Ammonium affects tight junctions and the cytoskeleton in MDCK cells.

In the kidney medulla, tubule cells are exposed not only to elevated NaCl but also to high NH(4)Cl concentrations. Although it is well known that long-term exposure to high NaCl concentrations leads to reorganization of the actin-based cytoskeleton and to altered transport properties of renal epithelial cells, there have been no comparable studies on the effects of elevated extracellular NH(4)Cl concentrations. We therefore examined the effect of prolonged (up to 72 h) exposure of Madin-Darby canine kidney (MDCK) cells to increased NH(4)Cl concentrations on the actin-based cytoskeleton, the transepithelial electrical resistance (TER) and the expression and intracellular distribution of the tight junction protein occludin. NH(4)Cl exposure resulted in rarefaction of cytoplasmic stress fibres, formation of intense peripheral actin bands and reduced abundance of both F- and G-actin. While under control conditions occludin staining was restricted to the tight junction region, ample dot-like intracellular staining was apparent after NH(4)Cl exposure. These changes in cell structure were associated with an increase in TER and the enhanced expression of an additional putative, 40-kDa occludin isoform. Exposure to elevated extracellular NH(4)Cl concentrations thus leads to distinct alterations in the architecture and transepithelial transport properties of MDCK cells that may also be relevant for the tubule cells of the renal inner medulla.

Recovery of cell volume and electrolytes of A6 cells after re-establishing isotonicity following hypotonic stress.

Cellular element concentrations and dry weight contents in A6 cells were determined using electron microprobe analysis to establish whether these cells exhibit a regulatory volume increase (post-RVD-RVI) when re-establishing isotonicity following a hypotonically induced regulatory volume decrease (RVD). Hypotonic stress was induced by reducing basolateral [NaCl], and hence, osmolarity fell from 260 to 140 mosmol/l. The alterations in cell volume after re-establishing isotonicity, calculated from the cellular dry weight changes, indicate within the first 2 min cell shrinkage from 120 to 76% of control, compatible with almost ideal osmometric behaviour of A6 cells, and thereafter a post-RVD-RVI to 94%. The cellular uptake of osmolytes necessary to explain the post-RVD-RVI could be accounted for solely by a gain in cellular K and Cl. The involvement of a Na-K-2Cl cotransporter in most of the KCl uptake seems plausible since basolateral bumetanide blocked KCl uptake and post-RVD-RVI. The net uptake of cations (K uptake of 185.2, Na loss of 8.2 mmol/kg dry wt) during the isotonic period exceeded the Cl uptake by 38.2 mmol/kg dry wt, suggesting the uptake of another anion and/or the alteration of cellular buffer capacity. The relatively low Na concentration maintained during the isotonic period (13.3 vs. 20.4 mmol/kg wet wt under control conditions) might favour electrolyte uptake via the Na-K-2Cl cotransporter.

Heat shock proteins and the cellular response to osmotic stress.

In antidiuresis, the intrarenal distribution of HSP25/27, alphaB-crystallin, HSP72, OSP94 and HSP110 corresponds to the osmotic gradient between cortex and papilla: low amounts in the cortex and high values in the inner medulla and papilla. In addition, medullary HSP72 levels change appropriately with the diuretic state. Studies on MDCK cells suggest that, in the renal medulla in vivo, stressors, such as NaCl and low pH, may act in concert to induce HSP72 expression. Urea, added to the medium at high concentrations (600 mM), causes the majority of MDCK cells to die. Prior exposure of these cells to hypertonic media (NaCl addition), a maneuver that induces HSP72, protects the cells against the deleterious effects of high urea concentrations. Inhibition of HSP72 expression by stable antisense transfection or SB203580 treatment abolishes the beneficial effects of prior hypertonic stress. Conversely, overexpression of HSP72 under isotonic conditions by a dexamethasone-driven vector confers substantial resistance against subsequent exposure to high urea concentrations. Taken together these results suggest that also in the renal inner medulla, NaCl-induced enhancement of HSP72 expression may help counteract the detrimental effects of high urea concentrations.

