Descending thin limb of the intermediate loop expresses both aquaporin 1 and urea transporter A2 in the mouse kidney.

A new intermediate type of Henle's loop has been reported that it extends into the inner medulla and turns within the first millimeter beyond the outer medulla. This study aimed to identify the descending thin limb (DTL) of the intermediate loop in the adult C57Bl/6 mouse kidney using aquaporin 1 (AQP1) and urea transporter A2 (UT-A2) antibodies. In the upper part of the inner stripe of the outer medulla (ISOM), AQP1 was expressed strongly in the DTL with type II epithelium of the long loop, but not in type I epithelium of the short loop. The DTL of the intermediate loop exhibited weak AQP1 immunoreactivity. UT-A2 immunoreactivity was not observed in the upper part of any DTL type. AQP1 expression was similar in the upper and middle parts of the ISOM. UT-A2 expression was variable, being expressed strongly in the DTL with type I epithelium of the short loop, but not in type II epithelium of the long loop. In the innermost part of the ISOM, AQP1 was expressed only in type III epithelium of the long loop. UT-A2-positive and UT-A2-negative cells were intermingled in type I epithelium of the intermediate loop, but were not observed in type III epithelium of the long loop. UT-A2-positive DTLs of the intermediate loop extended into the UT-A2/AQP1-negative type I epithelium in the initial part of the inner medulla. These results demonstrate that the DTL of the intermediate loop is composed of type I epithelium and expresses both AQP1 and UT-A2. The functional role of the DTL of the intermediate loop may be distinct from the short or long loops.

Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption.

Epithelia permit selective and regulated flux from apical to basolateral surfaces by transcellular passage through cells or paracellular flux between cells. Tight junctions constitute the barrier to paracellular conductance; however, little is known about the specific molecules that mediate paracellular permeabilities. Renal magnesium ion (Mg2+) resorption occurs predominantly through a paracellular conductance in the thick ascending limb of Henle (TAL). Here, positional cloning has identified a human gene, paracellin-1 (PCLN-1), mutations in which cause renal Mg2+ wasting. PCLN-1 is located in tight junctions of the TAL and is related to the claudin family of tight junction proteins. These findings provide insight into Mg2+ homeostasis, demonstrate the role of a tight junction protein in human disease, and identify an essential component of a selective paracellular conductance.

[Calcium oxalate stone formation. New pathogenetic aspects of an old disease]. / Kalziumoxalatsteinbildung. Neue pathogenetische Aspekte einer alten Erkrankung.

Calcium oxalate (CaOx) urolithiasis is a very common disorder. Surprisingly, the pathogenetic mechanisms leading to CaOx stone formation have been largely unknown so far. The long-accepted simple explanation by an exceeding of the solubility product of lithogenic substances in the urine cannot sufficiently describe the complex processes. Deviating from the hypothesis that proclaims that the initial crystal deposition takes place in the lumens of renal tubules, new insights suggest a primary plaque formation in the interstitial space of the renal papilla. Initially, calcium phosphate (CaPh) crystals and organic matrix are deposited along the basement membranes of the thin loops of Henle and extend further in the interstitial space to the urothelium, constituting the so-called Randall's plaques that can be regularly found during endoscopy of CaOx-stone-forming patients. These CaPh crystals seem to be the origin for the development of future CaOx stones, which form by the attachment of further matrix molecules and CaOx from the urine to the plaque. The driving forces, the exact pathogenetic mechanisms, and the involved matrix molecules remain largely unknown. Possibly, completely different pathomechanisms lead to the common clinical diagnosis of"CaOx stone former."

High frequency and wide range of human kidney papillary crystalline plugs.

