The intercalated cell Cl−/HCO3− exchanger, pendrin, modulates ENaC subunit abundance and function. Whether ENaC modulates pendrin abundance and function is however unknown. Because αENaC mRNA has been detected in pendrin-positive intercalated cells, we hypothesized that ENaC, or more specifically the αENaC subunit, modulates intercalated cell function. The purpose of this study was therefore to determine if αENaC is expressed at the protein level in pendrin-positive intercalated cells and to determine if αENaC gene ablation or constitutively upregulating ENaC activity changes pendrin abundance, subcellular distribution, and/or function. We observed diffuse, cytoplasmic αENaC label in pendrin-positive intercalated cells from both mice and rats, with much lower label intensity in pendrin-negative, type A intercalated cells. However, while αENaC gene ablation within principal and intercalated cells of the CCD reduced Cl− absorption, it did not change pendrin abundance or subcellular distribution in aldosterone-treated mice. Further experiments used a mouse model of Liddle’s syndrome to explore the effect of increasing ENaC channel activity on pendrin abundance and function. The Liddle’s variant did not increase either total or apical plasma membrane pendrin abundance in aldosterone-treated or in NaCl-restricted mice. Similarly, while the Liddle’s mutation increased total Cl− absorption in CCDs from aldosterone-treated mice, it did not significantly affect the change in Cl− absorption seen with pendrin gene ablation. We conclude that in rats and mice, αENaC localizes to pendrin-positive ICs where its physiological role remains to be determined. While pendrin modulates ENaC abundance, subcellular distribution, and function, ENaC does not have a similar effect on pendrin.

Intercalated cells make up about a third of all cells within the connecting tubule and the collecting duct and are subclassified as type A, type B and non-A, non-B based on the subcellular distribution of the H+-ATPase, which dictates whether it secretes H+or HCO3-. Type B intercalated cells mediate Cl-absorption and HCO3-secretion, which occurs largely through the anion exchanger pendrin. Pendrin is stimulated by angiotensin II via the angiotensin type 1a receptor and by aldosterone through MR (mineralocorticoid receptor). Aldosterone stimulates pendrin expression and function, in part, through the alkalosis it generates. Pendrin-mediated HCO3-secretion increases in models of metabolic alkalosis, which attenuates the alkalosis. However, pendrin-positive intercalated cells also regulate blood pressure, at least partly, through pendrin-mediated Cl-absorption and through their indirect effect on the epithelial Na+channel, ENaC. This aldosterone-induced increase in pendrin secondarily stimulates ENaC, thereby contributing to the aldosterone pressor response. This review describes the contribution of pendrin-positive intercalated cells to Na+, K+, Cl-and acid-base balance.

Patient: Male, 82-year-old Final Diagnosis: End stage renal disease • thrombocytopenia • co-existing disea Symptoms: Fatigue • melena • weakness Medication: — Clinical Procedure: — Specialty: Nephrology Objective: Background: Case Report: Conclusions: Unusual or unexpected effect of treatment Biocompatible hemodialysis membranes have greatly advanced the treatment of renal failure. Synthetic poly-sulfone dialysis membranes are considered to be very biocompatible because of their low propensity to activate complement. However, these membranes can reduce platelet count through platelet activation, although the mechanism of this activation is unknown. We report the case of an 82-year-old man with a history of chronic kidney disease with recurrent gastrointes-tinal bleeding and worsening renal function who was initiated on renal replacement therapy with polysulfone dialysis membranes. On admission, the patient’s platelet count was normal at 233×103/μL. A significant fall in platelet count was observed following most dialysis treatments, reaching a nadir of 37×103/μL. With occasional dialysis treatments, his platelet count did not change. This dialysis-induced thrombocytopenia resolved following substitution with Cellentia-H cellulose triacetate single-use, hollow-fiber, high-flux hemodialyzer membrane. Polysulfone membranes are capable of activating platelets, which can result in severe thrombocytopenia. However, the magnitude of dialysis-induced thrombocytopenia varies from treatment to treatment. As such, it may not be evident when the pre-and postdialysis platelet counts are measured for a single treatment. Because the etiology of this platelet activation is unknown, substitution with cellulose triacetate membranes should be considered. These membranes have an unrelated chemical composition and a very low propensity to activate platelets.

Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society. The mouse has become the most common mammalian animal model used in biomedical research. However, laboratory techniques used previously in rats and other larger animals to sample blood had to be adapted in mice due to their lower mouse plasma volume. Sampling is further confounded by the variability in plasma hormone and metabolite concentrations that can occur from the stress or the anesthesia that accompanies the collection. In this article, we describe in detail a protocol we developed for blood sampling in conscious, unrestrained mice. Our protocol implements the use of chronic indwelling catheters in the right external jugular vein, allowing the mice to recover fully in their home cages, untethered until the time of blood sampling. This protocol employs catheters that remain patent for days and does not require the purchase of expensive equipment. We validated this protocol by measuring the time course of plasma norepinephrine (NE) concentration during and after the relief of acute immobilization stress in wild type (WT) and pendrin knockout (KO) mice and compared these results with our previously published values. We found that following relief from immobilization stress, it takes longer for plasma NE concentration to return to basal levels in the pendrin KO than in the wild type mice. These results highlight the potential utility of this protocol and the potential role of pendrin in the neuroendocrine response to acute stress.

Pendrin is a Cl−/HCO3− exchanger, expressed in the apical regions of some intercalated cell subtypes, and is critical in the pressor response to angiotensin II. Since angiotensin type 1 receptor inhibitors reduce renal pendrin protein abundance in mice in vivo through a mechanism that is dependent on nitric oxide (NO), we asked if NO modulates renal pendrin expression in vitro and explored the mechanism by which it occurs. Thus we quantified pendrin protein abundance by confocal fluorescent microscopy in cultured mouse cortical collecting ducts (CCDs) and connecting tubules (CNTs). After overnight culture, CCDs maintain their tubular structure and maintain a solute gradient when perfused in vitro. Pendrin protein abundance increased 67% in CNT and 53% in CCD when NO synthase was inhibited (NG-nitro-l-arginine methyl ester, 100 μM), while NO donor (DETA NONOate, 200 μM) application reduced pendrin protein by ∼33% in the CCD and CNT. When CNTs were cultured in the presence of the guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (10 μM), NO donors did not alter pendrin abundance. Conversely, pendrin protein abundance rose when cAMP content was increased by the application of an adenylyl cyclase agonist (forskolin, 10 μM), a cAMP analog (8-bromo-cAMP, 1 mM), or a phosphodiesterase inhibitor (BAY60-7550, 50 μM). Since NO reduces cellular cAMP in the CNT, we asked if NO reduces pendrin abundance by reducing cAMP. With blockade of cGMP-stimulated phosphodiesterase II, NO did not alter pendrin protein abundance. We conclude that NO acts through cAMP to reduce pendrin total protein abundance by enhancing cAMP degradation.

Anemia is a major complication of chronic renal failure. To treat this anemia, prolylhydrox-ylase domain enzyme (PHD) inhibitors as well as erythropoiesis-stimulating agents (ESAs) have been used. Although PHD inhibitors rapidly stimulate erythropoietin (Epo) production, the precise sites of Epo production following the administration of these drugs have not been identified. We developed a novel method for the detection of the Epo protein that employs deglycosylation-coupled Western blotting. With protein deglycosylation, tissue Epo contents can be quantified over an extremely wide range. Using this method, we examined the effects of the PHD inhibitor, Roxadustat (ROX), and severe hypoxia on Epo production in various tissues in rats. We observed that ROX increased Epo mRNA expression in both the kidneys and liver. However, Epo protein was detected in the kidneys but not in the liver. Epo protein was also detected in the salivary glands, spleen, epididymis and ovaries. However, both PHD inhibitors (ROX) and severe hypoxia increased the Epo protein abundance only in the kidneys. These data show that, while Epo is produced in many tissues, PHD inhibitors as well as severe hypoxia regulate Epo production only in the kidneys.

In normal rats, vasopressin and hyperosmolality enhance urea permeability (Purea) in the terminal, but not in the initial IMCD, a process thought to occur through the UT-A1 urea transporter. In the terminal IMCD, UT-A1 is detected as 97 and 117 kDa glycoproteins. However, in the initial IMCD, only the 97 kDa form is detected. During streptozotocin induced diabetes mellitus, UT-A1 protein abundance is increased and the 117 kDa UT-A1 glycoprotein appears in the initial IMCD. We hypothesize that the 117 kDa glycoprotein mediates the vasopressin- and osmolality-induced changes in Purea. Thus in the present study, we measured Purea in in vitro perfused initial IMCDs from diabetic rats by imposing a 5 mM bath-to-lumen urea gradient without any osmotic gradient. Basal Purea was similar in control vs. diabetic rats (3±1 vs. 5±1 x10−5 cm/sec, n=4, p=NS). Vasopressin (10 nM) significantly increased Purea to 16±5 x10−5 cm/sec, n=4, p<0.05 in diabetic, but not in control rats. Forskolin (10 μM, adenylyl cyclase activator) also significantly increased Purea in diabetic rats. In contrast, increasing osmolality to 690 mOsm/kg H2O did not change Purea in diabetic rats. We conclude that initial IMCDs from diabetic rats have vasopressin- and forskolin-, but not hyperosmolality-stimulated Purea. The appearance of vasopressin-stimulated Purea in initial IMCDs correlates with an increase in UT-A1 protein abundance and the appearance of the 117 kDa UT-A1 glycoprotein in this region during diabetes. This suggests that the 117 kDa UT-A1 glycoprotein is necessary for vasopressin-stimulated urea transport.