Molecular chaperones in the kidney: distribution, putative roles, and regulation.

Molecular chaperones are intracellular proteins that prevent inappropriate intra- and intermolecular interactions of polypetide chains. A specific group of highly conserved molecular chaperones are the heat shock proteins (HSPs), many of which are constitutively expressed but most of which are inducible by diverse (in some cases specific) stress factors. HSPs, either alone or in cooperation with "partner" chaperones, are involved in cellular processes as disparate as correct folding and assembly of proteins, transport of proteins to specific intracellular locations, protein degradation, and preservation and restructuring of the cytoskeleton. The characteristic distribution of individual HSPs in the kidney, and their response to different challenges, suggests that a number of HSPs may fulfill specific, kidney-related functions. HSP72 and the osmotic stress protein 94 (Osp94) appear to participate in the adaptation of medullary cells to high extracellular salt and urea concentrations; the small HSPs (HSP25/27 and crystallins) may be involved in the function of mesangial cells and podocytes and contribute to the volume-regulatory remodeling of the cytoskeleton in medullary cells during changes in extracellular tonicity. HSP90 contributes critically to the maturation of steroid hormone receptors and may thus be a critical determinant of the aldosterone sensitivity of specific renal epithelial cells. Certain HSPs are also induced in various pathological states of the kidney. The observation that the expression of individual HSPs in specific kidney diseases often displays characteristic time courses and intrarenal distribution patterns supports the idea that HSPs are involved in the recovery but possibly also in the initiation and/or maintenance phases of these disturbances.

Inner-medullary organic osmolytes and inorganic electrolytes in K depletion.

The renal concentrating defect typical for chronic K depletion has been ascribed to malfunction of renomedullary cells caused by inadequate accumulation of organic osmolytes. A reduction in intracellular ionic strength, which is believed to influence decisively the accumulation of organic osmolytes, has been held responsible for insufficient osmolyte accumulation. To test this hypothesis, intra- and extracellular Na, Cl and K concentrations, the major determinants of ionic strength, were measured in the papilla by electron microprobe analysis and organic osmolytes (glycerophosphorylcholine, betaine, sorbitol, myo-inositol, free amino acids) in inner-medullary tissue by HPLC in antidiuretic rats kept on either a control (normal-K) or a K-deplete (low-K) diet and in euhydrated rats with free access to water and control diet. K depletion was associated with a reduced urine concentrating ability. Papillary interstitial ionic strength (sum of Na, Cl and K) in antidiuretic low-K rats was significantly reduced compared with antidiuretic normal-K rats (688+/-19 vs. 971+/-61 mmol/kg wet wt) but was similar to that in euhydrated normal-K rats (643+/-35 mmol/kg wet wt). The lower interstitial ionic strength in antidiuretic low-K and euhydrated normal-K rats was associated with a lower total content of organic osmolytes in the inner medulla (365+/-14 and 381+/-20, respectively, vs. 465+/-11 mmol/kg protein in antidiuretic normal-K rats). Intracellular ionic strength (sum of Na, Cl and K) of papillary collecting duct cells, however, was similar in antidiuretic normal-K and euhydrated normal-K rats (171+/-5 and 179+/-11 mmol/kg wet wt) but lower in antidiuretic low-K rats (138+/-9 mmol/kg wet wt). These results do not support the view that, in the steady state of osmotic adaptation of renomedullary cells in situ, intracellular ionic strength is the decisive factor for maintaining high levels of organic osmolytes. During chronic K depletion, reduced osmolyte accumulation by renomedullary cells may be the consequence, rather than the cause, of lower medullary interstitial tonicity.

Possible involvement of heat shock protein 25 in the angiotensin II-induced glomerular mesangial cell contraction via p38 MAP kinase.