Most of kidney stones are supposed to originate from Randall's plaque at the tip of the papilla or from papillary tubular plugs. Nevertheless, the frequency and the composition of crystalline plugs remain only partly described. The objective was to assess the frequency, the composition and the topography of papillary plugs in human kidneys. A total of 76 papillae from 25 kidneys removed for cancer and without stones were analysed by immunohistochemistry combined with Yasue staining, field emission-scanning electron microscopy and Fourier transformed infrared micro-spectroscopy. Papillary tubular plugs have been observed by Yasue staining in 23/25 patients (92%) and 52/76 papillae (68%). Most of these plugs were made of calcium phosphate, mainly carbonated apatite and amorphous calcium phosphate, and rarely octacalcium phosphate pentahydrate. Calcium and magnesium phosphate (whitlockite) have also been observed. Based upon immunostaining coupled to Yasue coloration, most of calcium phosphate plugs were located in the deepest part of the loop of Henle. Calcium oxalate monohydrate and dihydrate tubular plugs were less frequent and stood in collecting ducts. At last, we observed calcium phosphate plugs deforming and sometimes breaking adjacent collecting ducts. Papillary tubular plugging, which may be considered as a potential first step toward kidney stone formation, is a very frequent setting, even in kidneys of non-stone formers. The variety in their composition and the distal precipitation of calcium oxalate suggest that plugs may occur in various conditions of urine supersaturation. Plugs were sometimes associated with collecting duct deformation.

VSTM2A Overexpression Is a Sensitive and Specific Biomarker for Mucinous Tubular and Spindle Cell Carcinoma (MTSCC) of the Kidney.

Our recent study revealed recurrent chromosomal losses and somatic mutations of genes in the Hippo pathway in mucinous tubular and spindle cell carcinoma (MTSCC). Here, we performed an integrative analysis of 907 renal cell carcinoma (RCC) samples (combined from The Cancer Genome Atlas and in-house studies) and the Knepper data set of microdissected rat nephrons. We identified VSTM2A and IRX5 as novel cancer-specific and lineage-specific biomarkers in MTSCC. We then assessed their expression by RNA in situ hybridization (ISH) in 113 tumors, including 33 MTSCC, 40 type 1 papillary RCC, 8 type 2 papillary RCC, 2 unclassified RCC, 15 clear cell RCC, and 15 chromophobe RCC. Sensitivity and specificity were calculated as the area under the receiver operating characteristics curve (AUC). All MTSCC tumors demonstrated moderate to high expression of VSTM2A (mean ISH score=255). VSTM2A gene expression assessed by RNA sequencing strongly correlated with VSTM2A ISH score (r(2)=0.81, P=0.00016). The majority of non-MTSCC tumors demonstrated negative or low expression of VSTM2A. IRX5, nominated as a lineage-specific biomarker, showed moderate to high expression in MTSCC tumors (mean ISH score=140). IRX5 gene expression assessed by RNA sequencing strongly correlated with IRX5 ISH score (r(2)=0.69, P=0.00291). VSTM2A (AUC: 99.2%) demonstrated better diagnostic efficacy than IRX5 (AUC: 87.5%), and may thus serve as a potential diagnostic marker to distinguish tumors with overlapping histology. Furthermore, our results suggest MTSCC displays an overlapping phenotypic expression pattern with the loop of Henle region of normal nephrons.

Expression of the Na-K-2Cl cotransporter by macula densa and thick ascending limb cells of rat and rabbit nephron.

Sodium and chloride transport by the macula densa and thick ascending limb of Henle's loop participates importantly in extracellular fluid volume homeostasis, urinary concentration and dilution, control of glomerular filtration, and control of renal hemodynamics. Transepithelial Na and Cl transport across the apical membrane of thick ascending limb (TALH) cells is mediated predominantly by a loop diuretic sensitive Na-K-2Cl cotransport pathway. The corresponding transport protein has recently been cloned. Functional studies suggest that the cotransporter is expressed by macula densa cells as well as by TALH cells. The current studies were designed to identify sites of Na-K-2Cl cotransporter expression along distal nephron in rabbit and rat. Non-isotopic high-resolution in situ hybridization, using an antisense probe for the apical form of the Na-K-2Cl cotransporter identified expression throughout the TALH, from the junction between inner and outer medulla to the transition to distal convoluted tubule. Expression by macula densa cells was confirmed by colocalization using markers specific for macula densa cells. First, Na-K-2Cl cotransporter mRNA was detected in macula densa cells that did not stain with anti-Tamm-Horsfall protein antibodies. Second, Na-K-2Cl cotransporter mRNA was detected in macula densa cells that show positive NADPH-diaphorase reaction, indicating high levels of constitutive nitric oxide synthase activity. In rat, levels of Na-K-2Cl cotransporter mRNA expression were similar in TALH and macula densa cells. In rabbit, expression levels were higher in macula densa cells than in surrounding TALH cells. The present data provide morphological support for a previously established functional concept that Na-K-2Cl cotransport at the TALH is accomplished by the expression of a well-defined cotransporter. At the macula densa, this transporter may establish a crucial link between tubular salt load and glomerular vascular regulation.

