Immunocytochemical localization of gamma-glutamyl-transferase on isolated renal cortical tubular fragments

The localization of gamma-Glutamyltransferase (gamma-GT, E.C.2.3.2.2) was studied on isolated tubular fragments from rat kidney cortex immunocytochemically. Monospecific antibodies raised in the goat against rat kidney gamma-GT were used. Antigoat immunoglobulin from the rabbit conjugated with ferritin was used for visualisation of the antibody binding sites. The enzyme was found to be localized at the brush border membrane of proximal tubules, the luminal membrane of distal tubules and collecting duct segments. The enzyme could further be localized on the antiluminal or basolateral cell membranes of proximal and distal tubular fragments, whereas no such localization was verified for collecting duct segments. The role of this basolateral gamma-GT localization in context with the kidney's ability to extract over 83% of the renal arterial glutathione (GSH) input during a single passage is discussed.     

Beta-adrenoceptors in kidney tubules of spontaneously hypertensive and normotensive rats

Beta-adrenoceptor binding characteristics were determined in different fractions of rat kidney tubules using a [125Iodo]-(-)-cyanopindolol (ICYP) binding assay. The highest amount of binding sites was found in a fraction containing predominantly distal tubular fragments. In a separate series of experiments the ICYP binding characteristics were compared in whole tubular fractions from spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rats (WKY) of different ages. The maximum number of binding sites was significantly higher both in young (3 weeks) and adult (14 weeks) SHR when compared to age-matched WKY. These studies showed the presence of beta-adrenoceptor binding sites in rat kidney tubules and support the potential importance of tubular beta-adrenoceptors in the development of spontaneous hypertension and in the mechanism of antihypertensive action of beta-blockers.     

Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates

Paraffin sections of mouse and rat kidney were stained with a battery of ten lectin-horseradish peroxidase conjugates and lectin binding was correlated with the ultrastructural distribution of periodate-reactive sugar residues as determined by the periodic acid-thiocarbohydrazide-silver proteinate technique. Various segments of the uriniferous tubule in both species showed differential affinity for labelled lectins. Significant differences were also evident between comparable tubular segments in mouse and rat kidneys. Neutral glycoconjugates containing terminal beta-galactose and terminal alpha-N-acetylgalactosamine were prevalent on the luminal surface of the proximal convoluted tubule in the rat, but alpha-N-acetylgalactosamine was absent in this site in the mouse. In both species, terminal N-acetylglucosamine was abundant in the brush border of proximal straight tubules but absent in proximal convolutions. Fucose was demonstrated in both proximal and distal segments of mouse kidney tubules but only in the distal nephron and collecting ducts in the rat. Lectin staining revealed striking heterogeneity in the structure and distribution of cellular glycoconjugates. Such cellular heterogeneity was previously unrecognizable with earlier histochemical methods. The marked cellular heterogeneity observed with several lectin-conjugates in distal convoluted tubules and collecting ducts of both species raises a prospect that lectins can provide specific markers for intercalated and principal cells in the mammalian kidney. Glycoconjugates containing terminal sialic acid and penultimate beta-galactose were present on vascular endothelium in both rodent kidneys, as were terminal alpha-galactose residues; but both species lacked reactivity for Ulex europeus I lectin in contrast to human vascular endothelial cells. The constant binding pattern of lectin conjugates allows convenient and precise differentiation of renal tubular segments and should prove valuable in the study of changes in kidney morphology promoted by experimental manipulation or pathologic changes.     

Hemoglobin binding sites on renal brush-border membranes

Prolonged exposure of renal tubules to hemoglobin markedly reduces kidney function and eventually leads to acute renal failure called pigment nephropathy. Intracellular hemoglobin toxicity is one of main pathomechanisms involved in the disease development. However, the process in which hemoglobin is taken up by renal tubular epithelium has not been characterized so far. Isolated renal brush-border membranes of the rat and radioiodinated rat and human hemoglobins were used. Binding properties were examined by the use of rapid filtration technique. Partial isolation of hemoglobin binding proteins was achieved by affinity chromatography. Our experiments showed that both human and rat hemoglobins can be specifically bound to renal brush-border membranes by one class of low affinity (Kd, 7.7 microM) and high capacity (Bmax, 0.18 nmol/mg protein) binding sites. The sites were relatively selective for hemoglobin. Albumin did not compete with hemoglobin. Cationic molecules cytochrome C and lysine exhibited some competition while strong competition of myoglobin was observed. The binding was affected by EGTA indicating a Ca2+ requirement for the interaction. There was a rise in binding in pH 5.4. Fall in binding activity after preincubation of the membranes with peptidases suggested the proteinaceous nature of the binding sites. Affinity chromatography of membrane proteins extract yielded heterogeneous preparation consisting of proteins with molecular masses of 110, 72, 38 and 27 kDa respectively. The existence of binding sites for hemoglobin in renal brush-border membranes strongly suggests that uptake of the protein by tubular epithelia occurs via adsorptive endocytosis. Increased binding of hemoglobin to the membranes under acidic conditions may explain exacerbation of hemoglobinuric acute renal failure in aciduric states.     

