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.     

Tubular stress proteins and nitric oxide synthase expression in rat kidney exposed to mercuric chloride and melatonin.

Stress proteins such as HSP70 members (HSP72 and GRP75) and metallothionein (MT) protect the kidney against oxidative damage and harmful metals, whereas inducible nitric oxide synthase (iNOS) regulates tubular functions. A single dose of mercuric chloride (HgCl(2)) can cause acute renal failure in rats, its main target being the proximal tubule. Oxidative damage has been proposed as one of its pathogenic mechanisms. In this study we tested whether melatonin (MEL), a powerful antioxidant compound, is effective against HgCl(2) nephrotoxicity. Rats were treated with saline, HgCl(2) (3.5 mg/kg), MEL (5 mg/kg), and MEL + HgCl(2) and examined after 24 hr for HSP72, GRP75, MT, and iNOS by immunohistochemistry and immunoblotting. Tubular effects of the treatment were then characterized by ultrastructure. In the HgCl(2) group, all markers were overexpressed in convoluted proximal tubules and sometimes in distal tubules. In the MEL + HgCl(2) group, GRP75 and iNOS decreased in convoluted and straight proximal tubules, whereas HSP72 and MT persisted more than the saline and MEL-only groups. Tubular damage and mitochondrial morphometry were improved by MEL pretreatment. In conclusion, the beneficial effect of MEL against HgCl(2) nephrotoxicity was outlined morphologically and by the reduction of the tubular expression of stress proteins and iNOS. These markers could represent sensitive recovery index against mercury damage.     

Effects of nickel poisoning on expression pattern of the 72/73 and 94 kDa stress proteins in rat organs and in the COS-7, HepG2, and A549 cell lines

The present study deals with the effects of Ni on the expression level of three stress proteins, namely, the cytosolic HSP72 and HSP73, and the reticulum-associated GRP94. Experiments were carried out on "Wistar' female rats daily injected with 4 mg NiCl2 per kg body weight for 1, 3, 5, and 10 days. Another set of experiments were carried out using cell lines, derived from the monkey kidney (COS-7), and from human tumors of the lung (A549) and liver (HepG2). Cells were cultured for 4 days in the permanent presence of 100, 200, or 400 microM NiCl2. In control rats, stress proteins pattern was found to be tissue specific: two protein bands of 96 and 94 kDa were immunodetected with the anti-GRP94 antibody in kidney and liver extracts, whereas only the 96 kDa band was present in ovary extracts. HSP73 was present in kidney, liver, and ovary whereas HSP72 was only found in kidney. In kidney of nickel-treated animals, HSP73 and the 96 kDa proteins were overexpressed whereas HSP72 was strongly down regulated. No such effect was observed in liver or ovary. Similarly, in nickel-treated cell lines, HSP72 was downregulated and GRP94 (96 kDa protein) was overexpressed. HSP73 expression appeared moderately increased in A549 cells but decreased in COS-7 cells. Because long-term caloric restriction was reported to reduce free radical generation in cells, the effect of 1 month food restriction (50%) was tested in rats as a possible way to lower oxidative damages induced by Ni. No significant effect on HSP expression was observed.     

Immunohistochemical detection of betaine-homocysteine S-methyltransferase in human, pig, and rat liver and kidney

Betaine-homocysteine S-methyltransferase (BHMT) has been shown to be expressed at high levels in the livers of all vertebrate species tested. It has also been shown to be abundant in primate and pig kidney but notably very low in rat kidney and essentially absent from the other major organs of monogastric animals. We recently showed by enzyme activity and Western analysis that pig kidney BHMT was only expressed in the cortex and was absent from the medulla. Using immunohistochemical detection, we report here that in human, pig, and rat kidney, BHMT is expressed in the proximal tubules of the cortex. Immunohistochemical staining for BHMT in human, pig, and rat liver indicate high expression in hepatocytes. The staining patterns are consistent with cytosolic expression in both organs.     

