Thick Ascending Limb Of Henle Research Articles

Role of claudins in idiopathic hypercalciuria and renal lithiasis.

Paracellular transport in the kidney is mediated by a family of proteins located in the tight junctions called claudins which confers its ionic selectivity. Claudin-2 is highly expressed in the proximal tubule and descending limb of Henle and mediate paracellular reabsorption of sodium and calcium cations. In the thick ascending limb of Henle (TALH) calcium is reabsorbed by a paracellular channel formed by Claudin-16 and-19. Claudin-16 mediates cationic permeability while Claudin-19 increases the cationic selectivity of Claudin-16 by blocking anionic permeability. On the other hand, Claudin 14, that is also located in TALH, inhibits the paracellular permeability of Claudin-16 to calcium. Recent wide genomic association analysis studies have detected four common synonymous variants (genetic polymorphisms of a single nucleotide, SNPs) at the locus of Claudin-14 gene that were significantly associated with the presence of renal lithiasis. Another study of wide genomic association and nephrolithiasis was carried out in the general population but including chromosome X, where claudin-2 gene is located. They detected nine SNPs that had a significant association with renal lithiasis risk. A greater knowledge of the paracellular pathway controlled by claudins and its regulation will allow us to develop future new treatments for idiopathic hypercalciuria and renal lithiasis.

Magnesium and Calcium Homeostasis Depend on KCTD1 Function in the Distal Nephron

SUMMARYMagnesium (Mg2+) homeostasis depends on active transcellular Mg2+ reuptake from urine in distal convoluted tubules (DCTs) via the Mg2+ channel TRPM6, whose activity has been proposed to be regulated by EGF. Calcium (Ca2+) homeostasis depends on paracellular reabsorption in the thick ascending limbs of Henle (TALs). KCTD1 promotes terminal differentiation of TALs/DCTs, but how its deficiency affects urinary Mg2+ and Ca2+ reabsorption is unknown. Here, this study shows that DCT1-specific KCTD1 inactivation leads to hypomagnesemia despite normal TRPM6 levels because of reduced levels of the sodium chloride co-transporter NCC, whereas Mg2+ homeostasis does not depend on EGF. Moreover, KCTD1 deficiency impairs paracellular urinary Ca2+ and Mg2+ reabsorption in TALs because of reduced NKCC2/claudin-16/-19 and increased claudin-14 expression, leading to hypocalcemia and consequently to secondary hyperparathyroidism and progressive metabolic bone disease. Thus, KCTD1 regulates urinary reabsorption of Mg2+ and Ca2+ by inducing expression of NCC in DCTs and NKCC2/claudin-16/-19 in TALs.

Internalization of NKCC2 is impaired in thick ascending limb of Henle in moesin knockout mice.

Moesin is expressed in several types of cells including epithelial and endothelial cells. Several groups reported that moesin plays important roles in the regulation of the cellular motility, and the process of internalization of membrane proteins. However, the physiological roles of moesin in the kidney still remain unclear. Herein, we examined the physiological function of moesin in the kidney using moesin knockout (Msn -/y ) mice. There was no obvious abnormality in the renal morphology of Msn -/y mice. However, we found that Msn -/y mice exhibited mild hyperchloremia, and reduced glomerular filtration rate compared to wild type (WT) mice. Absolute electrolytes excretions of NaCl in Msn -/y mice were not significantly changed compared to WT mice. In the renal medulla, moesin was detected in thick ascending limb of Henle (TALH) as previously reported. To determine the physiological function of moesin in TALH, we examined the expression and subcellular localization of NKCC2 in Msn -/y mice. Interestingly, apical surface expression level, but not total expression of NKCC2 was increased in Msn -/y mice. Subcellular fractionation of renal medulla lysate and internalization assay using tubular suspension showed that the process of NKCC2 endocytosis is impaired. Since the distribution of NKCC2 in lipid raft fractions was decreased in Msn -/y mice, moesin may regulate the NKCC2 distribution to microdomain. These results suggest that moesin regulates the internalization of NKCC2. Furthermore, euhydration by water loading caused hyponatremina in Msn -/y mice, suggesting that dysfunction of moesin is associated with the nephrogenic syndrome of inappropriate antidiuresis (NSIAD).