Altered expression and localization of insulin receptor in proximal tubule cells from human and rat diabetic kidney

Diabetes is the major cause of end stage renal disease, and tubular alterations are now considered to participate in the development and progression of diabetic nephropathy (DN). Here, we report for the first time that expression of the insulin receptor (IR) in human kidney is altered during diabetes. We detected a strong expression in proximal and distal tubules from human renal cortex, and a significant reduction in type 2 diabetic patients. Moreover, isolated proximal tubules from type 1 diabetic rat kidney showed a similar response, supporting its use as an excellent model for in vitro study of human DN. IR protein down‐regulation was paralleled in proximal and distal tubules from diabetic rats, but prominent in proximal tubules from diabetic patients. A target of renal insulin signaling, the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), showed increased expression and activity, and localization in compartments near the apical membrane of proximal tubules, which was correlated with activation of the GSK3β kinase in this specific renal structure in the diabetic condition. Thus, expression of IR protein in proximal tubules from type 1 and type 2 diabetic kidney indicates that this is a common regulatory mechanism which is altered in DN, triggering enhanced gluconeogenesis regardless the etiology of the disease. J. Cell. Biochem. 114: 639–649, 2013. © 2012 Wiley Periodicals, Inc.     

Distal Renal Tubules Are Deficient in Aggresome Formation and Autophagy upon Aldosterone Administration

Prolonged elevations of plasma aldosterone levels are associated with renal pathogenesis. We hypothesized that renal distress could be imposed by an augmented aldosterone-induced protein turnover challenging cellular protein degradation systems of the renal tubular cells. Cellular accumulation of specific protein aggregates in rat kidneys was assessed after 7 days of aldosterone administration. Aldosterone induced intracellular accumulation of 60 s ribosomal protein L22 in protein aggregates, specifically in the distal convoluted tubules. The mineralocorticoid receptor inhibitor spironolactone abolished aldosterone-induced accumulation of these aggregates. The aldosterone-induced protein aggregates also contained proteasome 20 s subunits. The partial de-ubiquitinase ataxin-3 was not localized to the distal renal tubule protein aggregates, and the aggregates only modestly colocalized with aggresome transfer proteins dynactin p62 and histone deacetylase 6. Intracellular protein aggregation in distal renal tubules did not lead to development of classical juxta-nuclear aggresomes or to autophagosome formation. Finally, aldosterone treatment induced foci in renal cortex of epithelial vimentin expression and a loss of E-cadherin expression, as signs of cellular stress. The cellular changes occurred within high, but physiological aldosterone concentrations. We conclude that aldosterone induces protein accumulation in distal renal tubules; these aggregates are not cleared by autophagy that may lead to early renal tubular damage.     

High- and low-affinity binding sites for vasoactive intestinal peptide (VIP) in the rat kidney revealed by light microscopic autoradiography

The distribution of VIP binding sites in rat kidney and adrenal gland has been examined by light microscopic autoradiography. A fully characterized mono-iodinated molecular form of VIP (M-125-I-VIP) which maintains the biological activity of the native peptide, was used for this study. Two types of VIP binding sites, with high and low affinity, have been identified. High affinity sites are associated with (i) glomerular structures in the cortex, (ii) the inner stripe of the outer medulla, possibly corresponding to Henle's loops and distal tubules, (iii) radiated structures in the inner zone of the medulla, likely to represent labeling of collecting ducts and/or vascular bundles and (iv) the adrenal cortex. Autoradiographic grains associated with low affinity sites are present diffusely throughout the renal cortex, possibly corresponding to labeling of tubular and/or vascular structures, and throughout the adrenal gland. These observations further delineate a role of VIP in renal and neuroendocrine function.     