An investigation into the sensitivity of heat shock proteins as markers of cellular damage: a comparative study of hydrazine and cadmium chloride in primary rat hepatocyte cultures

Stress proteins have been proposed as markers of toxicity. This study investigated the sensitivity and specificity of stress proteins as markers of toxicity in primary hepatocyte cultures following exposure to two compounds, hydrazine and cadmium chloride (CdCl ). 2 Hepatocytes were exposed to increasing concentrations of hydrazine and CdCl for 2 h 2 and levels of the heat shock proteins HSP72/3, and HSP25 measured. In addition to this, ATP and GSH levels and LDH leakage were measured over the following 8 h. The results show that increasing concentrations of hydrazine caused dose-dependent decreases in ATP and GSH levels over 8 h. There was no change in the levels of HSP25 or HSP72/3 over that period. CdCl was found to significantly induce HSP72/3 at a concentration of 2 5 M when no other biochemical parameter was altered, levels were also elevated following administration of 10 M CdCl but ATP levels were found to be decreased at this 2 concentration. Levels of HSP25 were not increased following CdCl exposure at any 2 concentration. Higher concentrations of CdCl produced significant increases in LDH 2 leakage and depletion of intracellular levels of ATP and GSH. In addition to this levels of HSP25 and HSP72/3 were reduced to zero following administration of high concentrations of CdCl . In this study hydrazine does not induce either of the stress 2 proteins studied here whereas CdCl exposure causes the induction of HSP72/3 but not 2 HSP25. However it was determined that during the culture of primary hepatocytes basal levels of HSP25 and HSP72/3 were significantly increased when compared with levels determined in vivo . The results suggest that stress proteins may have the potential to be sensitive markers of toxicity in primary hepatocytes; however, the induction of individual stress proteins appears to be dependent upon the compound used. The apparent noninduction of the stress response by hydrazine and minor induction by CdCl might be 2 explained by the fact that whilst in culture the hepatocytes are under a continuous state of stress and therefore may not be able to elicit a full stress response following a chemical insult.     

Targeting GRP75 Improves HSP90 Inhibitor Efficacy by Enhancing p53-Mediated Apoptosis in Hepatocellular Carcinoma

Heat shock protein 90 (HSP90) inhibitors are potential drugs for cancer therapy. The inhibition of HSP90 on cancer cell growth largely through degrading client proteins, like Akt and p53, therefore, triggering cancer cell apoptosis. Here, we show that the HSP90 inhibitor 17-AAG can induce the expression of GRP75, a member of heat shock protein 70 (HSP70) family, which, in turn, attenuates the anti-growth effect of HSP90 inhibition on cancer cells. Additionally, 17-AAG enhanced binding of GRP75 and p53, resulting in the retention of p53 in the cytoplasm. Blocking GRP75 with its inhibitor MKT-077 potentiated the anti-tumor effects of 17-AAG by disrupting the formation of GRP75-p53 complexes, thereby facilitating translocation of p53 into the nuclei and leading to the induction of apoptosis-related genes. Finally, dual inhibition of HSP90 and GRP75 was found to significantly inhibit tumor growth in a liver cancer xenograft model. In conclusion, the GRP75 inhibitor MKT-077 enhances 17-AAG-induced apoptosis in HCCs and increases p53-mediated inhibition of tumor growth in vivo. Dual targeting of GRP75 and HSP90 may be a useful strategy for the treatment of HCCs.     

Cadmium-induced mRNA expression of Hsp32 is augmented in metallothionein-I and -II knock-out mice

