Marwa Eltoweissy

Secretion of ERP57 is important for extracellular matrix accumulation and progression of renal fibrosis, and is an early sign of disease onset.

Summary Renal fibrosis is characterized by excessive accumulation of extracellular matrix (ECM), which compromises organ function by replacing normal organ tissue. The molecular mechanisms leading to renal fibrosis are not fully understood. Here we demonstrated that TGF&bgr;1, AGT or PDGF stimulation of renal cells resulted in endoplasmic reticulum (ER) stress followed by activation of the protective unfolded protein response pathway and a high secretory level of protein disulfide isomerase ERP57 (also known as PDIA3). The TGF&bgr;1-induced impairment of ER function could be reversed by treatment with BMP7, suggesting a specific involvement in renal fibrosis. A clear correlation between the degree of fibrosis, ER stress and the level of ERP57 could be seen in fibrosis animal models and in biopsies of renal fibrosis patients. Protein interaction studies revealed that secreted ERP57 exhibits a strong interaction with ECM proteins. Knockdown of ERP57 or antibody-targeted inhibition of the secreted form significantly impaired the secretion and accumulation of ECM. Moreover, ERP57 was excreted in the early stages of chronic kidney disease, and its level in urine correlated with the degree of renal fibrosis, suggesting that the secretion of ERP57 represents one of the first signs of renal fibrosis onset and progression.

Calreticulin is crucial for calcium homeostasis mediated adaptation and survival of thick ascending limb of Henle's loop cells under osmotic stress

The thick ascending limb of Henles loop (TALH) is normally exposed to variable and often very high osmotic stress and involves different mechanisms to counteract this stress. ER resident calcium binding proteins especially calreticulin (CALR) play an important role in different stress balance mechanisms. To investigate the role of CALR in renal epithelial cells adaptation and survival under osmotic stress, two-dimensional fluorescence difference gel electrophoresis combined with mass spectrometry and functional proteomics were performed. CALR expression was significantly altered in TALH cells exposed to osmotic stress, whereas renal inner medullary collecting duct cells and interstitial cells exposed to hyperosmotic stress showed no significant changes in CALR expression. Moreover, a time dependent downregulation of CALR was accompanied with continuous change in the level of free intracellular calcium. Inhibition of the calcium release, through IP3R antagonist, prevented CALR expression alteration under hyperosmotic stress, whereas the cell viability was significantly impaired. Overexpression of wild type CALR in TALH cells resulted in significant decrease in cell viability under hyperosmotic stress. In contrast, the hyperosmotic stress did not have any effect on cells overexpressing the CALR mutant, lacking the calcium-binding domain. Silencing CALR with siRNA significantly improved the cell survival under osmotic stress conditions. Taken together, our data clearly highlight the crucial role of CALR and its calcium-binding role in TALH adaptation and survival under osmotic stress.

Proteomics analysis identifies PARK7 as an important player for renal cell resistance and survival under oxidative stress

Renal fibrosis is a process that is characterized by declining excretory renal function. The molecular mechanisms of fibrosis are not fully understood. Oxidative stress pathways were reported to be involved in renal tissue deterioration and fibrosis progression. In order to identify new molecular targets associated with oxidative stress and renal fibrosis, differential proteomics analysis was performed with established renal cell lines (TK173 and HK-2). The cells were treated with oxidative stress triggering factor H(2)O(2) and the proteome alterations were investigated. Two dimensional protein maps were generated and differentially expressed proteins were processed and identified using mass spectrometry analysis combined with data base search. Interestingly the increase of ROS in the renal cell lines upon H(2)O(2) treatment was accompanied by alteration of a large number of proteins, which could be classified in three categories: the first category grouped the proteins that have been described to be involved in fibrogenesis (e.g. ACTA2, VIN, VIM, DES, KRT, COL1A1, COL4A1), the second category, which was more interesting involved proteins of the oxidative stress pathway (PRDX1, PRDX2, PRDX6, SOD, PARK7, HYOU1), which were highly up-regulated under oxidative stress, and the third category represented proteins, which are involved in different other metabolic pathways. Among the oxidative stress proteins the up-regulation of PARK7 was accompanied by a shift in the pI as a result of oxidation. Knockdown of PARK7 using siRNA led to significant reduction in renal cell viability under oxidative stress. Under H(2)O(2) treatment the PARK7 knockdown cells showed up to 80% decrease in cell viability and an increase in apoptosis compared to the controls. These results highlight for the first time the important role of PARK7 in oxidative stress resistance in renal cells.