Localization and functional characterization of rat kidney-specific chloride channel, ClC-K1.

To investigate the physiological role of a kidney-specific chloride channel (ClC-K1), we sought to determine its exact localization by immunohistochemistry and its functional regulation using Xenopus oocyte expression system. The antiserum specifically recognized a 70-kD protein in SDS-PAGE of membrane protein from rat inner medulla and an in vitro translated ClC-K1 protein. Immunohistochemistry revealed that ClC-K1 was exclusively localized to the thin limb of Henle's loop in rat inner medulla. In comparison with the immunostaining with anti-aquaporin-CHIP antibody that only stains the descending thin limb of Henle's loop (tDL), ClC-K1 was found to be localized only in the ascending limb (tAL) which has the highest chloride permeability among nephron segments. Immunoelectron microscopy confirmed that the staining of ClC-K1 in tAL was observed in the region of both apical and basolateral plasma membranes. Expressed chloride current in Xenopus oocytes by ClC-K1 cRNA was regulated by extracellular pH and extracellular calcium. Furosemide inhibited the expressed current (Ki = 100 microM), whereas N-ethyl-maleimide stimulated the current. These functional characteristics were consistent with the in vitro perfusion studies of chloride transport in tAL. The localization and the functional characteristics described here indicate that ClC-K1 is responsible for the transepithelial chloride transport in tAL.

Hyposmolality stimulates apical membrane Na(+)/H(+) exchange and HCO(3)(-) absorption in renal thick ascending limb.

The regulation of epithelial Na(+)/H(+) exchangers (NHEs) by hyposmolality is poorly understood. In the renal medullary thick ascending limb (MTAL), transepithelial bicarbonate (HCO(3)(-)) absorption is mediated by apical membrane Na(+)/H(+) exchange, attributable to NHE3. In the present study we examined the effects of hyposmolality on apical Na(+)/H(+) exchange activity and HCO(3)(-) absorption in the MTAL of the rat. In MTAL perfused in vitro with 25 mM HCO(3)(-) solutions, decreasing osmolality in the lumen and bath by removal of either mannitol or sodium chloride significantly increased HCO(3)(-) absorption. The responses to lumen addition of the inhibitors ethylisopropyl amiloride, amiloride, or HOE 694 are consistent with hyposmotic stimulation of apical NHE3 activity and provide no evidence for a role for apical NHE2 in HCO(3)(-) absorption. Hyposmolality increased apical Na(+)/H(+) exchange activity over the pH(i) range 6.5-7.5 due to an increase in V(max). Pretreatment with either tyrosine kinase inhibitors or with the tyrosine phosphatase inhibitor molybdate completely blocked stimulation of HCO(3)(-) absorption by hyposmolality. These results demonstrate that hyposmolality increases HCO(3)(-) absorption in the MTAL through a novel stimulation of apical membrane Na(+)/H(+) exchange and provide the first evidence that NHE3 is regulated by hyposmotic stress. Stimulation of apical Na(+)/H(+) exchange activity in renal cells by a decrease in osmolality may contribute to such pathophysiological processes as urine acidification by diuretics, diuretic resistance, and renal sodium retention in edematous states.

A molecular map of G protein alpha chains in microdissected rat nephron segments.

Membrane-associated guanine nucleotide binding proteins regulate many receptor-mediated signals. Heterogeneity of biochemical and functional properties in nephron segments could be due to differences in G protein expression. To ascertain whether such heterogeneity of G proteins is present in various nephron segments, this study examines the distribution and relative abundance of G protein alpha chains in microdissected medullary thick ascending limb, cortical collecting tubules, outer medullary collecting tubules, proximal inner medullary tubules, and distal inner medullary tubules. Reverse transcription and polymerase chain reactions were employed using oligonucleotides encoding highly conserved regions of all known alpha chains. The cDNA was sequenced for alpha chain identification. The alpha i2 versus alpha s distribution was different in the outer medullary collecting tubules, when compared with the medullary thick ascending limb (P < 0.001) or the cortical collecting tubule, the proximal inner medullary tubules, and the distal inner medullary tubules (P < 0.05). These latter four segments did not significantly differ from each other. A similar analysis was applied to the frequently used line of kidney cells, LLC-PK1, whose exact cellular origin remains unclear. Interestingly, we detected both alpha i2 and alpha i3, while only alpha i2 was detected in the rat distal nephron. No alpha o or alpha z reverse transcription PCR products were detected. In contrast alpha 11 and alpha 14 members of the more recently described alpha q family were detected in the outer medullary collecting tubules and the proximal inner medullary tubules, respectively. We conclude that the majority of nephron segments have a relatively constant distribution of G protein alpha chains.