In the rat kidney, mesangial cells (MCs), especially those in the extraglomerular mesangium (EGM) region of the juxtagomerular apparatus, express high amounts of heat shock protein 25 (HSP25). Because MCs are contractile in vivo and HSP25 is known to modulate polymerization/depolymerization of F-actin and to be involved in smooth muscle contraction, it is possible that HSP25 participates in the contraction process of MCs. We analyzed a permanent mouse MC line using Northern and Western blot analyses, and observed that similar to the MCs in the glomerulus, these cells also express high amounts of HSP25 constitutively. Exposure of these cells to angiotensin II (ANG II: 2 x 10(-7) M) evoked contraction and a concomitant increase in HSP25 phosphorylation, while the cytoplasmic fraction of HSP25 was transiently reduced. Because phosphorylation of HSP25 is essential for its actin-modulating function, we suppressed the activity of p38 MAP kinase, the major upstream activator of HSP25 phosphorylation, with the specific inhibitor SB 203580. This maneuver reduced HSP25 phosphorylation dramatically, abolished cell contraction, and prevented the decrease of the cytoplasmic HSP25 content. This suggests that HSP25 might be a component of the contraction machinery in MCs and that this process depends on p38 MAP kinase-mediated HSP25 phosphorylation. The decrease of cytoplasmic HSP25 content observed after ANG II exposure is probably the result of a transient redistribution of HSP25 into a buffer-insoluble fraction, because the whole cell content of HSP25 did not change, a phenomenon known to be related to the actin-modulating activity of HSP25. The fact that this function requires phosphorylation of HSP25 would explain the observation that HSP25 does not redistribute in SB 203580-pretreated cells.

Hypertonicity-induced accumulation of organic osmolytes in papillary interstitial cells.

BACKGROUND: Medullary cells of the concentrating kidney are exposed to high extracellular solute concentrations. It is well established that epithelial cells in this kidney region adapt osmotically to hypertonic stress by accumulating organic osmolytes. Little is known, however, of the adaptive mechanisms of a further medullary cell type, the papillary interstitial cell [renal papillary fibroblast (RPF)]. We therefore compared the responses of primary cultures of RPFs and papillary collecting duct (PCD) cells exposed to hypertonic medium. METHODS: In RPFs and PCD cells, organic osmolytes were determined by high-performance liquid chromatography; mRNA expression for organic osmolyte transporters [Na+/Cl(-)-dependent betaine transporter (BGT), Na(+)-dependent myo-inositol transporter (SMIT)], and the sorbitol synthetic and degrading enzymes [aldose reductase (AR) and sorbitol dehydrogenase (SDH), respectively] was determined by Northern blot analysis. RESULTS: Exposure to hypertonic medium (600 mOsm/kg by NaCl addition) caused intracellular contents of glycerophosphorylcholine, betaine, myo-inositol, and sorbitol, but not free amino acids, to increase significantly in both RPFs and PCD cells. The rise in intracellular contents of these organic osmolytes was accompanied by enhanced expression of mRNAs coding for BGT, SMIT, and AR in both RPFs and PCD cells. SDH mRNA abundance, however, was unchanged. Nonradioactive in situ hybridization studies on sections from formalin-fixed and paraffin-embedded, normally concentrating kidneys showed strong expression of BGT, SMIT, and AR mRNAs in interstitial and collecting duct cells of the papilla, whereas expression of SDH mRNA was much weaker in both cell types. CONCLUSIONS: These results suggest that both RPFs and PCD cells use similar strategies to adapt osmotically to the high interstitial NaCl concentrations characteristic for the inner medulla and papilla of the concentrating kidney.

Inhibition of NaCl-induced heat shock protein 72 expression renders MDCK cells susceptible to high urea concentrations.