Cadmium is toxic and carcinogenic to humans and animals. The testis and lung are the target organs for cadmium carcinogenesis. Heat shock proteins (HSPs) as well as metallothionein (MT) and glutathione (GSH) play an important role in protection against its toxicity. HSP32, also known as heme oxygenase-1, is a 32-kDa protein induced by heme, heavy metals, oxidative stresses, and heat. We investigated expression of the Hsp32 gene of various organs (the liver, lung, heart, stomach, kidney, and testis) in transgenic mice deficient in the MT-I and -II genes (MT-KO) and in control mice (MT-W) after an injection of cadmium chloride (CdCl2). Survival of MT-W mice after a subcutaneously injection of CdCl2 was higher than that of MT-KO mice, while no significant difference was observed in the level of GSH in each organ between MT-W and MT-KO mice. Northern blot analysis showed that the MT-I mRNA was more extensively induced in the liver, kidney, and heart than other organs 6 h after an injection of CdCl2 (30 micromol/kg body wt, sc). There was little increase of the MT-I mRNA in the testis when induced by CdCl2. Expression of the Hsp32 gene in the liver and kidney in response to CdCl2 was more extensively augmented in MT-KO mice than in MT-W mice. In the lung and testis, there was little induction and no augmentation in expression of the Hsp32 gene induced by CdCl2 in both MT-W and MT-KO mice. In the stomach, there was little induction of the Hsp32 mRNA in MT-W mice, but was increased in MT-KO mice. Immunohistochemical staining revealed that the HSP32 protein was strongly expressed in the kidney and liver of MT-W mice 24 h after an injection of CdCl2 (20 micromol/kg body wt, sc), while the expression of HSP32 protein was not increased in the testis. In metabolically active organs such as the liver and kidney, expression of the Hsp32 gene as well as the MT-I gene was extensively induced by cadmium in MT-W mice, and more eminently induced in MT-KO mice. We suggest that organs of low stress response to cadmium such as the testis and lung may be vulnerable target sites for cadmium toxicity and carcinogenesis.     

Comparative characterization of the 21-kD and 26-kD gap junction proteins in murine liver and cultured hepatocytes

Affinity-purified antibodies to mouse liver 26- and 21-kD gap junction proteins have been used to characterize gap junctions in liver and cultured hepatocytes. Both proteins are colocalized in the same gap junction plaques as shown by double immunofluorescence and immunoelectron microscopy. In the lobules of rat liver, the 21-kD immunoreactivity is detected as a gradient of fluorescent spots on apposing plasma membranes, the maximum being in the periportal zone and a faint reaction in the perivenous zone. In contrast, the 26-kD immunoreactivity is evenly distributed in fluorescent spots on apposing plasma membranes throughout the rat liver lobule. Immunoreactive sites with anti-21 kD shown by immunofluorescence are also present in exocrine pancreas, proximal tubules of the kidney, and the epithelium of small intestine. The 21-kD immunoreactivity was not found in thin sections of myocardium and adult brain cortex. Subsequent to partial rat hepatectomy, both the 26- and 21-kD proteins first decrease and after approximately 2 d increase again. By comparison of the 26- and 21-kD immunoreactivity in cultured embryonic mouse hepatocytes, we found (a) the same pattern of immunoreactivity on apposing plasma membranes and colocalization within the same plaque, (b) a similar decrease after 1 d and subsequent increase after 3 d of both proteins, (c) cAMP-dependent in vitro phosphorylation of the 26-kD but not of the 21-kD protein, and (d) complete inhibition of intercellular transfer of Lucifer Yellow in all hepatocytes microinjected with anti-26 kD and, in most cases, partial inhibition of dye transfer after injection of anti-21 kD. Our results indicate that both the 26-kD and the 21-kD proteins are functional gap junction proteins.     

Immunohistochemical Localization of Arginase II and Other Enzymes of Arginine Metabolism in Rat Kidney and Liver

