Eva Müller

Cellular response to osmotic stress in the renal medulla

Abstract 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.

HEAT SHOCK PROTEINS HSP25, HSP60, HSP72, HSP73 IN ISOOSMOTIC CORTEX AND HYPEROSMOTIC MEDULLA OF RAT KIDNEY

The distribution of heat shock proteins (HSP) HSP60, HSP73, HSP72 and HSP25 in the isoosmotic cortex and the hyperosmotic medulla of the rat kidney was investigated using Western blot analysis and immunohistochemistry. HSP73 was homogeneously distributed throughout the whole kidney. The level of HSP60 was high in the renal cortex and low in the medulla. HSP25 and HSP72 were present in large amounts in the medulla. Only low levels of HSP25 and almost undetectable amounts of HSP72 were found in the cortex. HSP25 exists in one nonphosphorylated and several phosphorylated isoforms. Western blot analysis preceded by isoelectric focussing showed that HSP25 predominates in its nonphosphorylated form in the outer medulla but in its phosphorylated form in cortex and inner medulla. Although this intrarenal distribution pattern was not changed during prolonged anaesthesia (thiobutabarbital sodium), a shift from the nonphosphorylated to the phosphorylated isoforms of HSP25 occurred in the medulla. The characteristic intrarenal distribution of the constitutively expressed HSPs (HSP73, HSP60, HSP25) may reflect different states of metabolic activity in the isoosmotic (cortex) and hyperosmotic (medulla) zones of the kidney. The high content of inducible HSP72 in the medulla most likely is a consequence of the osmotic stress imposed upon the cells by the high urea and salt concentrations in the hyperosmotic medullary environment.

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

Abstract 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 µM) 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.

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

Abstract 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.

Effects of long-term changes in medullary osmolality on heat shock proteins HSp25, HSP60, HSP72 and HSP73 in the rat kidney.

Abstract The influence of diuresis and antidiuresis on the expression of heat shock proteins (HSP) 25, 60, 72 and 73 in the renal cortex and outer and inner medulla of Wistar rats was analysed. Medullary osmolality was reduced by long-term diuresis (3% sucrose in the drinking water for 3 weeks) and subsequently enhanced by transition to a concentrating state by giving normal drinking water again in combination with deamino-D-arginine vasopressin (dDAVP) for 5 days. Western blot analyses revealed that neither HSP73 nor HSP60 was influenced by any treatment. The HSP72 level in the medulla was markedly reduced (50%) when osmolality was lowered and increased when tonicity was high. RNAse protection assays showed that the effects on HSP72 are parallelled in general by changes in HSP72 mRNA. While levels of HSP25 were not influenced, isoelectric focusing revealed that the degree of phosphorylation of outer and inner medullary HSP25 increased following both treatments. It thus seems that HSP73 and HSP60 are not directly involved in the long-term adaptation to varying medullary osmolalities. The correlation between changes in osmolality and amounts of the major stress-inducible HSP72 in the medulla implies that medullary hypertonicity is stressful for kidney cells. Furthermore, adaptation to pronounced changes in the osmolality of the environment most likely involves phosphorylation of HSP25.

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 × 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. J. Cell. Physiol. 181:462–469, 1999.

Ifosfamide induced stress response in human lymphocytes

Ifosfamide, an isomer of cyclophosphamide, has been shown to be one of the most effective antineoplastic agents for the treatment of human malignancies. There is considerable evidence that the intracellular status of glutathione (GSH) plays a major role in modifying the cytotoxicity of ifosfamide in cells and tissues. We have studied the effects of 4-hydroperoxy-ifosfamide (4-OOH-IF) upon the proliferation of human peripheral blood lymphocytes (PBL) and the intracellular GSH content. The major finding was that occurrence of significant inhibition of [3H]-thymidine incorporation in interleukin-2 (IL-2) expanded PBL after exposure with 4-OOH-IF was accompanied by substantial depletion of intracellular GSH content in these cells. PBL seemed to be more sensitive to this drug induced effect comparing our results obtained in other cells (e.g. Ewing sarcoma, Chinese hamster ovary). In PBL 4-OOH-IF also induced rapid phosphorylation of the small heat shock protein (HSP27) signaling a similar type of stress response as reported for several other agents (e.g. arsenite, phorbol ester, tumor necrosis factor). Reconstitution of the depleted GSH content in PBL after treatment with 4-OOH-IF could be achieved by GSH-monoethylester and mesna within 24 hours of postincubation time. From these results we conclude that human lymphocytes are sensitive targets for ifosfamide induced metabolic stress during treatment. This might have further importance in regard to the immunological function of these cells.

Experimentelle Untersuchungen zur Pathogenese des mikrobiellen Ekzems

ZusammenfassungAnhand von 162 Excisaten wird das histologische Bild epicutaner Testreaktionen beim Meerschweinchen dargestellt, wie sie nach erstmaliger Applikation von lebenden Staphylococcus aureus (haem.)-Stämmen bzw. daraus hergestelltem Cellophankulturfiltrat auf die normale Haut gesunder Tiere auftreten. Die hierbei nach Anwendung lebender Keime erzielten feingeweblichen Veränderungen scheinen komplexer Natur zu sein, während die nach Auftragen des Kulturfiltrates zu beobachtende Reaktion einen mehr einheitlichen Charakter erkennen läßt. Das Studium des zeitlichen Ablaufs dieser Reaktion zeigt so weitgehende Parallelen zu den auslösenden Testreaktionen beim tierexperimentellen DNCB-Ekzem, daß sie nicht davon abgetrennt werden kann.

Interaction between tumor necrosis factor-α and HSP 70 in human leukemia cells

Abstract We confirm here by immunoblotting that a cell line resistant to clonal inhibition from tumor necrosis factor (K562) expresses high levels of HSP 70, whereas two sensitive cell lines (HL60 blasts and EM3) express low levels. When sensitive cell lines are exposed to tumor necrosis factor, HSP 70 is not generally induced; when tumor necrosis factor is removed, however, HSP 70 is transiently induced. In five bone marrow samples from patients, generally low levels of HSP 70 were detected.

Solute composition and heat shock proteins in rat renal medulla

Abstract 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.