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.

Renal Cells Express Different Forms of Vimentin: The Independent Expression Alteration of these Forms is Important in Cell Resistance to Osmotic Stress and Apoptosis

Osmotic stress has been shown to regulate cytoskeletal protein expression. It is generally known that vimentin is rapidly degraded during apoptosis by multiple caspases, resulting in diverse vimentin fragments. Despite the existence of the known apoptotic vimentin fragments, we demonstrated in our study the existence of different forms of vimentin VIM I, II, III, and IV with different molecular weights in various renal cell lines. Using a proteomics approach followed by western blot analyses and immunofluorescence staining, we proved the apoptosis-independent existence and differential regulation of different vimentin forms under varying conditions of osmolarity in renal cells. Similar impacts of osmotic stress were also observed on the expression of other cytoskeleton intermediate filament proteins; e.g., cytokeratin. Interestingly, 2D western blot analysis revealed that the forms of vimentin are regulated independently of each other under glucose and NaCl osmotic stress. Renal cells, adapted to high NaCl osmotic stress, express a high level of VIM IV (the form with the highest molecular weight), besides the three other forms, and exhibit higher resistance to apoptotic induction with TNF-α or staurosporin compared to the control. In contrast, renal cells that are adapted to high glucose concentration and express only the lower-molecular-weight forms VIM I and II, were more susceptible to apoptosis. Our data proved the existence of different vimentin forms, which play an important role in cell resistance to osmotic stress and are involved in cell protection against apoptosis.

The antioxidant protein PARK7 plays an important role in cell resistance to cisplatin induced apoptosis in case of clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most malignant tumor in the adult kidney. Many factors are responsible for the development and progression of this tumor. Increased reactive oxygen species accumulation and altered redox status have been observed in cancer cells and this biochemical property of cancer cells can be exploited for therapeutic benefits. In earlier work we identified and characterize Protein DJ-1 (PARK7) as an oxidative stress squevenger in renal cells exposed to oxidative stress. To investigate whether the PARK7 or other oxidative stress proteins play a role in the renal cell carcinoma and its sensitivity or resistance to cytostatic drug treatment, differential proteomics analysis was performed with a cell model for clear cell renal carcinoma (Caki-2 and A498). Caki-2 cells were treated with cisplatin and differentially expressed proteins were investigated. The cisplatin treatment resulted in an increase in reactive oxygen species accumulation and ultimately apoptosis of Caki-2 and A498 cells. In parallel, the apoptotic effect was accompanied by a significant downregulation of antioxidant proteins especially PARK7. Knockdown of PARK7 using siRNA and overexpression using plasmid highlights the role of PARK7 as a key player in renal cell carcinoma response to cisplatin induced apoptosis. Overexpression of PARK7 resulted in significant decrease in apoptosis, whereas knockdown of the protein was accompanied by an increase in apoptosis in Caki-2 and A498 cells treated with cisplatin. These results highlights for the first time the important role of PARK7 in cisplatin induced apoptosis in clear renal cell carcinoma cells.

Short-term increase of glucose concentration in PDS results in extensive removal and high glycation level of vital proteins during continuous ambulatory peritoneal dialysis