A Na+- and Cl- -activated K+ channel in the thick ascending limb of mouse kidney.

This study investigates the presence and properties of Na+-activated K+ (K(Na)) channels in epithelial renal cells. Using real-time PCR on mouse microdissected nephron segments, we show that Slo2.2 mRNA, which encodes for the K(Na) channels of excitable cells, is expressed in the medullary and cortical thick ascending limbs of Henle's loop, but not in the other parts of the nephron. Patch-clamp analysis revealed the presence of a high conductance K+ channel in the basolateral membrane of both the medullary and cortical thick ascending limbs. This channel was highly K+ selective (P(K)/P(Na) approximately 20), its conductance ranged from 140 to 180 pS with subconductance levels, and its current/voltage relationship displayed intermediate, Na+-dependent, inward rectification. Internal Na+ and Cl- activated the channel with 50% effective concentrations (EC50) and Hill coefficients (nH) of 30 +/- 1 mM and 3.9 +/- 0.5 for internal Na+, and 35 +/- 10 mM and 1.3 +/- 0.25 for internal Cl-. Channel activity was unaltered by internal ATP (2 mM) and by internal pH, but clearly decreased when internal free Ca2+ concentration increased. This is the first demonstration of the presence in the epithelial cell membrane of a functional, Na+-activated, large-conductance K+ channel that closely resembles native K(Na) channels of excitable cells. This Slo2.2 type, Na+- and Cl--activated K+ channel is primarily located in the thick ascending limb, a major renal site of transcellular NaCl reabsorption.

Accumulation of indoxyl sulfate in OAT1/3-positive tubular cells in kidneys of patients with chronic renal failure.

Indoxyl sulfate shows nephrotoxicity and is a stimulating factor for progression of chronic renal failure (CRF). Indoxyl sulfate is taken up by renal proximal tubular cells through organic anion transporters 1 and 3 (OAT1/3), and is accumulated in the renal proximal tubular cells of uremic rats. To determine whether indoxyl sulfate is accumulated in human OAT1/3 (hOAT1/3)-positive renal proximal tubular cells, localization of indoxyl sulfate and hOAT1/3 in the kidneys of CRF patients was determined by immunohistochemistry. Kidney samples were obtained by autopsy from 9 CRF patients (mean serum creatinine 4.7 mg/dL, ranging from 2.0 to 14.5 mg/dL) and 9 patients with non-kidney disease (mean serum creatinine 0.6 mg/dL, ranging from 0.4 to 0.9 mg/dL). Immunohistochemistry was performed using antibodies against indoxyl sulfate, hOAT1, and hOAT3. Indoxyl sulfate was localized in the hOAT1- and hOAT3-positive renal tubular cells in the kidneys of CRF patients. The indoxyl sulfate-positive area in the kidneys was markedly increased in the kidneys of CRF patients compared with patients with non-kidney disease. The indoxyl sulfate-positive area was positively correlated with serum creatinine. In conclusion, in CRF patients, indoxyl sulfate is accumulated in the tubular cells with hOAT1 and/or hOAT3 localized at the basolateral membrane. The extent of indoxyl sulfate accumulation in the kidneys is more prominent in those patients with more severe CRF.

Restricted localization of claudin-16 at the tight junction in the thick ascending limb of Henle's loop together with claudins 3, 4, and 10 in bovine nephrons.