Mercuric chloride-induced alterations in stress protein distribution in rat kidney

Mercuric chloride (HgCl2) induces acute renal failure associated to tubular impairment in experimental animals and humans. Stress proteins are a superfamily of proteins, comprising heat- shock proteins (HSP) and glucose-regulated proteins (GRP), enhanced or induced in the kidney in response to stress. They act as molecular chaperones that protect organelles and repair essential proteins which have been denatured during adverse conditions. The involvement of stress proteins in mercury-nephrotoxicity has not yet been well clarified. This study was undertaken to detect the tubular distribution of four stress proteins (HSP25, HSP60, GRP75, HSP72) in the rat kidney injected with HgCl2 and to quantify lysosomal and mitochondrial changes in straight proximal tubules, the main mercury target. Sprague-Dawley rats were administered i.p. with progressive sublethal doses of HgCl2 (0.25 mg/kg, 0.5 mg/kg, 1 mg/kg and 3.5 mg/kg) or saline (as controls) and sacrificed after 24 h. In dosages over 0.50 mg/kg, stress proteins increased and changed localization in a dose-dependent manner. HSP25 was focally expressed in altered proximal tubules at 1 mg/kg but in the macula densa it was at 3.5 mg/kg. HSP60 and GRP75 were intense in the nucleus and cytoplasm of proximal tubules but moderate in distal tubules. HSP72 was induced in distal tubules after low exposures but in proximal tubules it happened at the highest dose. Moreover, a significant increase in lysosomal and total mitochondria (normal and with broken cristae) area and density were progressively found after HgCl2 treatments. Stress proteins could represent sensitive biomarkers that strongly correlate with the degree of oxidative injury induced by HgCl2 in the rat proximal tubules.     

Immunocytochemical localization of Na+, K+-ATPase in the rat kidney

To determine if rat kidney Na+, K+-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified lamb kidney medulla Na+, K+-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified lamb kidney Na+, K+-ATPase and rat kidney microsomes were treated with antiserum (1:200), followed by [125I]-Protein A and autoradiography, the rat kidney microsomes showed a prominent radioactive band coincident with the alpha-subunit of the purified lamb kidney enzyme and a fainter radioactive band which corresponded to the beta-subunit. When the Na+, K+-ATPase antiserum was used for immunoperoxidase staining of paraffin and plastic sections of rat kidney fixed with Bouin's, glutaraldehyde, or paraformaldehyde, intense immunoreactive staining was present in the distal convoluted tubules, subcapsular collecting tubules, thick ascending limb of the loops of Henle, and papillary collecting ducts. Proximal convoluted tubules stained faintly, and the thin portions of the loops of Henle, straight descending portions of proximal tubules, and outer medullary collecting ducts did not stain. Staining was confined to basolateral surfaces of tubular epithelial cells. No staining was obtained with preimmune serum or primary antiserum absorbed with purified lamb kidney Na+, K+-ATPase, or with osmium tetroxide postfixation. We conclude that the basolateral membranes of the distal convoluted tubules and ascending thick limb of the loops of Henle are the major sites of immunoreactive Na+, K+-ATPase concentration in the rat kidney.     

Expression of metallothionein in renal tubules of rats exposed to acute and endurance exercise

The induction of exercise-induced apoptosis in not actively involved in exercise organs, such as kidney could be a result of oxidative stress. Metallothionein (MT) exerts a protective effect in the cell against oxidative stress and apoptosis. We have previously demonstrated an increased incidence of apoptosis in distal tubular cells and collecting ducts in rat kidney after acute exercise. The present study was designed to test the hypothesis that MT may play a protective role in rat renal tubules against exercise-induced apoptosis after the acute exercise and regular training. Male Wistar rats were divided into control, acute exercised and 8-wk regularly trained groups. The kidneys were removed after a rest period of 6 h and 96 h. The ultrastructure of renal tubular cells was examined by electron microscopy. Apoptosis was detected in paraffin sections by the TUNEL technique. Expression of MT was examined by immunohistochemistry. The level of lipid peroxidation (thiobarbituric acid reactive substances - TBARS) was assayed in renal tissue homogenates. After acute exercise, the occurrence of apoptosis was restricted to distal tubules and collecting ducts of rat kidney, whereas the proximal tubules remained unaffected. The 8-wk training did not result in increased apoptosis in tubular cell. MT expression was confined exclusively to proximal tubules in all groups. However, it was significantly increased in acutely exercised animals, as compared to control and trained rats. After the 8-wk training, MT expression remained unaltered as compared to the control group. TBARS levels were significantly increased after acute exercise, while after regular training they remained unchanged. A significant correlation between TBARS level and MT expression was demonstrated. The findings could suggest a protective role of MT against oxidative stress and apoptosis in proximal tubular cells.     