Arginine is a precursor for the synthesis of urea, polyamines, creatine phosphate, nitric oxide and proteins. It is synthesized from ornithine by argininosuccinate synthetase and argininosuccinate lyase and is degraded by arginase, which consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Recently, cDNAs for human and rat arginase II have been isolated. In this study, immunocytochemical analysis showed that human arginase II expressed in COS-7 cells was localized in the mitochondria. Arginase II mRNA was abundant in the rat small intestine and kidney. In the kidney, argininosuccinate synthetase and lyase were immunostained in the cortex, intensely in proximal tubules and much less intensely in distal tubules. In contrast, arginase II was stained intensely in the outer stripes of the outer medulla, presumably in the proximal straight tubules, and in a subpopulation of the proximal tubules in the cortex. Immunostaining of serial sections of the kidney showed that argininosuccinate synthetase and arginase II were collocalized in a subpopulation of proximal tubules in the cortex, whereas only the synthetase, but not arginase II, was present in another subpopulation of proximal tubules. In the liver, all the enzymes of the urea cycle, i.e. carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase and lyase and arginase I, showed similar zonation patterns with staining more intense in periportal hepatocytes than in pericentral hepatocytes, although zonation of ornithine transcarbamylase was much less prominent. The implications of these results are discussed.     

Differences in the incorporation of L- and DL-Amino acids into renal tubular cells. An autoradiographic study.

The cytoplasmic uptake of 3H-L-leucine and 3H-L-proline by hepatocytes and cells of the proximal and distal convoluted and of the collecting tubules of the kidney was compared with that of 3H-DL-leucine and 3H-DL-proline in an autoradiographic study. 34 male white Sprague-Dawley rats were killed 1, 2, 6, and 24 hours after the intraperitoneal injection of these amino acids. The rate of incorporation of 3H-L-leucine in the liver and in the renal tubules, as judged by the number of silver grains counted, was about twice that of 3H-L-proline. In the tubules of the kidney the intensity of labelling progressively declined from the proximal convoluted to the collecting tubules. When the two 3H-DL-amino acids were used, almost identical rates of incorporation were found in the liver as well as in the kidney. The only exception was the pars recta of the proximal tubule: Here there could be found an unusually high uptake of 3H-DL-proline. The values were not only higher than those found for the uptake of 3DL-leucine in this particular segment, but they also surpassed those due to 3H-DL-proline and 3DL-leucine in the other parts of the renal tubules, as well as in the liver. The conspicuously high labelling seen in the pars recta after the injection of 3H-DL-proline suggests that there is present in the cells of this segment a d-amino acid oxidase, which may be relatively specific for D-proline. The possibility is considered that this enzyme may participate in a detoxifying function of the pars recta.     

Immunocytochemical localization of cathepsin B in rat kidney. I. Light microscopic study using the indirect immunoenzyme technique

Localization of cathepsin B in rat kidney was studied using immunocytochemical techniques. Cathepsin B was purified from rat liver and antibody to it was raised in rabbits. The antibody reacted with a lysosomal extract of rat kidney to form a single precipitin line in a double-diffusion test. Immunoblot analysis of lysosomal cathepsin B of rat kidney showed two species of 29K and 25K MW. After removal of Epon, semi-thin sections of glutaraldehyde-fixed tissue were stained by the indirect immunoenzyme technique. Dark-brown reaction product, indicating the antigenic sites for cathepsin B, was found in cytoplasmic granules throughout the nephron. Staining intensity and size of the positive granules varied widely in each segment of the nephron. In the glomeruli and distal tubules, a few small cytoplasmic granules were stained. In the proximal tubules, the S1 segment exhibited many large granules which were most heavily stained, whereas the S2 and S3 segments contained few positive granules. All segments of the distal tubules showed the smallest amount of positive granules. A few positive granules were also noted in the cortical and medullary collecting tubules. Control experiments confirmed the specificity of the staining. The results indicate that the major site for cathepsin B in rat kidney is the S1 segment of the proximal tubule which is known to actively take up proteins leaked through the glomerulus.     