Claudin-16 is one of the tight junction protein claudins and has been shown to contribute to reabsorption of divalent cations in the human kidney. In cattle, total deficiency of claudin-16 causes severe renal tubular dysplasia without aberrant metabolic changes of divalent cations, suggesting that bovine claudin-16 has some roles in renal tubule formation and paracellular transport that are somewhat different from those expected from the pathology of human disease. As the first step to clarify these roles, we examined the expression and distribution of claudin-16 and several other major claudin subtypes, claudins 1-4 and 10, in bovine renal tubular segments by immunofluorescence microscopy. Claudin-16 was exclusively distributed to the tight junction in the tubular segment positive for Tamm-Horsfall glycoprotein, the thick ascending limb (TAL) of Henle's loop, and was found colocalized with claudins 3, 4, and 10. This study also demonstrates that bovine kidneys possess segment-specific expression patterns for claudins 2-4 and 10 that are different from those reported for mice. Particularly, distribution of claudin-4 in the TAL and distal convoluted tubules was characteristic of bovine nephrons as were differences in the expression patterns of claudins 2 and 3. These findings demonstrate that the total lack of claudin-16 in the TAL segment is the sole cause of renal tubular dysplasia in cattle and suggest that the tight junctions in distinct tubular segments including the TAL have barrier functions in paracellular permeability that are different among animal species.

An NMR spectroscopic characterization of a new epithelial cell line, TALH-SVE, with properties of the renal medullary thick ascending limb of Henle's loop.

NMR spectroscopy has been used to characterize a new renal cell line, TALH-SVE.1, which is derived from the medullary thick ascending limb of Henle's loop. From the 31P-NMR spectrum of a suspension of TALH-SVE cells using the chemical shift of the intracellular inorganic phosphate a value of 7.24 +/- 0.04 for the steady-state intracellular pH (pHi) was determined at pHo = 7.40. In addition, the 31P-NMR spectrum indicated rather high levels of UDPG, a finding confirmed by 1H-NMR spectra of perchloric acid extracts. The 1H-NMR data also demonstrate the presence of 'organic osmolytes' such as inositol, sorbitol, choline and glycerophosphoryl choline (GPC). 13C-NMR spectra of perchloric acid extracts of TALH-SVE cells incubated with [2-13C]- and [3-13 C]alanine were used to determine the relative influx in the Krebs cycle via pyruvate carboxylase (PCB) versus the influx via pyruvate dehydrogenase (PDH). The ratio was 0.41, while about 52% of all acetyl-CoA entering the Krebs cycle was unlabeled. 13C-NMR experiments also indicated that TALH-SVE cells lack gluconeogenic activity. The NMR study presented indicates that TALH-SVE cells possess metabolic pathways similar to those of the parental cells.

Immunocytochemical localization of stanniocalcin cells in the rat kidney.

Stanniocalcin (STC) is a polypeptide hormone that was first discovered in fishes, where it functions as a regulator of calcium and phosphate homoeostasis. Recently, complementary DNAs encoding human STC (hSTC) have been characterized, and recombinant hSTC has been synthesized in a bacterial expression system. In preliminary studies, STC-immunoreactive cells have already been identified in human kidney tubules with antibodies to recombinant hSTC. The purpose of this study was to map the overall spatial distribution of STC cells in mammalian kidney, using the rat as a model system. Immunocytochemistry was performed on fixed sections of rat kidney tissue using hSTC antiserum in conjunction with fluorescein isothiocyanate-conjugated second antibodies. STC-immunoreactive cells were found in cortical thick ascending limb, in macula densa, in distal convoluted tubules, and in the cortical and medullary collecting ducts. All cortical thick ascending limb cells contained immunoreactive STC. Most distal convoluted tubules cells contained STC, and these were identified as principal cells. The distribution of STC cells in cortical and medullary collecting ducts also corresponded closely to the known frequently of principle cells in these segments, suggesting that principal cells are the site of STC storage and/or synthesis in both distal convoluted tubules and collecting ducts. Some collecting duct intercalated cells contained STC as well, and these were tentatively identified as alpha-type intercalated cells. As all tubular segments containing STC are known to be involved in regulated ion transport, renally derived STC may be acting in an autocrine, paracrine and/or endocrine fashion to regulate one or more of these transport processes.

Localization of Na(+)-dependent active type and erythrocyte/HepG2-type glucose transporters in rat kidney: immunofluorescence and immunogold study.