Exposure of Madin-Darby canine kidney (MDCK) cells to elevated extracellular NaCl concentrations is associated with increased heat shock protein 72 (HSP72) expression and improved survival of these pretreated cells upon exposure to an additional 600 mM urea in the medium. To establish a causal relationship between HSP72 expression and cell protection against high urea concentrations, two approaches to inhibit NaCl-induced HSP72 synthesis prior to exposure to 600 mM urea were employed. First, the highly specific p38 kinase inhibitor SB203580 was added (100 microM) to the hypertonic medium (600 mosm/kg H2O by NaCl addition, 2 days of exposure), which significantly reduced HSP72 mRNA abundance and HSP72 content. Survival of these cells after a 24-h urea treatment (600 mM) was markedly curtailed compared with appropriate controls. Second, a pcDNA3-based construct, containing 322 bases of the HSP72 open reading frame in antisense orientation and the geneticine resistance gene, was transfected into MDCK cells. Clones with strong inhibition of HSP72 synthesis and others which express the protein at normal levels (comparable to nontransfected MDCK cells) after heat shock treatment or hypertonic stress were established. When these transformants were subjected to hypertonic stress for 2 days prior to exposure to an additional 600 mM urea for 24 h, cell survival was significantly reduced in those clones in which HSP72 expression was strongly inhibited. These results provide further evidence for the protective function of HSP72 against high urea concentrations in renal epithelial cells.

Influence of NaCl, urea, potassium and pH on HSP72 expression in MDCK cells.

The renal inner medulla is characterised by elevated extracellular concentrations of NaCl, urea, potassium and hydrogen ions, an environment that may affect cell viability negatively. High amounts of HSP72, a stress protein allowing cells to resist harmful situations, are also observed in this region. The present study examined HSP72 induction by various medullary stress factors, individually or in combination, in MDCK cells, a renal epithelial cell line expressing characteristics of the medullary collecting duct. MDCK cells were incubated for 3 days in media containing elevated concentrations of NaCl, urea, potassium and hydrogen ions individually or in combination. HSP72 mRNA and protein expression were determined by Northern and Western blot analyses, respectively. HSP72 expression was enhanced moderately by addition of 50 mM NaCl to normal medium at pH 7.4 but enhanced strongly when added at pH 6.5. The latter degree of HSP72 induction was comparable to that observed when 150 mM NaCl was added at pH 7.4. In normal medium (pH 7.4) containing 300 mM urea, MDCK HSP72 expression was not different from controls. In contrast, urea-induced HSP72 expression was clearly evident when medium pH was lowered to 6.5. Potassium at 20 or 40 mM induced HSP72 only slightly. These results indicate that expression of HSP72 in renal epithelial cells is regulated synergistically by NaCl, urea and pH. Since HSP72 is only slightly induced by increased potassium, this probably reflects the changes in medium osmolality rather than a specific effect of potassium. The high medullary HSP72 content observed even in diuresis may be due to co-operative effects of medullary solutes on HSP72 expression.

Cellular response to osmotic stress in the renal medulla.

Cells of the renal medulla, which are exposed under normal physiological conditions to widely fluctuating extracellular solute concentrations, respond to hypertonic stress by accumulating the organic osmolytes glycerophosphorylcholine (GPC), betaine, myo-inositol, sorbitol and free amino acids. Increased intracellular contents of these osmolytes are achieved by a combination of increased uptake (myo-inositol and betaine) and synthesis (sorbitol, possibly GPC), decreased degradation (GPC) and reduced osmolyte release. In the medulla of the concentrating kidney, accumulation of organic osmolytes, which do not perturb cell function even at high concentrations, allows the maintenance of "normal" intracellular concentrations of inorganic electrolytes. Adaptation to decreasing extracellular solute concentrations, e.g. diuresis, is achieved primarily by activation of pathways allowing the efflux of organic osmolytes, and secondarily by inactivation of production (sorbitol) and uptake (betaine, myo-inositol) and stimulation of degradation (GPC). Apart from modulation of the osmolyte content, osmolality-dependent reorganization of the cytoskeleton and expression of specific stress proteins (heat shock proteins) may be further, as yet poorly characterized, components of the regulatory systems involved in the adaptation of medullary cells to osmotic stress.