Immunohistochemical studies on the expression pattern of molecular chaperones HSC70 and HSP25 and cell cycle-related proteins cyclin D1 and PCNA in rat liver after thioacetamide intoxication

Intoxication of rats with thioacetamide (TAA) is a model system to investigate mechanisms involved in liver cell death and tissue reconstitution. Our study was undertaken to determine by immunohistochemistry the expression pattern of the cytoprotective chaperone proteins HSC70 and HSP25 and proliferation markers cyclin D1 and PCNA in livers of Wistar rats intraperitoneally injected with TAA at a single dose of 50 mg/kg. For each protein studied we observed distinct dynamic changes in appearance and localization in liver lobules. During 24-36 h after TAA injection the HSC70 cytoplasmic immunoreaction gradually disappeared from hepatocytes localized around central veins and a shift of immunostaining to cell nuclei took place. Then, 36-48 h after TAA injection the HSC70 cytoplasmic immunoreaction reappeared with the highest intensity in hepatocytes surrounding the areas of inflammatory cells. HSP25, undetectable in control hepatocytes began to appear at approximately 36 h after TAA injection and HSP25-immunopositive cells formed a characteristic ring around areas of inflammation. Of the proteins studied, the most rapid reaction to TAA was observed for cyclin D1. As early as 15 min after TAA administration cyclin D1-positive hepatocytes appeared in intermediate and periportal areas of liver lobules and a subsequent shift of staining to centrilobular hepatocytes took place at 36 and 48 h. There was no correlation of cyclin D1 localization either with PCNA-positive cells or mitotic cells. Our observations suggest that in TAA-treated livers HSP25 and HSC70 proteins can play an anti-inflammatory role, and the early and distinct cyclin D1 expression is not related to proliferation of hepatocytes.     

Enzyme cytochemistry combined with electron microscopy, pharmacokinetics, and clinical chemistry for the evaluation of the effects of steady-state valproic acid concentrations on the mouse

A number of organs from adult female mice were investigated after continuous application of the anticonvulsant drug valproic acid (VPA) by enzyme cytochemistry, light and electron microscopy, pharmacokinetics and clinical chemistry. VPA plasma levels were maintained between 55 micrograms/ml and 67 micrograms/ml for three days following subcutaneous implantation of drug reservoirs. Effects detectable by enzyme cytochemical or electron microscopical means were mainly observed in liver, kidney, thymus and spleen. A strict concentration-dependency of drug effects could not be found. In the liver, the activities of some surface-membrane hydrolases were increased at the biliary pole; the activities of other hydrolases were decreased or unchanged. Electron microscopically, number and length of microvilli of hepatocytes were increased and many of them showed fat inclusions, mitochondrial swellings and autophagic vacuoles. In some of the proximal convoluted tubules of the kidney, the reaction product originating from microvillous and lysosomal hydrolases was diffusely distributed and its amount lowered. This was paralleled by tubular cells with an increased number of fat droplets and swollen mitochondria or destroyed tubular cells, as demonstrated by electron microscopy. Additionally, peritubular endothelial cells were arranged in a garland-like pattern. Alkaline phosphatase was activated in the straight portion of the proximal tubules. Increased glucose, creatinine and total protein concentrations and increased gamma-glutamyl transpeptidase and alkaline phosphatase activities in the urine reflected well the damage of the proximal renal tubules. Cortical and medullary morphology varied considerably in the thymus. In extreme cases, the cortical zone was either reduced in size or the medulla showed a cortex-like structure or vice versa (inverted type of thymus). The thymic cortical reticular cells showed increased aminopeptidase A activity accompanied by a generalized aminopeptidase M and alkaline phosphatase reaction. Our data indicate that--in addition to the liver--also the kidney, thymus and spleen are target organs of VPA-induced toxicity in the mouse.     

An investigation into the sensitivity of heat shock proteins as markers of cellular damage: a comparative study of hydrazine and cadmium chloride in primary rat hepatocyte cultures