Glucose is actively taken up from the glomerular filtrate into the tubule cells by the Na(+)-dependent active glucose transporter (GT), and passively crosses the basolateral membrane via facilitated diffusion GT. With the use of antibodies directed against two types of GTs, we show the immunocytochemical localization of the Na(+)-dependent active GT (SGLT1) and the erythrocyte/HepG2-type facilitated diffusion GT (GLUT1). For light microscopic observation, frozen sections were stained by the rhodamine labeling method. Counterstaining with fluorescein-phalloidin and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) was employed to facilitate cell type identification. Immunogold staining was carried out on ultra-thin frozen sections for electron microscopy. The antibody to SGLT1 reacted with a 77 KD protein in immunoblotting of a kidney lysate. By immunocytochemistry, SGLT1 was localized in the microvillous plasma membrane in the apical brush borders of the cells of all three proximal tubule segments (S1, S2, and S3). The antibodies to GLUT1, a member of the facilitated diffusion GT family, were raised against human erythrocyte GT or synthetic oligopeptides derived from HepG2 GT, which reacted with a 48 KD protein in immunoblotting of the kidney lysate. GLUT1 was found at the basolateral plasma membranes of S3 proximal tubule cells, cells of the thick limb of Henle's loop, and collecting duct cells. Combined with known physiological data, our findings suggest that SGLT1 in the apical plasma membrane of the proximal tubule cells is responsible for the Na(+)-dependent active reabsorption of glucose from the glomerular filtrate. GLUT1 in the basolateral plasma membrane of S3 cells may transport reabsorbed glucose to the blood vessels. GLUT1 in the basolateral plasma membranes of cells of the thick limb of Henle's loop and of the collecting duct, on the other hand, may nourish these metabolically active cells by facilitating the diffusion of extracellular glucose provided from blood through the basolateral side of the cells.

Expression of the Na+/myo-inositol cotransporter in the juxtaglomerular region.

Myo-inositol is a major compatible osmolyte in the renal medulla and is accumulated in cells under hypertonic conditions by uptake via a Na+/myo-inositol cotransporter (SMIT). SMIT is regulated by extracellular osmolarity at the transcription level. We investigated localization of SMIT in rat kidney by immunohistochemical staining using an anti-SMIT-antibody raised against a synthetic peptide corresponding to part of SMIT and by in situ hybridization. SMIT protein localized predominantly to the basolateral membranes of cells of the thick ascending limb of Henle (TAL) and inner medullary collecting duct (IMCD). Macula densa (MD) cells, identified as the Tamm-Horsfall-protein (THP)-unreactive cells surrounded by THP-reactive TAL cells, also stained for anti-SMIT. In situ hybridization yielded the intense SMIT signals in the TAL and IMCD and also in the juxtaglomerular (JG) region. Prior loading of the animal with a high concentration of NaCl rapidly induced SMIT mRNA; furosemide down-regulated it. The high level of SMIT expression suggests that MD cells are exposed to hypertonicity at the basolateral surface. Because SMIT expression seemed to be proportional to the magnitude of NaCl reabsorption, it may be a good marker for examination of the tubuloglomerular feedback mechanism in vivo.

Tamm-Horsfall protein in human renal tumours.

Tamm-Horsfall Protein (THP) is found to be localised in the ascending loop of Henle and the distal tubules of normal and foetal kidneys. We have used an anti-THP monoclonal antibody (D2) to stain foetal, normal and kidney tumours. The dinitrophenyl hapten staining technique was the most specific and sensitive staining procedure. The staining of Tamm-Horsfall Protein in foetal and normal kidney was in agreement with most other investigators. The majority of renal cell carcinoma (22/24) and rhabdoid renal tumours (3/4) stained positively for THP. None of the Wilms' tumours, mesoblastic nephromas or bone metastasising renal tumours of childhood gave positive staining. The findings would suggest that renal cell carcinomas and rhabdoid kidney tumours arise from ascending loop of Henle or distal tubules. The lack of positive staining in the majority of childhood renal tumours raises the possibility of a different histogenesis for these neoplasms.

1,25-Dihydroxyvitamin D3 stimulates osteopontin expression in rat kidney.