Parathyroid hormone controls paracellular Ca2+ transport in the thick ascending limb by regulating the tight-junction protein Claudin14

Renal Ca2+ reabsorption is essential for maintaining systemic Ca2+ homeostasis and is tightly regulated through the parathyroid hormone (PTH)/PTHrP receptor (PTH1R) signaling pathway. We investigated the role of PTH1R in the kidney by generating a mouse model with targeted deletion of PTH1R in the thick ascending limb of Henle (TAL) and in distal convoluted tubules (DCTs): Ksp-cre;Pth1rfl/fl Mutant mice exhibited hypercalciuria and had lower serum calcium and markedly increased serum PTH levels. Unexpectedly, proteins involved in transcellular Ca2+ reabsorption in DCTs were not decreased. However, claudin14 (Cldn14), an inhibitory factor of the paracellular Ca2+ transport in the TAL, was significantly increased. Analyses by flow cytometry as well as the use of Cldn14-lacZ knock-in reporter mice confirmed increased Cldn14 expression and promoter activity in the TAL of Ksp-cre;Pth1rfl/fl mice. Moreover, PTH treatment of HEK293 cells stably transfected with CLDN14-GFP, together with PTH1R, induced cytosolic translocation of CLDN14 from the tight junction. Furthermore, mice with high serum PTH levels, regardless of high or low serum calcium, demonstrated that PTH/PTH1R signaling exerts a suppressive effect on Cldn14. We therefore conclude that PTH1R signaling directly and indirectly regulates the paracellular Ca2+ transport pathway by modulating Cldn14 expression in the TAL. Finally, systemic deletion of Cldn14 completely rescued the hypercalciuric and lower serum calcium phenotype in Ksp-cre;Pth1rfl/fl mice, emphasizing the importance of PTH in inhibiting Cldn14. Consequently, suppressing CLDN14 could provide a potential treatment to correct urinary Ca2+ loss, particularly in patients with hypoparathyroidism.

Targeting gene expression to specific cells of kidney tubules in vivo, using adenoviral promoter fragments.

Although techniques for cell-specific gene expression via viral transfer have advanced, many challenges (e.g., viral vector design, transduction of genes into specific target cells) still remain. We investigated a novel, simple methodology for using adenovirus transfer to target specific cells of the kidney tubules for the expression of exogenous proteins. We selected genes encoding sodium-dependent phosphate transporter type 2a (NPT2a) in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) in the collecting duct. The promoters of the three genes were linked to a GFP-coding fragment, the final constructs were then incorporated into an adenovirus vector, and this was then used to generate gene-manipulated viruses. After flushing circulating blood, viruses were directly injected into the renal arteries of rats and were allowed to site-specifically expression in tubule cells, and rats were then euthanized to obtain kidney tissues for immunohistochemistry. Double staining with adenovirus-derived EGFP and endogenous proteins were examined to verify orthotopic expression, i.e. “adenovirus driven NPT2a-EGFP and endogenous NHE3 protein”, “adenovirus driven NKCC2-EGFP and endogenous NKCC2 protein” and “adenovirus driven AQP2-EGFP and endogenous AQP2 protein”. Owing to a lack of finding good working anti-NPT2a antibody, an antibody against a different protein (sodium-hydrogen exchanger 3 or NHE3) that is also specifically expressed in the proximal tubule was used. Kidney structures were well-preserved, and other organ tissues did not show EGFP staining. Our gene transfer method is easier than using genetically engineered animals, and it confers the advantage of allowing the manipulation of gene transfer after birth. This is the first method to successfully target gene expression to specific cells in the kidney tubules. This study may serve as the first step for safe and effective gene therapy in the kidney tubule diseases.

Acute alloxan renal toxicity in the rat initially causes degeneration of thick ascending limbs of Henle.

Alloxan (AL) is a material well-known to induce diabetes. Prior to inducing a prolonged diabetic state, AL causes acute tubulointerstitial nephritis. However, the precise primary target site and mechanism of its nephrotoxicity remain unclear. The objective of this study was to evaluate the morphological characteristics relevant to acute renal toxicity following AL administration. Rats were intravenously treated with AL. Eight hours after AL treatment, aquaporin 1-negative and Na/K pump-positive thick ascending limbs of Henle (TAL) were degenerated in the outer medulla. These tubular lesions progressed from the outer medulla to the cortex. At day 2 after AL treatment, the lesions reached a peak, then both proximal and distal tubules also showed degeneration and necrosis, and tubular regeneration was seen in TAL. Immunohistochemically, damaged tubular epithelium included slightly enlarged prohibitin-positive granules, but it expressed no GLUT2, which is an AL transporter. Ultrastructurally, cytoplasmic and mitochondrial swelling was detected in degenerated cells of TAL. These findings suggest that AL initially causes degeneration of TAL, and induces mitochondrial and cellular damage in the tubular epithelium without involving GLUT2.