Accumulation of 8-oxoguanine in the cellular DNA and the alteration of the OGG1 expression during ischemia-reperfusion injury in the rat kidney

During ischemia-reperfusion (I/R) injury in the rat kidney, apoptosis was observed in the distal tubules of the cortico-medullary region and outer medulla (OM) while severe necrosis was seen in the proximal straight tubules of the OM. The majority of these changes disappeared within 2 weeks. We examined the contents of 8-oxo-2'-deoxyguanosine (8-oxo-dG), which is a major type of oxidative damage in DNA, in the rat kidney during I/R injury, and also investigated the expression level of the OGG1 gene encoding the 8-oxoguanine DNA glycosylase. High-performance liquid chromatography with an MS/MS analysis of the nuclear DNA revealed an immediate accumulation of 8-oxo-dG in the nuclear DNA prepared from the cortex and OM of the kidney 1h after I/R, and an immunohistochemical analysis demonstrated the immediate accumulation of 8-oxo-dG in the nuclei of renal tubular cells both in the cortex and OM. A delayed increase of cytoplasmic staining with anti-8-oxo-dG was observed only in the cortico-medulla and OM, where the cytoplasmic staining in the proximal tubular cells is higher than in the distal tubular cells. The level of cytoplasmic staining representing 8-oxo-dG in mitochondrial DNA, peaked at 6h after I/R and preceded the necrosis of proximal tubular cells in the OM. An RNase protection assay showed a high level of OGG1 mRNA in the normal kidney, and the level decreased within 3h only in the OM, and increased thereafter 1-7 days of I/R both in the cortex and OM. In situ hybridization showed higher levels of OGG1 mRNA expression in the renal tubules in the OM than in the cortex of the normal kidney, which decreased rapidly within 3h of I/R. Thus, the accumulation of 8-oxo-dG in the mitochondrial DNA rather than in nuclear DNA is likely to be involved in the pathogenic responses such as necrosis of renal tubular cells during I/R injury of the kidney, together with an altered level of OGG1 expression.     

Immunocytochemical localization of γ-glutamyl-transferase on isolated renal cortical tubular fragments

Summary The localization of -Glutamyltransferase (-GT, E.C.2.3.2.2.) was studied on isolated tubular fragments from rat kidney cortex immunocytochemically. Monospecific antibodies raised in the goat against rat kidney -GT were used. Antigoat immunoglobulin from the rabbit conjugated with ferritin was used for visualisation of the antibody binding sites. The enzyme was found to be localized at the brush border membrane of proximal tubules, the luminal membrane of distal tubules and collecting duct segments.The enzyme could further be localized on the antiluminal or basolateral cell membranes of proximal and distal tubular fragments, whereas no such localization was verified for collecting duct segments. The role of this basolateral -GT localization in context with the kidney's ability to extract over 83% of the renal arterial glutathione (GSH) input during a single passage is discussed.     

Metabolic heterogeneity of the proximal and distal kidney tubules

Proximal and distal tubule suspensions were prepared from kidneys of Sprague-Dawley rats by an isolation procedure on a Percoll^R gradient. The marker enzymes alkaline phosphatase (brush border) and hexokinase (cytoplasmic) as well as p-aminohippurate transport capacity, gluconeogenic activity and electron microscopy were used to characterize the two kidney tubule suspensions. The results of this study indicate that cytochrome P-450 is localized to the proximal tubular cells and that the O-deethylation of 7- ethoxycoumarin was higher in the proximal than distal fraction. Both proximal and distal tubules showed glucuronidation and deacetylation capacities and a relatively equal distribution of non-protein sulfhydryls. These studies demonstrate metabolic heterogeneity of the nephron, the proximal tubule being the main site of renal xenobiotic metabolism. Understanding of metabolic heterogeneity of proximal and distal kidney tubules should provide important information regarding cell specific mechanisms of nephrotoxicity.     