Osteopontin (OPN) is a secreted phosphoprotein expressed constitutively in the descending thin limb (DTL) and papillary surface epithelium (PSE) of the kidney. Although its function is not fully established, a role for OPN in the regulation of calcium-mediated or calcium-dependent processes has been proposed. The aim of this study was to examine the effects of 1,25-dihydroxyvitamin D(3) (vitD), a hormone involved in the regulation of calcium homeostasis, on renal OPN expression. Four groups of rats were studied: acute vehicle (single intraperitoneal [i.p.] injection of 0.1 ml 10% ethanol-90% propylene glycol, 12 h before being killed); acute vitD (single injection of vitD, 2 ng/g i.p., 12 h before being killed); chronic vehicle (daily subcutaneous [s.c.] injection of 0.1 ml 10% ethanol-90% propylene glycol for 7 days); and chronic vitD (daily s.c. injection of vitD, 0.5 ng/g, for 7 days). Kidneys were processed for light and electron microscope immunocytochemistry, in situ hybridization, and Western blot analysis. In vehicle-treated animals, OPN mRNA and protein were expressed primarily in the DTL and PSE. In the acute vitD group, OPN mRNA and immunoreactivity appeared in the thick ascending limb (TAL) of the inner stripe of the outer medulla, and increased slightly in the DTL and PSE. The proximal tubules exhibited strong OPN immunoreactivity, but no hybridization signal. In the chronic vitD group, there was a marked increase in OPN mRNA and immunoreactivity in the distal tubule, including the TAL, as well as in the DTL and PSE. A weak hybridization signal and immunostaining were also observed in some proximal tubules. Administration of vitD causes a marked increase in OPN mRNA and protein in the rat kidney, mainly in the distal nephron, but also in the DTL, PSE, and proximal tubules. These results indicate that vitD is involved in the regulation of OPN expression in the kidney.

Immunolocalization of anion transporter Slc26a7 in mouse kidney.

Previous studies have indicated that a major fraction of the filtered Cl(-) is reabsorbed via apical membrane Cl(-)/base exchange in the proximal tubule. Recent studies in Slc26a6 null mice have suggested that this transporter mediates only a portion of proximal tubule Cl(-)/base exchange, raising the possibility that one or more unidentified apical membrane transporters may additionally contribute. Recent studies have identified Slc26a7 as another Cl(-)/base exchanger expressed in the kidney. We therefore generated Slc26a7-specific polyclonal and monoclonal antibodies to examine cellular and subcellular sites of expression in mouse kidney. The specificity of each antibody was verified by immunoblotting and immunofluorescence of COS-7 cells transiently transfected with mouse Slc26a7. Immunofluorescence microscopy of mouse kidney detected the expression of Slc26a7 subapically in proximal tubule cells, and on the basolateral surface of thick ascending limb cells. Similar staining patterns were demonstrated with two antibodies shown to react with different epitopes on Slc26a7. Immunolocalization of Slc26a7 to proximal tubule and thick ascending limb was also observed in rat kidney. We conclude that Slc26a7 is expressed in the proximal tubule and thick ascending limb of the loop of Henle, and it may therefore contribute to anion transport in these nephron segments.

Changes in subcellular distribution of the ammonia transporter, Rhcg, in response to chronic metabolic acidosis.

The primary mechanism by which the kidneys mediate net acid excretion is through ammonia metabolism. In the current study, we examined whether chronic metabolic acidosis, which increases ammonia metabolism, alters the cell-specific and/or the subcellular expression of the ammonia transporter family member, Rhcg, in the outer medullary collecting duct in the inner stripe (OMCDi). Chronic metabolic acidosis was induced in normal SD rats by HCl ingestion for 7 days; controls were pair-fed. The subcellular distribution of Rhcg was determined using immunogold electron microscopy and morphometric analyses. In intercalated cells, acidosis increased total Rhcg, apical plasma membrane Rhcg, and the proportion of total cellular Rhcg in the apical plasma membrane. Intracellular Rhcg decreased significantly, and basolateral Rhcg was unchanged. Because apical plasma membrane length increased in parallel with apical Rhcg immunolabel, apical plasma membrane Rhcg density was unchanged. In principal cells, acidosis increased total Rhcg, apical plasma membrane Rhcg, and the proportion of total cellular Rhcg in the apical plasma membrane while decreasing the intracellular proportion. In contrast to the intercalated cell, chronic metabolic acidosis did not significantly alter apical boundary length; accordingly, apical plasma membrane Rhcg density increased. In addition, basolateral Rhcg immunolabel increased in response to chronic metabolic acidosis. These results indicate that in the rat OMCDi 1) chronic metabolic acidosis increases apical plasma membrane Rhcg in both the intercalated cell and principal cell where it may contribute to enhanced apical ammonia secretion; 2) increased apical plasma membrane Rhcg results from both increased total protein and changes in the subcellular distribution of Rhcg; 3) the mechanism of Rhcg subcellular redistribution differs in intercalated and principal cells; and 4) Rhcg may contribute to regulated basolateral ammonia transport in the principal cell.