated cells in freshly isolated mouse cortical collecting duct (CCD) cells. The channel has characteristics attributable to the ClC-K2: slow gating kinetics, conductance ∼10 pS, voltage independence, Cl(-)>NO3 (-) anion selectivity, and inhibition/activation by low/high pH, respectively. IGF-1 (100 and 500 nM) acutely stimulates ClC-K2 activity in a reversible manner. Inhibition of PI3-kinase (PI3-K) with LY294002 (20 µM) abrogates activation of ClC-K2 by IGF-1. Interestingly, insulin (100 nM) reversibly decreases ClC-K2 activity in CCD cells. This inhibitory action is independent of PI3-K and is mediated by stimulation of a mitogen-activated protein kinase-dependent cascade. We propose that IGF-1, by stimulating ClC-K2 channels, promotes net Na(+) and Cl(-) reabsorption, thus reducing driving force for potassium secretion by the CCD. In contrast, inhibition of ClC-K2 by insulin favors coupling of Na(+) reabsorption with K(+) secretion at the apical membrane contributing to kaliuresis.

Emerging role of the calcium-activated, small conductance, SK3 K+ channel in distal tubule function: regulation by TRPV4.

The Ca2+-activated, maxi-K (BK) K+ channel, with low Ca2+-binding affinity, is expressed in the distal tubule of the nephron and contributes to flow-dependent K+ secretion. In the present study we demonstrate that the Ca2+-activated, SK3 (KCa2.3) K+ channel, with high Ca2+-binding affinity, is also expressed in the mouse kidney (RT-PCR, immunoblots). Immunohistochemical evaluations using tubule specific markers demonstrate significant expression of SK3 in the distal tubule and the entire collecting duct system, including the connecting tubule (CNT) and cortical collecting duct (CCD). In CNT and CCD, main sites for K+ secretion, the highest levels of expression were along the apical (luminal) cell membranes, including for both principal cells (PCs) and intercalated cells (ICs), posturing the channel for Ca2+-dependent K+ secretion. Fluorescent assessment of cell membrane potential in native, split-opened CCD, demonstrated that selective activation of the Ca2+-permeable TRPV4 channel, thereby inducing Ca2+ influx and elevating intracellular Ca2+ levels, activated both the SK3 channel and the BK channel leading to hyperpolarization of the cell membrane. The hyperpolarization response was decreased to a similar extent by either inhibition of SK3 channel with the selective SK antagonist, apamin, or by inhibition of the BK channel with the selective antagonist, iberiotoxin (IbTX). Addition of both inhibitors produced a further depolarization, indicating cooperative effects of the two channels on Vm. It is concluded that SK3 is functionally expressed in the distal nephron and collecting ducts where induction of TRPV4-mediated Ca2+ influx, leading to elevated intracellular Ca2+ levels, activates this high Ca2+-affinity K+ channel. Further, with sites of expression localized to the apical cell membrane, especially in the CNT and CCD, SK3 is poised to be a key pathway for Ca2+-dependent regulation of membrane potential and K+ secretion.