Cationic amino acid transporter mRNA expression in rat kidney and liver.

Expression of rat cationic amino acid transporter 2 (r-CAT-2) mRNA was studied in kidney and liver using Northern blot analysis and nonradioactive in situ hybridization with a probe identifying both the r-CAT-2alpha and -2beta splice variants. Expression of r-CAT-2 mRNA was higher in the liver than in the kidney. Within the kidney, r-CAT-2 mRNA was more abundant in the outer and inner medulla than in the cortex. In the liver lobule, the intensity of the hybridization signal in hepatocytes decreased between the portal area and the central vein. In the kidney, hybridization signals were detected in parietal cells of Bowman's capsule, various tubule cells of outer and inner medulla, in endothelial and interstitial cells of inner medulla, and in papillary epithelial cells.

Influence of osmotic stress on heat shock proteins 25 and 72 in mouse mesangial cells.

Previous studies have shown intense staining for heat shock protein 25 (HSP25) in the extraglomerular mesangium (EGM). Because relationships are believed to exist between osmotic stress, expression of HSP25, and protection against stress and because the EGM may be exposed to high local tonicity, we examined the expression of HSP25 and the major stress-inducible and cytoprotective HSP72 in mouse mesangial cells and embryonic lung fibroblasts (3T3) after exposure to hypertonic stress (addition of 150 mM NaCl to the medium for two to seven days). Mesangial, but not 3T3, cells expressed high levels of HSP25 already under control conditions, whereas neither cell line contained HSP72. Hypertonic treatment neither enhanced (mesangial cells) or induced (3T3 cells) HSP25 expression. HSP72, however, was induced strongly in 3T3 cells, but only minimally in mesangial cells. The high level of HSP25 in mesangial cells thus seems not to be a consequence of high tonicity in the EGM because cultured mesangial cells express HSP25 already under control conditions, and osmotic stress did not induce HSP25 in either cell line. Furthermore, high amounts of HSP25 seem to reduce the requirement for HSP72 after stress exposure, suggesting that, in mesangial cells, HSP25 might assume some functions of HSP72.

Hypertonicity affects heat shock protein 27 and F-actin localization in Madin-Darby canine kidney cells.

High concentrations of NaCl are known to perturb the cytoskeleton. In this study, expression and intracellular localization of actin, an important component of the cytoskeleton and of heat shock protein (HSP)27, which promotes the assembly of F-actin, were examined in Madin-Darby canine kidney (MDCK) cells grown chronically in hypertonic medium. HSP27 mRNA abundance was increased twofold compared with wild-type MDCK cells. Chronic hypertonic stress led to enrichment of HSP27 in the insoluble component of the cell lysate and colocalization with cortical F-actin. These results support the notion that HSP27 participates in the modulation of actin dynamics following hypertonic stress.

Effect of ischemia on localization of heat shock protein 25 in kidney.

The effects of renal ischemia on the intracellular distribution of the low-molecular weight heat shock protein (HSP)25 were examined using immunofluorescence microscopy. In all kidney zones, ischemia decreased HSP25 in the supernatant of the tissue homogenates and increased it in the pellet fraction (containing mainly nuclei and cytoskeletal components). This was associated with disappearance of HSP25 staining from the brush border of proximal convoluted tubule (PCT) cells. Because no nuclear staining of cortical tubule cells was apparent either in control or ischemic kidneys, ischemia seems to cause a closer association of HSP25 with cytoskeletal components. HSP25 probably participates in the postischemic restructuring of the cytoskeleton of PCT cells.

Quantitative morphology of renal cortical structures during compensatory hypertrophy.