BACKGROUND The effect of clearance of so-called middle- and high-molecular weight proteins on clinical outcome of patients treated by peritoneal dialysis is still a matter of debate. In our present study, we investigated the impact of short-time alteration of the glucose concentration and the osmolarity of the peritoneal dialysis solution (PDS) on protein removal. METHODS Peritoneal dialysis liquids (PDL) were collected from 19 well-characterized continuous ambulatory peritoneal dialysis (CAPD) patients treated with two types of PDS: Baxter (n = 10) and Fresenius (n = 9). The patients were treated with two different glucose concentration of each PDS in 4-h cycles. The depletion of the six interfering high-abundant proteins from the PDL samples was performed with the Multiple Affinity Removal LC Column-Human 6. The resulting protein fractions were analysed by 2D gel electrophoresis, differential in gel electrophoresis, mass spectrometry and 2D western blot. RESULTS Proteomics investigation of the PDL fractions after depletion allowed the identification of 198 polypeptides of 424 excised spots. These polypeptides equates to 48 non-redundant proteins. Comparative analyses of 2D gel electrophoresis protein pattern revealed a clear correlation between protein removal and PDS glucose concentration and osmolarity. An increase for 4 h in the PDS osmolarity (with 43-51 mosmol/L) resulted qualitatively in 18-23% more protein removal in PDL. Moreover, 2D western blot analyses of the protein glycation pattern showed that the short-time increase in PDS glucose concentration (45-50 mM) resulted in significant alteration of the advanced glycosylation end products (AGEs) pattern. CONCLUSIONS The data presented in this study demonstrate a clear correlation between the short-time changes in glucose concentration and osmolarity of the PDS, and the augmentation of the protein removal and the appearance of AGEs during CAPD.

Proteomic characterization of adrenal gland embryonic development reveals early initiation of steroid metabolism and reduction of the retinoic acid pathway

BackgroundAdrenal glands are essential endocrine organs composed of two embryological distinct tissues. Morphological changes during their development are well described, but less understood with regard to their molecular mechanisms. To identify proteins and pathways, which drive the initial steps of the specification of the endocrine function of the adrenal gland, rat’s adrenal glands were isolated at different embryonic days (E): E14, E16, E18, E19 and postnatal day 1 (P1).ResultsThe alteration of the proteome during the stages E16, E19 and P1 was investigated by combining two dimensional gel electrophoresis and mass spectrometric analysis. Out of 594 excised protein spots, 464 spots were identified, resulting in 203 non-redundant proteins. The ontogenic classification of the identified proteins according to their molecular function resulted in 10 different categories, whereas the classification of their biological processes resulted in 19 different groups. This gives an insight into the complex mechanisms underlying adrenal gland development. Interestingly, the expression of retinoic acid pathway proteins was decreased during the development of the adrenal gland, suggesting that this pathway is only important at early stages. On the other hand, key proteins of the cholesterol synthesis increased their expression significantly at E19 revealing the initiation of the endocrine specialization of the adrenal glands.ConclusionsThis study presents the first comprehensive wide proteome analysis of three different stages of embryonic adrenal gland development. The identified proteins, which were expressed in early stages of development, will shed light on the molecular mechanisms underlying embryonic development of the adrenal gland.

Cellulose membranes are more effective in holding back vital proteins and exhibit less interaction with plasma proteins during hemodialysis.

The vast majority of patients with end-stage renal disease are treated with intermittent hemodialysis as a form of renal replacement therapy. To investigate the impact of hemodialysis membrane material on vital protein removal, dialysates from 26 well-characterized hemodialysis patients were collected 5 min after beginning, during 5h of treatment, as well as 5 min before ending of the dialysis sessions. Dialysis sessions were performed using either modified cellulose (n=12) (low-flux and high flux) or synthetic Polyflux (n=14) (low-flux and high-flux) dialyzer. Protein removal during hemodialysis was quantified and the dialysate proteome patterns were analyzed by 2-DE, MS and Western blot. There was a clear correlation between the type of membrane material and the amount of protein removed. Synthetic Polyflux membranes exhibit strong interaction with plasma proteins resulting in a significantly higher protein loss compared to modified cellulosic membrane. Moreover, the proteomics analysis showed that the removed proteins represented different molecular weight range and different functional groups: transport proteins, protease inhibitors, proteins with role in immune response and regulations, constructive proteins and as a part of HLA immune complex. The effect of this protein removal on hemodialysis treatment outcome should be investigated in further studies.

Correction: Secretion of ERP57 is important for extracellular matrix accumulation and progression of renal fibrosis, and is an early sign of disease onset (doi:10.1242/jcs.125088)

There was an error published in J. Cell Sci. (2013) 126, [3649–3663][1] ([doi:10.1242/jcs.125088][2]). The affiliations for Marwa Eltoweissy were incorrect. The correct affiliations are as given below. Hassan Dihazi1, Gry Helene Dihazi1, Asima Bibi1, Marwa Eltoweissy1,2, Claudia A. Mueller3,