Immunocytochemical localization of cathepsin B in rat kidney. II. Electron microscopic study using the protein A-gold technique

Thin sections of Lowicryl K4M-embedded materials were labeled with protein A-gold complex. Gold particles representing the antigen sites for cathepsin B were exclusively confined to lysosomes of each segment of the nephron. The heaviest labeling was noted in the lysosomes of the S1 segment of the proximal tubules. Labeling intensity varied considerably with the individual lysosomes. Lysosomes of the other tubular segments, such as the S2 and S3 segments of the proximal tubules, distal convoluted tubules, and collecting tubules were weakly labeled by gold particles. Quantitative analysis of labeling density also confirmed that lysosomes in the S1 segment have the highest labeling density and that approximately 65% of labeling in the whole renal segments, except for the glomerulus, was found in the S1 segment. These results indicate that in rat kidney the lysosomes of the S1 segment are a main location of cathepsin B. Further precise observations on lysosomes of the S1 segment revealed that apical vesicles, tubules, and vacuoles were devoid of gold particles, but when the vacuoles contained fine fibrillar materials, gold labeling was detectable in such vacuoles. As the lysosomal matrix becomes denser, the labeling density is increased. Some small vesicles around the Golgi complex were also labeled. These results indicate that the endocytotic apparatus including the apical vesicles, tubules, and vacuoles contains no cathepsin B. When the vacuoles develop into phagosomes, they acquire this enzyme to digest the absorbed proteins.     

Altered renal morphology in transgenic mice with cholecystokinin overexpression

Although cholecystokinin is a regulatory peptide with a predominant role in the brain and the gastrointestinal tract, there is an increasing evidence for its role in the kidney. The aim of this study was to reveal morphological changes in the structure of kidney of mice with cholecystokinin overexpression by means of light, transmission and scanning electron microscope, and atomic force microscopy. Using immunohistochemistry the expression of important basement membrane proteins collagen IV, laminin and fibronectin, as well the distribution of cholecystokinin-8 in the renal structures was evaluated. The altered morphology of kidneys of mice with cholecystokinin overexpression was seen by all microscopic techniques used. The renal corpuscles were relatively small with narrow capsular lumen. The basement membranes of renal tubules were thickened and the epithelial cells were damaged, which was more pronounced for distal tubules. Characteristic feature was the increased number of vesicles seen throughout the epithelial cells of proximal and especially in distal tubules reflecting to the enhanced cellular degeneration. The relative expression of laminin but not collagen IV in the glomerular basement membrane was higher than in the tubular basement membranes. The content of fibronectin, in opposite, was higher in tubular membranes. Cholecystokinin-8 was clearly expressed in the glomeruli, in Bowman’s capsule, in proximal and distal tubules, and in collecting ducts. Ultrastructural studies showed irregularly thickened glomerular basement membranes to which elongated cytopodia of differently shaped podocytes were attached. As foot processes were often fused the number of filtration pores was decreased. In conclusion, cholecystokinin plays important role in renal structural formation and in functioning as different aspects of urine production in mice with cholecystokinin overexpression are affected-the uneven glomerular basement membrane thickening, structural changes in podocytes and in filtration slits affect glomerular filtration, while damaged tubular epithelial cells and changed composition of thickened tubular basement membranes affect reabsorption.     

Effect of epsilon toxin-GFP on MDCK cells and renal tubules in vivo.

Epsilon toxin (epsilon-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxemia, also known as pulpy kidney disease, in livestock. Recombinant epsilon-toxin-green fluorescence protein (epsilon-toxin-GFP) and epsilon-prototoxin-GFP were successfully expressed in Escherichia coli. MTT assays on MDCK cells confirmed that recombinant epsilon-toxin-GFP retained the cytotoxicity of the native toxin. Direct fluorescence analysis of MDCK cells revealed a homogeneous peripheral pattern that was temperature sensitive and susceptible to detergent. epsilon-Toxin-GFP and epsilon-prototoxin-GFP bound to endothelia in various organs of injected mice, especially the brain. However, fluorescence mainly accumulated in kidneys. Mice injected with epsilon-toxin-GFP showed severe kidney alterations, including hemorrhagic medullae and selective degeneration of distal tubules. Moreover, experiments on kidney cryoslices demonstrated specific binding to distal tubule cells of a range of species. We demonstrate with new recombinant fluorescence tools that epsilon-toxin binds in vivo to endothelial cells and renal tubules, where it has a strong cytotoxic effect. Our binding experiments indicate that an epsilon-toxin receptor is expressed on renal distal tubules of mammalian species, including human.     

