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.

Pretreatment with hypertonic NaCl protects MDCK cells against high urea concentrations.

In antidiuresis, the cells of the renal medulla are exposed to high extracellular concentrations of NaCl and urea. Since urea equilibrates with the intracellular compartment and is known to perturb intracellular macromolecules, high urea concentrations may well disturb the structure and function of cell proteins. Two types of organic substances are believed to counteract the adverse effects of high intracellular urea concentrations: specific organic osmolytes of the trimethylamine family [betaine and glycerophosphorylcholine (GPC)], which accumulate in renal medullary cells during prolonged periods of antidiuresis and cytoprotective heat shock proteins (HSPs), the tissue content of two of which (HSPs 27 and 72) is much higher in the inner medulla than in the iso-osmotic renal cortex. To evaluate the contribution of trimethylamines and HSPs to cytoprotection in the presence of high urea concentrations, the effect of HSP induction and osmolyte accumulation prior to exposure to high urea concentrations was examined in Madin-Darby canine kidney (MDCK) cells. Accumulation of organic osmolytes and synthesis of HSP27 and HSP72 was initiated by hypertonic stress (increasing the osmolality of the medium from 290 to 600 mosmol/kg H2O by NaCl addition). Control, non-conditioned cells remained in the isotonic medium for the same period. Upon subsequent exposure to an additional 600 mM urea in the medium for 24 h, 90% of the osmotically conditioned cells but only 15% of non-conditioned cells survived. The HSP72 and trimethylamine contents of the NaCl-conditioned MDCK cells, but not HSP27 content, correlated positively with cell survival. To separate the effects of organic osmolytes and HSP72, chronically NaCl-adapted MDCK cells were returned to isotonic medium for 1 or 2 days, so depleting them of trimethylamine osmolytes. HSP72, with its longer half life, remained elevated. Subsequent exposure of these cells to 600 mM urea in the medium resulted in about 80% survival. These results suggest that in MDCK cells and probably in the renal medulla, HSP72 and perhaps additional protective factors contribute substantially to the resistance against high urea concentrations.

Solute composition and heat shock proteins in rat renal medulla.

The high content of heat shock proteins (HSPs) 25 and 72 in the hyperosmotic inner medulla of the concentrating kidney has been ascribed to the high NaCl and urea concentrations in this kidney zone. To assess the effects of variations in the composition of solutes in the renal medulla on the intrarenal distribution of HSPs, rats were fed either a high- or low-Na diet for 3 weeks. These diets result in greatly differing urine and inner medullary solute composition. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot techniques were used to analyse HSP25 and HSP72 in the cortex, outer medulla and inner medulla. In addition, the amounts of organic osmolytes (sorbitol, myo-inositol, betaine and glycerophosphorylcholine) and urea in the tissue were determined by high-performance liquid chromatography. Intra- and extracellular electrolyte concentrations at the papillary tip were measured by electron microprobe analysis. In the high-Na group, urine osmolality was about 1000 mosmol/kg lower than in rats fed a low-Na diet, due to lower urea concentrations. The sum of urine sodium and potassium concentrations, however, did not differ between the two groups. Neither in the outer nor in the inner medulla was the sum of the concentrations of organic osmolytes affected by the dietary treatment. The sum of sodium, potassium and chloride concentrations did not differ between the two experimental groups, neither in the interstitial nor in the intracellular compartments. However, the urea content and the amounts of HSP25 and HSP72 were significantly lower in the inner medulla of the group of rats fed a high-Na diet. Our results suggest that urea participates in the regulation of the medullary levels of the HSPs and that both HSP25 and HSP72 are components of mechanisms protecting medullary cells against the deleterious effects of high urea concentrations.

Ketoconazole inhibits organic osmolyte efflux and induces heat shock proteins in rat renal medulla.

Although ketoconazole (KC) is known to inhibit the cellular efflux of organic osmolytes in vitro, it is not known whether this effect can also be shown in vivo. Inhibition of osmolyte efflux by KC would impair osmotic adaptation and result in stress to the cells of the renal medulla when extracellular osmolality falls. Stress-inducible heat shock proteins (HSPs) may also participate in this response to osmotic stress. The aim of the present study was thus to establish whether KC inhibits organic osmolyte efflux from the cells of the renal medulla in vivo in response to a furosemide diuresis, and to establish whether HSPs are involved. A 20-minute furosemide infusion reduced urine osmolality and medullary urea content in control and KC-treated rats similarly. However, the efflux of methylamines (glycerophosphorylcholine, betaine) and polyols (myo-inositol, sorbitol) was attenuated in KC-treated rats while the efflux of amino acids was not significantly affected. Phosphorylation of HSP25 after the 20-minute furosemide diuresis was increased in KC rats. With continuing diuresis this returned to control levels after three hours. While short-term (up to 3 hr) diuresis did not alter the absolute amounts of HSPs in the renal medulla, long-term (24 or 48 hr) diuresis was associated with significantly increased amounts of HSP25 and HSP72 in KC-treated rats compared with control. These results suggest that KC inhibits the efflux of methylamines and polyols, thus impeding osmoadaptation of renal medullary cells during the onset of diuresis. This situation apparently increases the osmotic stress experienced by the cells of the renal medulla and provokes expression of HSP25 and HSP72.