Thiazide diuretics are used to treat hypertension; however, compensatory processes in the kidney can limit antihypertensive responses to this class of drugs. Here, we evaluated compensatory pathways in SPAK kinase-deficient mice, which are unable to activate the thiazide-sensitive sodium chloride cotransporter NCC (encoded by Slc12a3). Global transcriptional profiling, combined with biochemical, cell biological, and physiological phenotyping, identified the gene expression signature of the response and revealed how it establishes an adaptive physiology. Salt reabsorption pathways were created by the coordinate induction of a multigene transport system, involving solute carriers (encoded by Slc26a4, Slc4a8, and Slc4a9), carbonic anhydrase isoforms, and V-type H⁺-ATPase subunits in pendrin-positive intercalated cells (PP-ICs) and ENaC subunits in principal cells (PCs). A distal nephron remodeling process and induction of jagged 1/NOTCH signaling, which expands the cortical connecting tubule with PCs and replaces acid-secreting α-ICs with PP-ICs, were partly responsible for the compensation. Salt reabsorption was also activated by induction of an α-ketoglutarate (α-KG) paracrine signaling system. Coordinate regulation of a multigene α-KG synthesis and transport pathway resulted in α-KG secretion into pro-urine, as the α-KG-activated GPCR (Oxgr1) increased on the PP-IC apical surface, allowing paracrine delivery of α-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides insight into thiazide diuretic efficacy.

Pendrin is expressed in the apical regions of type B and non-A, non-B intercalated cells, where it mediates Cl− absorption and HCO3− secretion through apical Cl−/HCO3− exchange. Since pendrin is a robust I− transporter, we asked whether pendrin is upregulated with dietary I− restriction and whether it modulates I− balance. Thus I− balance was determined in pendrin null and in wild-type mice. Pendrin abundance was evaluated with immunoblots, immunohistochemistry, and immunogold cytochemistry with morphometric analysis. While pendrin abundance was unchanged when dietary I− intake was varied over the physiological range, I− balance differed in pendrin null and in wild-type mice. Serum I− was lower, while I− excretion was higher in pendrin null relative to wild-type mice, consistent with a role of pendrin in renal I− absorption. Increased H2O intake enhanced differences between wild-type and pendrin null mice in I− balance, suggesting that H2O intake modulates pendrin abundance. Raising water intake from ∼4 to ∼11 ml/day increased the ratio of B cell apical plasma membrane to cytoplasm pendrin label by 75%, although circulating renin, aldosterone, and serum osmolality were unchanged. Further studies asked whether H2O intake modulates pendrin through the action of AVP. We observed that H2O intake modulated pendrin abundance even when circulating vasopressin levels were clamped. We conclude that H2O intake modulates pendrin abundance, although not likely through a direct, type 2 vasopressin receptor-dependent mechanism. As water intake rises, pendrin becomes increasingly critical in the maintenance of Cl− and I− balance.

Urea plays a critical role in the concentration of urine, thereby regulating water balance. Vasopressin, acting through cAMP, stimulates urea transport across rat terminal inner medullary collecting ducts (IMCD) by increasing the phosphorylation and accumulation at the apical plasma membrane of UT-A1. In addition to acting through protein kinase A (PKA), cAMP also activates Epac (exchange protein activated by cAMP). In this study, we tested whether the regulation of urea transport and UT-A1 transporter activity involve Epac in rat IMCD. Functional analysis showed that an Epac activator significantly increased urea permeability in isolated, perfused rat terminal IMCD. Similarly, stimulating Epac by adding forskolin and an inhibitor of PKA significantly increased urea permeability. Incubation of rat IMCD suspensions with the Epac activator significantly increased UT-A1 phosphorylation and its accumulation in the plasma membrane. Furthermore, forskolin-stimulated cAMP significantly increased ERK 1/2 phosphorylation, which was not prevented by inhibiting PKA, indicating that Epac mediated this phosphorylation of ERK 1/2. Inhibition of MEK 1/2 phosphorylation decreased the forskolin-stimulated UT-A1 phosphorylation. Taken together, activation of Epac increases urea transport, accumulation of UT-A1 at the plasma membrane, and UT-A1 phosphorylation, the latter of which is mediated by the MEK–ERK pathway.