The compensatory hypertrophy in different renal cortical structures was studied in rats 10 and 21 days after unilateral nephrectomy (UNX). Quantitative morphological/stereological analysis revealed significant increases in total renal cortical volume--33% on day 10 and 48% on day 21--after UNX. These changes were paralleled by significant increments in the volumes of proximal convoluted tubule (PCT, 55%), distal convoluted tubule (DCT, 114%), and cortical collecting duct (CCD, 106%) segments on day 10. The corresponding changes on day 21 were 76, 122, and 212%, respectively. These alterations were accompanied by increases in segment length; 3% PCT, 23% DCT, and 50% CCD on day 10 and 9% PCT, 30% DCT, and 142% CCD on day 21 after UNX. The total luminal and basolateral cell membrane surface areas also exhibited a time-dependent increase after UNX. The increments in both luminal and basolateral membrane domains in PCT and DCT after 10 days were not significant, but reached significance after 21 days (PCT: luminal membrane 21%, basolateral membrane 63%; DCT: luminal membrane 98%, basolateral membrane 63%). In contrast, CCD membrane areas had increased substantially already 10 days after UNX (luminal membrane 92%, basolateral membrane 71%). It declined subsequently by day 21 (luminal membrane 57%, basolateral membrane 32%). The cell rubidium concentration after a 30-second rubidium infusion, an index of Na-K-ATPase activity, as well as sodium concentrations were unaltered in cells of all nephron segments investigated. Altogether the stereological analysis shows that the compensatory increase in organ volume can be attributed primarily to an increase in nephron epithelial volume. The PCT responds with 'radial' hypertrophy (thickening of the tubular epithelial wall), while the DCT undergoes 'length' hypertrophy (increase of tubular length without thickening of the tubular wall and without an increase in number of cells). This type of hypertrophy is especially prominent on day 21 after UNX for the CCD which doubles in length. Only on day 10 does the CCD seem to respond with hyperplasia. Adaptive changes in response to UNX develop gradually. Only a few of the morphological parameters studied had completed their change by 10 days, the majority required longer.

Expression of Na+/Cl-/betaine and Na+/myo-inositol transporters, aldose reductase and sorbitol dehydrogenase in macula densa cells of the kidney.

It has been suggested that macula densa cells may be exposed to hyperosmotic stress. Since chronic exposure to hypertonic stress causes the amount of intracellular organic osmolytes to increase, the expression of transporters and enzymes that participate in the intracellular accumulation of organic osmolytes was examined using non-radioactive in situ hybridization in the macula densa region of control rats and furosemide-treated animals. Both the sodium- and chloride-dependent betaine transporter (BGT) and sodium-dependent myo-inositol transporter (SMIT) were expressed preferentially in macula densa cells and for both mRNAs the signal intensity was visibly reduced by furosemide. The enzymes aldose reductase (which mediates the conversion of glucose to sorbitol) and sorbitol dehydrogenase (which converts sorbitol into fructose) were expressed not only in macula densa cells but also in the surrounding tubular cells, and the expression was insensitive to furosemide. Thus it remains unclear whether the expression of BGT and SMIT is related to a putative hypertonic juxtaglomerular region.

Cellular site of active K absorption in the guinea-pig distal colonic epithelium.