Immunolocalization of hyperpolarization-activated cationic HCN1 and HCN3 channels in the rat nephron: regulation of HCN3 by potassium diets.

Hyperpolarization-activated cationic and cyclic nucleotide-gated channels (HCN) comprise four homologous subunits (HCN1-HCN4). HCN channels are found in excitable and non-excitable tissues in mammals. We have previously shown that HCN2 may transport ammonium (NH4 (+)), besides sodium (Na(+)), in the rat distal nephron. In the present work, we identified HCN1 and HCN3 in the proximal tubule (PT) and HCN3 in the thick ascending limb of Henle (TALH) of the rat kidney. Immunoblot assays detected HCN1 (130kDa) and HCN3 (90KDa) and their truncated proteins C-terminal HCN1 (93KDa) and N-terminal HCN3 (65KDa) in enriched plasma membranes from cortex (CX) and outer medulla (OM), as well as in brush-border membrane vesicles. Immunofluorescence assays confirmed apical localization of HCN1 and HCN3 in the PT. HCN3 was also found at the basolateral membrane of TALH. We evaluated chronic changes in mineral dietary on HCN3 protein abundance. Animals were fed with three different diets: sodium-deficient (SD) diet, potassium-deficient (KD) diet, and high-potassium (HK) diet. Up-regulation of HCN3 was observed in OM by KD and in CX and OM by HK; the opposite effect occurred with the N-terminal truncated HCN3 in CX (KD) and OM (HK). SD diet did not produce any change. Since HCN channels activate with membrane hyperpolarization, our results suggest that HCN channels may play a role in the Na(+)-K(+)-ATPase activity, contributing to Na(+), K(+), and acid-base homeostasis in the rat kidney.

Localizations of Na(+)-D-glucose cotransporters SGLT1 and SGLT2 in human kidney and of SGLT1 in human small intestine, liver, lung, and heart.

Novel affinity-purified antibodies against human SGLT1 (hSGLT1) and SGLT2 (hSGLT2) were used to localize hSGLT2 in human kidney and hSGLT1 in human kidney, small intestine, liver, lung, and heart. The renal locations of both transporters largely resembled those in rats and mice; hSGLT2 and SGLT1 were localized to the brush border membrane (BBM) of proximal tubule S1/S2 and S3 segments, respectively. Different to rodents, the renal expression of hSGLT1 was absent in thick ascending limb of Henle (TALH) and macula densa, and the expression of both hSGLTs was sex-independent. In small intestinal enterocytes, hSGLT1 was localized to the BBM and subapical vesicles. Performing double labeling with glucagon-like peptide 1 (GLP-1) or glucose-dependent insulinotropic peptide (GIP), hSGLT1 was localized to GLP-1-secreting L cells and GIP-secreting K cells as has been shown in mice. In liver, hSGLT1 was localized to biliary duct cells as has been shown in rats. In lung, hSGLT1 was localized to alveolar epithelial type 2 cells and to bronchiolar Clara cells. Expression of hSGLT1 in Clara cells was verified by double labeling with the Clara cell secretory protein CC10. Double labeling of human heart with aquaporin 1 immunolocalized the hSGLT1 protein in heart capillaries rather than in previously assumed myocyte sarcolemma. The newly identified locations of hSGLT1 implicate several extra renal functions of this transporter, such as fluid absorption in the lung, energy supply to Clara cells, regulation of enteroendocrine cells secretion, and release of glucose from heart capillaries. These functions may be blocked by reversible SGLT1 inhibitors which are under development.