Stress proteins have been proposed as markers of toxicity. This study investigated the sensitivity and specificity of stress proteins as markers of toxicity in primary hepatocyte cultures following exposure to two compounds, hydrazine and cadmium chloride (CdCl) . 2 Hepatocytes were exposed to increasing concentrations of hydrazine and CdCl for 2 h 2 and levels of the heat shock proteins HSP72/3, and HSP25 measured. In addition to this, ATP and GSH levels and LDH leakage were measured over the following 8 h. The results show that increasing concentrations of hydrazine caused dose-dependent decreases in ATP and GSH levels over 8 h. There was no change in the levels of HSP25 or HSP72/3 over that period. CdCl was found to significantly induce HSP72/3 at a concentration of 2 5 M when no other biochemical parameter was altered, levels were also elevated following administration of 10 M CdCl but ATP levels were found to be decreased at this 2 concentration. Levels of HSP25 were not increased following CdCl exposure at any 2 concentration. Higher concentrations of CdCl produced significant increases in LDH 2 leakage and depletion of intracellular levels of ATP and GSH. In addition to this levels of HSP25 and HSP72/3 were reduced to zero following administration of high concentrations of CdCl. In this study hydrazine does not induce either of the stress 2 proteins studied here whereas CdCl exposure causes the induction of HSP72/3 but not 2 HSP25. However it was determined that during the culture of primary hepatocytes basal levels of HSP25 and HSP72/3 were significantly increased when compared with levels determined in vivo. The results suggest that stress proteins may have the potential to be sensitive markers of toxicity in primary hepatocytes; however, the induction of individual stress proteins appears to be dependent upon the compound used. The apparent noninduction of the stress response by hydrazine and minor induction by CdCl might be 2 explained by the fact that whilst in culture the hepatocytes are under a continuous state of stress and therefore may not be able to elicit a full stress response following a chemical insult.     

Gender differences in the expression of heat shock proteins: the effect of estrogen

The heat shock proteins (HSPs) are an important family of endogenous, protective proteins that are found in all tissues. In the heart, HSP72, the inducible form of HSP70, has been the most intensely studied. It is well established that HSP72 is induced with ischemia and is cardioprotective. Overexpression of other HSPs also is protective against cardiac injury. Recently, we observed that 17beta-estradiol increases levels of HSPs in male rat cardiac myocytes. We hypothesized that there were gender differences in HSP72 expression in the heart secondary to estrogen. To test this hypothesis, we examined cardiac levels of HSP72 by ELISA in male and female Sprague-Dawley rats. In addition, three other HSPs were assessed by Western blot (HSP27, HSP60, and HSP90). To determine whether estrogen status affected HSP72 expression in other muscles or tissues, two other muscle tissues, slow twitch muscle (soleus muscle) and fast twitch muscle (gastrocnemius muscle), were studied as well as two other organs, the kidney and liver. Because HSP72 is cardioprotective, and females are known to have less cardiovascular disease premenopause, the effects of ovariectomy were examined. We report that female Sprague-Dawley rat hearts have twice as much HSP72 as male hearts. Ovariectomy reduced the level of HSP72 in female hearts, and this could be prevented by estrogen replacement therapy. These data show that the expression of cardiac HSP72 is greater in female rats than in male rats, due to upregulation by estrogen.     

Localization of L-arginine-glycine amidinotransferase protein in rat tissues by immunofluorescence microscopy

Creatine is a major component of energy metabolism and enzymes involved in its synthesis have therefore been of considerable interest. L-arginine-glycine amidinotransferase, commonly called transamidinase, catalyzes the first reaction in the biosynthesis of creatine. This first reaction is believed to occur in the kidney because of the high concentration of transamidinase in that tissue. Transamidinase activity is also found in many other tissues of the rat, but its role in these tissues is not known. Immunochemical studies with antisera and monoclonal antibodies were used to confirm and refine our understanding of the presence of transamidinase in rat tissues. Immunofluorescence histochemistry was performed to localize transamidinase immunoreactivity within specific tissues including cells in the proximal tubules of the kidney, hepatocytes of the liver, and alpha cells of the pancreatic islet. Immunochemical studies with monoclonal antibodies confirm localization of transamidinase immunoreactivity in the proximal tubules of the kidney. The localization of such immunoreactivity in specialized cells yields insight into possible physiological role(s) of transamidinase in the rat.     