Recurrence of posterior polymorphous corneal dystrophy is caused by the overgrowth of the original diseased host endothelium

Peplomycin (PEP), an anti-tumor antibiotic related structurally to bleomycin, is widely used, especially for squamous cell carcinoma but shows renal toxicity. We prepared monoclonal antibodies (mAbs) against N-(γ-maleimidobutyryloxy)succinimide-conjugated PEP. The mAbs were monospecific for PEP, but did not react with bleomycin and other anticancer antibiotics. The mAbs enabled us to develop an immunocytochemical (ICC) method for detecting the uptake of PEP in the rat kidney. Two hours after a single i.v. administration of PEP, ICC revealed immunostaining for PEP in irregularly shaped cytoplasmic granules of the proximal tubules in which the microvilli were also stained. Also, staining occurred in the distal tubules and collecting ducts, in both of which we observed scattered swollen cells, reminiscent of necrotic cells, in which both the nuclei and cytoplasm reacted strongly with the antibody. Twenty-four hours after injection, PEP in the proximal tubules completely vanished, but yet significant amounts of PEP remained in both the distal tubules and collecting ducts. Distribution patterns of PEP in cells of the kidneys resembled, in some ways, those of our recent ICC studies for an organic cation aminoglycoside antibiotic gentamicin. This ICC suggests that PEP taken up in the proximal tubule cells is localized in the lysosomes, and organic cation transporters and bleomycin hydrolase might be involved in entrance and/or disappearance of PEP in this cell type. Furthermore, the distal tubules and collecting ducts may be the sites readily affected by some chemotherapeutic agents.     

Species Diversity Regarding the Presence of Proximal Tubular Progenitor Cells of the Kidney

The cellular source for tubular regeneration following kidney injury is a matter of dispute, with reports suggesting a stem or progenitor cells as the regeneration source while linage tracing studies in mice seemingly favor the classical theory, where regeneration is performed by randomly surviving cells. We, and others have previously described a scattered cell population localized to the tubules of human kidney, which increases in number following injury. Here we have characterized the species distribution of these proximal tubular progenitor cells (PTPCs) in kidney tissue from chimpanzee, pig, rat and mouse using a set of human PTPC markers. We detected PTPCs in chimpanzee and pig kidneys, but not in mouse tissue. Also, subjecting mice to the unilateral urethral obstruction model, caused clear signs of tubular injury, but failed to induce the PTPC phenotype in renal tubules.Key words: Acute tubular necrosis, tubular regeneration, species diversity, proximal tubules     

High dietary potassium chloride intake augments rat renal mineralocorticoid receptor selectivity via 11beta-hydroxysteroid dehydrogenase

Glucocorticoid access to renal corticosteroid receptors is regulated by 11beta-hydroxysteroid dehydrogenases (11beta-HSDs), converting 11beta-hydroxyglucocorticoids into inactive 11-ketones. This mechanism plays a key role in maintaining normal salt-water homeostasis and blood pressure. To study whether renal cortical proximal and distal tubular 11beta-HSDs are modulated, upon shifting the electrolyte status (and may thereby contribute to adjusting the salt-water homeostasis), rats were treated for 14 days with diets with low (0.058 w/w%), normal (0.58%, which is the KCl content of standard European laboratory rat food) or high (5.8%) potassium chloride content. In proximal tubules, dietary KCl had no effect regarding corticosterone 11beta-oxidation in intact cells as well as 11beta-HSD1 and 11beta-HSD2 protein (Western blotting) and mRNA levels (semi-quantitative RT-PCR). In distal tubules, the low KCl diet also had no effect. However, distal tubules of rats fed the high KCl diet showed increased corticosterone 11beta-oxidation rates (1.6-fold, P<0.01) and 11beta-HSD2 protein (4-fold, P<0.01), whereas 11beta-HSD1 protein was decreased (no longer detected, P<0.05). Distal tubular 11beta-HSD mRNA levels were not changed upon dietary treatment. Our results suggest that upon dietary KCl loading distal tubular mineralocorticoid receptor selectivity for aldosterone is increased because of enhanced corticosterone 11beta-oxidation. This may contribute to the fine-tuning of salt-water homeostasis by the kidney.