Proteolytic cleavage of SPAK by a kidney protease (893.36)

The Ste20 kinase SPAK is a major regulator of sodium reabsorption in the distal nephron. Recently, SPAK fragments have been detected in the mouse kidney, and particularly in the thick ascending limb of Henle (TAL). These truncated fragments have been demonstrated to inhibit the activity of the Na+‐K+‐2Cl‐ cotransporter, NKCC2, and thereby decrease sodium transport across the TAL. Here we show that mouse kidney lysates possess protease activity that leads to the specific cleavage of a SPAK, as we observed a precise and reproducible cleavage pattern with a GST‐SPAK fusion protein, but not a GST‐OSR1 fusion protein. This activity is not observed with brain, liver, or spleen lysates. The activity is insensitive to PMSF, leupeptin, aprotinin, and pepstatin, but inhibited by DTT and EDTA. In an effort to identify the protease, we performed ion exchange chromatography. The proteolytic activity was detected in an elution fraction corresponding to 250 mM NaCl. The fraction was subjected to MS/MS analysis and 1119 proteins were identified at 95% threshold. Among these proteins, we identified 44 peptidases and proteases. Gel permeation chromatography experiments determined that the protease is larger than 100 kDa. Effort will be made to determine the identity of the protein involved in the cleavage of SPAK and to identify the sites of cleavage.Grant Funding Source: RO1 DK0935010

Increased expression of renal TRPM6 compensates for Mg(2+) wasting during furosemide treatment.

BackgroundFurosemide is a loop diuretic, which blocks the Na+, K+, 2Cl− cotransporter (NKCC2) in the thick ascending limb of Henle (TAL). By diminishing sodium (Na+) reabsorption, loop diuretics reduce the lumen-positive transepithelial voltage and consequently diminish paracellular transport of magnesium (Mg2+) and calcium (Ca2+) in TAL. Indeed, furosemide promotes urinary Mg2+ excretion; however, it is unclear whether this leads, especially during prolonged treatment, to hypomagnesaemia. The aim of the present study was, therefore, to determine the effect of chronic furosemide application on renal Mg2+ handling in mice.MethodsTwo groups of 10 mice received an osmotic minipump subcutaneously for 7 days with vehicle or 30 mg/kg/day furosemide. Serum and urine electrolyte concentrations were determined. Next, renal mRNA levels of the epithelial Mg2+ channel (TRPM6), the Na+, Cl− cotransporter (NCC), the epithelial Ca2+ channel (TRPV5), the cytosolic Ca2+-binding protein calbindin-D28K, as well parvalbumin (PV), claudin-7 (CLDN7) and claudin-8 (CLDN8), the epithelial Na+ channel (ENaC) and the Na+–H+ exchanger 3 (NHE3) were determined by real-time quantitative polymerase chain reaction. Renal protein levels of NCC, TRPV5, calbindin-D28K and ENaC were also measured using semi-quantitative immunohistochemistry and immunoblotting.ResultsThe mice chronically treated with 30 mg/kg/day furosemide displayed a significant polyuria (2.1 ± 0.3 and 1.3 ± 0.2 mL/24 h, furosemide versus control respectively, P < 0.05). Furosemide treatment resulted in increased serum concentrations of Na+ [158 ± 3 (treated) and 147 ± 1 mmol/L (control), P < 0.01], whereas serum K+, Ca2+ and Mg2+ values were not significantly altered in mice treated with furosemide. Urinary excretion of Na+, K+, Ca2+ and Mg2+ was not affected by chronic furosemide treatment. The present study shows specific renal upregulation of TRPM6, NCC, TRPV5 and calbindin-D28K.ConclusionsDuring chronic furosemide treatment, enhanced active reabsorption of Mg2+ via the epithelial channel TRPM6 in DCT compensates for the reduced reabsorption of Mg2+ in TAL.

Lecture

The physiology of paracellular permeation of ions and solutes in the kidney is pivotally important but poorly understood. Claudins are the key components of the paracellular pathway. Defects in claudin function result in a broad range of renal diseases, including hypomagnesemia, hypercalciuria and nephrolithiasis. This review describes recent findings on the physiological function of claudins underlying paracellular transport mechanisms with a focus on renal Ca2+ handling. We have uncovered a molecular mechanism underlying paracellular Ca2+ transport in the thick ascending limb of Henle (TAL) that involves the functional interplay of three important claudin genes: claudin-14, -16 and -19, all of which are associated with human kidney diseases with hypercalciuria, nephrolithiasis and bone mineral loss. The Ca2+ sensing receptor (CaSR) signaling in the kidney has long been a mystery. By analyzing small non-coding RNA molecules in the kidney, we have uncovered a novel microRNA based signaling pathway downstream of CaSR that directly regulates claudin-14 gene expression and establishes the claudin-14 molecule as a key regulator for renal Ca2+ homeostasis. The molecular cascade of CaSR-microRNAs-claudins forms a regulatory loop to maintain proper Ca2+ homeostasis in the kidney.