Purification, characterization, molecular cloning, and subcellular distribution of neutral ceramidase of rat kidney

Previously, we reported two types of neutral ceramidase in mice, one solubilized by freeze-thawing and one not. The former was purified as a 94-kDa protein from mouse liver, and cloned (Tani, M., Okino, N., Mori, K., Tanigawa, T., Izu, H., and Ito, M. (2000) J. Biol. Chem. 275, 11229--11234). In this paper, we describe the purification, molecular cloning, and subcellular distribution of a 112-kDa membrane-bound neutral ceramidase of rat kidney, which was completely insoluble by freeze-thawing. The open reading frame of the enzyme encoded a polypeptide of 761 amino acids having nine putative N-glycosylation sites and one possible transmembrane domain. In the ceramidase overexpressing HEK293 cells, 133-kDa (Golgi-form) and 113-kDa (endoplasmic reticulum-form) Myc-tagged ceramidases were detected, whereas these two proteins were converted to a 87-kDa protein concomitantly with loss of activity when expressed in the presence of tunicamycin, indicating that the N-glycosylation process is indispensable for the expression of the enzyme activity. Immunohistochemical analysis clearly showed that the ceramidase was mainly localized at the apical membrane of proximal tubules, distal tubules, and collecting ducts in rat kidney, while in liver the enzyme was distributed with endosome-like organelles in hepatocytes. Interestingly, the kidney ceramidase was found to be enriched in the raft microdomains with cholesterol and GM1 ganglioside.     

Implication of free radicals and glutathione in the mechanism of cadmium-induced expression of stress proteins in the A549 human lung cell-line

Cellular mechanisms underlying the expression of stress proteins (HSP) were studied in the human cell-line A549 submitted to a pollutant, cadmium, in the presence of several agents which modulate the glutathione level and, supposedly, the effects of this metal in the cell. It was observed that HSP 90, HSP 72 and HSP 27 are significantly over-expressed after exposure to cadmium chloride for 24 h. Low cadmium concentrations (i.e. from 1 to 10 microM) also triggered a slight accumulation of glutathione, whereas this compound was depleted after exposure to higher cadmium concentrations (25-100 microM). When 50 microM diethyl-maleate, which traps glutathione, was added together with cadmium, the over-expression of HSP 72 and HSP 90 was much stronger. Treatment of cells with 20 or 40 mM N-acetyl-L-cysteine, which traps free radicals, was found to increase by 30% the glutathione level and to suppress the HSP over-expression. From our results, it is suggested that HSP induction by cadmium in A549 cells is due, at least in part, to the oxidative stress consisting in formation of reactive oxygen species and inhibition of peroxides detoxification. Due to this oxidative status within the cell, more proteins would be damaged inducing the HSP over-expression.     

Proteomics characterization of cell model with renal fibrosis phenotype: osmotic stress as fibrosis triggering factor

Renal fibroblasts are thought to play a major role in the development of renal fibrosis (RF). The mechanisms leading to this renal alteration remain poorly understood. We performed differential proteomic analyses with two established fibroblast cell lines with RF phenotype to identify new molecular pathways associated with RF. Differential 2-DE combined with mass spectrometry analysis revealed the alteration of more than 30 proteins in fibrotic kidney fibroblasts (TK188) compared to normal kidney fibroblast (TK173). Among these proteins, markers of the endoplasmic reticulum (ER) stress- and the unfolded protein response (UPR) pathway (GRP78, GRP94, ERP57, ERP72, and CALR) and the oxidative stress pathway proteins (PRDX1, PRDX2, PRDX6, HSP70, HYOU1) were highly up-regulated in fibrotic cells. Activation of these stress pathways through long time exposition of TK173, to high NaCl or glucose concentrations resulted in TK188 like phenotype. Parallel to an increase in reactive oxygen species, the stressed cells showed significant alteration of fibrosis markers, ER-stress and oxidative stress proteins. Similar effects of osmotic stress could be also observed on renal proximal tubule cells. Our data suggest an important role of the ER-stress proteins in fibrosis and highlights the pro-fibrotic effect of osmotic stress through activation of oxidative stress and ER-stress pathways.