The mammalian distal colon, which is composed of different cell types, actively transports Na, K and Cl in absorptive and K and Cl in secretory directions. To further characterize the K absorption process and to identify the cells involved in K absorption, unidirectional Rb fluxes and luminal Rb uptake into different epithelial cell types were determined in isolated guinea-pig distal colon. Net Rb absorption (1.5-2.5 micromol.h-1.cm-2) was not influenced by inhibition of Na transport with amiloride or by incubating both sides of the epithelium with Na-free solutions, but was almost completely abolished by luminal ouabain, ethoxzolamide or by incubating both sides of the epithelium with Cl-free solutions. Luminal Rb uptake, blockable by luminal ouabain, preferentially occurred in columnar surface and neck cells, to a lesser extent in surface goblet cells and to an insignificant degree in lower crypt cells. Employing a luminal Rb-Ringer (5.4 mM Rb) the Rb concentration increased within 10 min in columnar surface and neck, surface goblet and lower crypt cells to 70, 32 and about 10 mmol. kg-1 wet weight, respectively. The presence of 5.4 mM K in the luminal incubation solution reduced Rb uptake almost completely indicating a much higher acceptance of the luminal H-K-ATPase for K than for Rb. The increase in Na and decrease in K concentrations in surface and neck cells induced by luminal ouabain might indicate inhibition of the basolateral Na-K-ATPase or drastic enhancement of cellular Na uptake by the Na-H exchanger. Bilateral Na-free incubation did not alter Rb uptake, but bilateral Cl-free incubation drastically reduced it. Inhibition of net Rb absorption by ethoxzolamide and inhibition of both Rb absorption and Rb uptake by bilateral Cl-free incubation support the notion that cellular CO2 hydration is a necessary prerequisite for K absorption and that HCO3 leaves the cell via a Cl-HCO3 exchanger. Since ouabain-inhibitable transepithelial Rb flux and luminal Rb uptake rate by surface and neck cells were about the same, Rb(K) absorption seems to be accomplished mainly by columnar surface cells.

The response of heat shock proteins 25 and 72 to ischaemia in different kidney zones.

Induction of heat shock proteins (HSPs) following cell injury contributes to the protection of vital cell functions. It was, therefore, of interest to study the effects of transient renal ischaemia on the abundance and distribution of two HSPs, HSP25 and HSP72, in renal tissue using Western-blot techniques. Analyses were performed on the supernatant (HSP25, HSP72) and pellet (HSP25) of homogenates obtained from cortex (CX) and outer (OM) and inner (IM) medulla of the rat kidney immediately after 60 min of ischaemia followed by varying periods of reperfusion. Ischaemia of the left kidney caused HSP25 contents to decrease in CX, OM and IM by 73, 89 and 54% respectively, compared with the corresponding zones of the contralateral control kidney. This initial decrease in supernatant HSP25 was accompanied by an increased abundance of HSP25 in the pellet. Following reperfusion, HSP25 contents in the supernatant gradually increased in CX and OM, reaching, after 24 h, values that were 5.4- and 2.5-fold higher, respectively, than those in the control kidneys. After 7 or 14 days of reperfusion, HSP25 contents had not completely normalised in CX, but had reached control levels in OM. In IM, the HSP25 content remained below control throughout the entire reperfusion period. HSP72 (supernatant) was below the detection limit in the CX of the control kidney. Similar to the level of HSP25, that of HSP72 was also markedly lower in OM and IM immediately after ischaemia. The intrarenal distribution of HSP72 and the sequence of zonal changes in HSP72 contents were similar to those observed for HSP25. These results are compatible with the view that, during ischaemia and the initial reperfusion period, HSP25 migrates from the cytoplasmic compartment (supernatant) into the nucleus and/or associates with cytoskeletal structures. The observation that both HSP25 and HSP72 are transiently induced in CX and OM, but not in IM, may be explained by the fact that, while all kidney cells are exposed to ischaemic stress, only inner medullary cells experience a major postischaemic attenuation of osmotic stress.

Tracing the Na/K-ATPase with rubidium.

Rubidium (Rb) was used as a marker ion for K to assess Na/K(Rb)-ATPase activity in single renal tubule cells. Initial Rb uptake rates were measured by electron microprobe analysis in individual tubule cells of the rat kidney during acute stimulation or during inhibition of transepithelial Na absorption. Under these conditions, Rb uptake closely correlates with intracellular Na concentrations, indicating that the intracellular Na concentration is a major determinant in the precise adjustment of basolateral, Na/K(Rb)-ATPase-dependent Na extrusion to Na entry across the apical cell membrane. Chronically increased distal Na delivery induced by loop diuretics triggers adaptive processes which allow increased transcellular Na movement at normal or near-normal intracellular Na concentrations.