Determinations of Renal Cortical and Medullary Oxygenation Using Blood Oxygen Level-Dependent Magnetic Resonance Imaging and Selective Diuretics

This study was undertaken to test the hypothesis that blood O2 level-dependent magnetic resonance imaging (BOLD MRI) can detect changes in cortical proximal tubule (PT) and medullary thick ascending limb of Henle (TAL) oxygenation consequent to successive administration of furosemide and acetazolamide (Az). Assessment of PT and TAL function could be useful to monitor renal disease states in vivo. Therefore, the adjunct use of diuretics that inhibit Na reabsorption selectively in PT and TAL, Az and furosemide, respectively, may help discern tubular function by using BOLD MRI to detect changes in tissue oxygenation. BOLD MRI signal R2* (inversely related to oxygenation) and tissue oxygenation with intrarenal O2 probes were measured in pigs that received either furosemide (0.05 mg/kg) or Az (15 mg/kg) alone, Az sequentially after furosemide (n = 6 each, 15-minute intervals), or only saline vehicle (n = 3). R2* decreased in the cortex of Az-treated and medulla of furosemide-treated kidneys, corresponding to an increase in their tissue O2 assessed with probes. However, BOLD MRI also showed decreased cortical R2* following furosemide that was additive to the Az-induced decrease. Az administration, both alone and after furosemide, also decreased renal blood flow (-26% ± 3.5% and -29.2% ± 3%, respectively, P < 0.01). These results suggest that an increase in medullary and cortical tissue O2 elicited by selective diuretics is detectable by BOLD MRI, but may be complicated by hemodynamic effects of the drugs. Therefore, the BOLD MRI signal may reflect functional changes additional to oxygenation, and needs to be interpreted cautiously.

P.2.b.023 Rodent magnesium-deficiency models for depression and their relationship to NMDA receptor/Nitric Oxide pathways

Colocalization of demethylating enzymes and NOS and functional effects of methylarginines in rat kidney. NG-monomethylarginine (L-NMA) and asymmetric NG, NG-dimethylarginines (ADMA) are endogenous inhibitors of cellular L-arginine uptake and/or nitric oxide (NO) synthesis that are implicated in renal parenchymal and Dahl salt-sensitive hypertension. Since the L-arginine:(L-NMA+ADMA) ratio determines NO synthase (NOS) activity, we compared the immunohistochemical distribution of NOS with NG, NG-dimethylarginine dimethylaminohydrolase (DDAH), which inactivates dimethylarginines (DMA) and L-NMA by hydrolysis to L-citrulline. Neuronal NOS (nNOS) was expressed predominantly in tubular epithelial cells of macula densa (MD), endothelial NOS (eNOS) in vascular endothelial cells (EC), and inducible NOS (iNOS) quite widely in tubular epithelium, including proximal tubules (PT), thick ascending limbs of Henle (TAL), distal convoluted tubule and intercalated cells (IC) of the collecting duct. Immunostaining for DDAH was present in PT, TAL, MD, and IC, and was also present in the glomerulus, Bowman's capsule, and endothelium of blood vessels. DDAH was detected in small vesicles of TAL and PT by electron microscopic (EM) immunocytochemistry. To study the effects of methylarginines on tubuloglomerular feedback (TGF) response, vehicle or methylarginines (10−3 M) were added to artificial tubular fluid (ATF) perfused orthogradely from the late PT at 40 nl. min−1 while assessing changes in glomerular capillary pressure from proximal stop flow pressure (PSF). Whereas the maximal TGF responses were unchanged by vehicle (ΔTGF 0 ± 0%) or symmetric DMA (SDMA; +1 ± 2%, NS), they were enhanced by L-NMA (+22 ± 4%, P < 0.001) and asymmetric DMA (ADMA; +28 ± 3%, P < 0.001). Since L-arginine transport can regulate renal epithelial NO generation, methylarginines (10−3 M) or vehicle were co-perfused orthogradely with [3H]-L-arginine from the late PT and collected at the early distal tubule to study arginine uptake from the perfused loop of Henle. All methylarginines reduced fractional loop [3H] absorption significantly (P < 0.001; vehicle, 84 ± 6; ADMA, 49 ± 6; SDMA, 56 ± 6; L-NMA, 41 ± 6%). In conclusion, sites of DDAH expression in the vasculature or nephron are all sites of expression of an isoform of NOS. L-NMA, ADMA, and SDMA all inhibit renal tubular L-arginine uptake, whereas L-NMA and ADMA, but not SDMA, enhance TGF responses. Therefore, DDAH may regulate the cellular L-arginine: methylarginine levels in specific renal cells, thereby governing cell-specific L-arginine uptake and NO generation in renal tubular epithelium.

A possible catalytic role for NH4+in Na+reabsorption across the thick ascending limb

A possible catalytic role for NH₄⁺in Na⁺reabsorption across the thick ascending limb

Renal Function in Mice with Targeted Disruption of the A Isoform of the Na-K-2Cl Co-Transporter

Three different full-length splice isoforms of the Na-K-2Cl co-transporter (NKCC2/BSC1) are expressed along the thick ascending limb of Henle (TAL), designated NKCC2A, NKCC2B, and NKCC2F. NKCC2F is expressed in the medullary, NKCC2B mainly in the cortical, and NKCC2A in medullary and cortical portions of the TAL. NKCC2B and NKCC2A were shown to be coexpressed in the macula densa (MD) segment of the mouse TAL. The functional consequences of the existence of three different isoforms of NKCC2 are unclear. For studying the specific role of NKCC2A in kidney function, NKCC2A-/- mice were generated by homologous recombination. NKCC2A-/- mice were viable and showed no gross abnormalities. Ambient urine osmolarity was reduced significantly in NKCC2A-/- compared with wild-type mice, but water deprivation elevated urine osmolarity to similar levels in both genotypes. Baseline plasma renin concentration and the effects of a high- and a low-salt diet on plasma renin concentration were similar in NKCC2A+/+ and -/- mice. However, suppression of renin secretion by acute intravenous saline loading (5% of body weight), a measure of MD-dependent inhibition of renin secretion, was reduced markedly in NKCC2A-/- mice compared with wild-type mice. Cl and water absorption along microperfused loops of Henle of NKCC2A-/- mice were unchanged at normal flow rates but significantly reduced at supranormal flow. Tubuloglomerular feedback function curve as determined by stop flow pressure measurements was left-shifted in NKCC2A-/- compared with wild-type mice, with maximum responses being significantly diminished. In summary, NKCC2A activity seems to be required for MD salt sensing in the high Cl concentration range. Coexpression of both high- and low-affinity isoforms of NKCC2 may permit transport and Cl-dependent tubuloglomerular feedback regulation to occur over a wider Cl concentration range.

Activation of NAD(P)H oxidase by outward movements of H+ ions in renal medullary thick ascending limb of Henle

The present study was designed to test the hypothesis that the production of superoxide (O(2)-* by NAD(P)H oxidase is coupled to tubular metabolic activity through ionic activation mediated by H(+) movement across cell membrane. Using dual fluorescent microscopic imaging analysis, intracellular O(2)-* levels and pH (pH(i)) in renal medullary thick ascending limb of Henle (TALH) cells were simultaneously measured. It was found that intracellular O(2)-* levels in these cells were increased in parallel to the elevation of pH(i) by outflow of H(+) induced via NH(4)Cl loading followed by rapid removal. This increase in intracellular O(2)-* levels was substantially blocked by an inhibitor of Na(+)/H(+) exchanger, methylisobutyl-amiloride (MIA; 100 microM), a chemical SOD mimetic, Tiron (1 mM) or an inhibitor of NAD(P)H oxidase, diphenylene iodonium (DPI; 100 microM). In additional groups of TALHs, a proton ionophore, carbonylcyanide m-chlorophenylhydrazone (10 microM) was used to produce H(+) conductance, leading to H(+) flux across cell membrane depending on extracellular pH. The efflux of H(+) increased both pH(i) and intracellular O(2)-* levels, but the influx of H(+) did not increase intracellular O(2)-* levels. The H(+) efflux-induced increase in intracellular O(2)-* levels was completely blocked by DPI and another NAD(P)H oxidase inhibitor, apocynin (100 microM). In in invo experiments, renal medullary infusion of MIA (100 microM) was found to significantly decrease the concentrations of H(2)O(2) in the renal medullary interstitium. These results suggest that it is the outward movements of H(+) ions that activates NAD(P)H oxidase to produce O(2)-* in TALH cells. This H(+) outflow-associated activation of NAD(P)H oxidase importantly contributes to tissue levels of reactive oxygen species in the renal medulla.

Polyductin, the PKHD1 gene product, comprises isoforms expressed in plasma membrane, primary cilium, and cytoplasm

PKHD1, the autosomal-recessive polycystic kidney disease (ARPKD) gene, encodes multiple alternatively spliced transcripts predicted to generate membrane-bound and secreted proteins. The longest open reading frame encodes polyductin (fibrocystin), a putative 4074 amino acid protein with a single transmembrane domain and an intracellular C-terminus. To characterize the PKHD1 products and their expression profile, we raised polyclonal antibodies against different portions of polyductin and analyzed different organs using various methods. Western blot analyses demonstrated specific bands of >440 kD in human adult kidney, liver, and pancreas and approximately 230 kD in kidney and liver, predominantly observed in membrane fractions. The >440-kD putative membrane protein was immunoprecipitated from kidney and subsequently detected by Western blotting using two distinct antisera. An additional product of approximately 140 kD was specifically recognized by affinity-purified antisera predominantly in soluble fractions. Immunohistochemistry studies revealed specific staining in cortical and medullary collecting ducts and thick ascending limbs of Henle (TALH). Serial sections were stained with antibodies against aquaporin-2 and Tamm-Horsfall protein to confirm the nephron segment localization. Positive staining was also detected in biliary and pancreatic duct epithelia. Analyses of mouse developing tissues showed specific staining in the ureteric bud branches, intra- and extrahepatic biliary ducts, pancreatic ducts, and salivary glands. Immunofluorescence studies in inner medullary collecting duct cultured cells and immunoelectron microscopy analysis of medullary collecting ducts demonstrated that the protein localizes to the primary cilium. Positive signal was also detected in the apical membrane and in cytoplasm. The results indicate that polyductin is part of the group of polycystic kidney disease (PKD)-related proteins expressed in primary apical cilia. Our data also suggest that, in addition to its likely involvement in cilia function, polyductin probably serves in other subcellular functional roles. The detection of three different products using two antisera, with evidence for distinct subcellular localizations, suggests that PKHD1 encodes membrane-bound and soluble isoforms.

Calcium and salinity sensing by the thick ascending limb: A journey from mammals to fish and back again

The roles of the CaSR in endocrine, epithelial, CNS, and other cells have been reviewed previously [17-19, 20, 27-30, 31-33]. This brief review focuses on the roles of the CaSR in the thick ascending limb of Henle (TAL), and is written in honor of my mentor and long-term friend and colleague, Thomas E. Andreoli, on the occasion of his retirement. My early studies of TAL function with Tom Andreoli were the inspiration for this work.

Renal Sodium Handling for Body Fluid Maintenance and Blood Pressure Regulation

AbstractFor Abstract see ChemInform Abstract in Full Text.

Differential Expression of Calcineurin A Isoforms in the Diabetic Kidney

Calcineurin is an important signaling molecule in mesangial cells in vitro and is involved in some manifestations of diabetic nephropathy in vivo. However, calcineurin acts in a cell-specific and tissue-specific manner in the kidney, and mechanisms of specificity are unknown. Three closely related isoforms of the calcineurin A (CnA) subunit are expressed in a tissue-specific manner. This study was undertaken to determine if specificity of calcineurin action is linked to regulation of CnA isoforms in the diabetic kidney. After induction of diabetes with streptozotocin, expression of all three CnA isoforms rapidly increased, primarily in the thick ascending limb of Henle (TAL). After prolonged diabetes, increase specifically of the alpha isoform was observed in collecting ducts (CD) and in endothelial cells of glomeruli. Aquaporin 2 (AQP2), a putative substrate of calcineurin phosphatase in the kidney, is also involved in diabetic nephropathy. Co-localization of CnA isoforms with AQP2 revealed that CnA-alpha is the predominant isoform that associates with AQP2 in the diabetic kidney. Furthermore, inhibition of calcineurin with cyclosporin A (CsA) alters AQP2 localization and phosphorylation in principal cells of CD. Alterations in subcellular localization of AQP2 were parallel with CnA-alpha. Similarly, CsA treatment results in a further increase in urine output compared with diabetes alone, suggesting a functional consequence of inhibiting calcineurin-mediated regulation of AQP2. In conclusion, all three isoforms of CnA are upregulated in the diabetic kidney. Increased expression of CnA-alpha, in particular, is observed in glomeruli and CD and participates in regulation of AQP2 expression, phosphorylation, and function.