Chloride Intracellular Channel 4 Overexpression in the Proximal Tubules of Kidneys from the Spontaneously Hypertensive Rat: Insight from Proteomic Analysis.

BACKGROUND: Hypertensive nephropathy, a leading cause of declining kidney function, is a multifactorial process not well understood. In order to elucidate biological processes and identify novel macromolecular components crucially involved in the process of kidney damage, the application of system biology approaches, like proteomics, is required. METHODS: Proteomic studies were performed using the renal parenchyma of spontaneously hypertensive rats (SHR) and their normotensive Wistar Kyoto controls. Animals were sacrificed at early time intervals (6, 13, and 20 weeks after birth), the renal tissue extract was subjected to two-dimensional gel electrophoresis, differential expressed proteins were identified, and altered pathways were evaluated. One specific protein, chloride intracellular channel 4 (CLIC4), not implicated so far in the development of hypertension and nephrosclerosis, was further studied by Western blotting, immunohistochemistry and immunofluorescence. RESULTS: Proteomic analysis identified several pathways/processes and organelles (mitochondria) as being affected from the early stages of hypertension. CLIC4 was overexpressed in SHR at all 3 time intervals examined. This finding was confirmed by Western blotting and by immunohistochemistry and immunofluorescence; these morphological techniques demonstrated that CLIC4 was almost exclusively localized at the apical surface of the proximal tubular epithelial cells. CONCLUSIONS: Our studies provide evidence that major changes occur in the renal parenchyma from early stages of the development of hypertension. The overexpression of CLIC4 suggests that alterations in the proximal tubular compartment during hypertension should be further examined and that CLIC4 may be a useful early marker of renal tubular alterations due to elevated blood pressure.

Vitamin D3 ameliorates podocyte injury through the nephrin signalling pathway.

Renal podocytes form the main filtration barrier possessing unique phenotype maintained by proteins including podocalyxin and nephrin, which are modulated in pathological conditions. In diabetic nephropathy (DN), podocytes become structurally and functionally compromised. Nephrin, a structural backbone protein of the slit diaphragm, acts as regulator of podocyte intracellular signalling with renoprotective role. Vitamin D3 through its receptor, VDR, provides renal protection in DN but limited data exist about its effect on podocytes. In this study, we used isolated rat glomeruli to assess podocalyxin and nephrin expression after treatment with the 1,25-dihydroxyvitamin D3 analogue paricalcitol in the presence of normal and diabetic glucose levels. The role of 1,25-dihydroxyvitamin D3 (calcitriol) and its analogue, paricalcitol, on podocyte morphology and survival was also investigated in the streptozotocin (STZ)-diabetic animal model. In our ex vivo model, glomeruli exhibited high glucose-mediated down-regulation of podocalyxin, and nephrin, while paricalcitol reversed the high glucose-induced decrease of nephrin and podocalyxin expression. Paricalcitol treatment enhanced VDR expression and promoted VDR and RXR co-localization in the nucleus. Our data also indicated that hyperglycaemia impaired survival of cultured glomeruli and suggested that the implemented nephrin down-regulation was reversed by paricalcitol treatment, initiating Akt signal transduction which may be involved in glomerular survival. Our findings were further verified in vivo, as in the STZ-diabetic animal model, calcitriol and paricalcitol treatment resulted in significant amelioration of hyperglycaemia and restoration of nephrin signalling, suggesting that calcitriol and paricalcitol may provide molecular bases for protection against loss of the permselective renal barrier in DN.

High Glucose Impairs Insulin Signaling in the Glomerulus: An In Vitro and Ex Vivo Approach.

OBJECTIVE: Chronic hyperglycaemia, as seen in type II diabetes, results in both morphological and functional impairments of podocytes in the kidney. We investigated the effects of high glucose (HG) on the insulin signaling pathway, focusing on cell survival and apoptotic markers, in immortalized human glomerular cells (HGEC; podocytes) and isolated glomeruli from healthy rats. METHODS AND FINDINGS: HGEC and isolated glomeruli were cultured for various time intervals under HG concentrations in the presence or absence of insulin. Our findings indicated that exposure of HGEC to HG led to downregulation of all insulin signaling markers tested (IR, p-IR, IRS-1, p-Akt, p-Fox01,03), as well as to increased sensitivity to apoptosis (as seen by increased PARP cleavage, Casp3 activation and DNA fragmentation). Short insulin pulse caused upregulation of insulin signaling markers (IR, p-IR, p-Akt, p-Fox01,03) in a greater extent in normoglycaemic cells compared to hyperglycaemic cells and for the case of p-Akt, in a PI3K-dependent manner. IRS-1 phosphorylation of HG-treated podocytes was negatively regulated, favoring serine versus tyrosine residues. Prolonged insulin treatment caused a significant decrease of IR levels, while alterations in glucose concentrations for various time intervals demonstrated changes of IR, p-IR and p-Akt levels, suggesting that the IR signaling pathway is regulated by glucose levels. Finally, HG exerted similar effects in isolated glomeruli. CONCLUSIONS: These results suggest that HG compromises the insulin signaling pathway in the glomerulus, promoting a proapoptotic environment, with a possible critical step for this malfunction lying at the level of IRS-1 phosphorylation; thus we herein demonstrate glomerular insulin signaling as another target for investigation for the prevention and/ or treatment of diabetic nephropathy.

Glucose-induced gradual phenotypic modulation of cultured human glomerular epithelial cells may be independent of Wilms' tumor 1 (WT1).

BACKGROUND: Renal podocytes form the main filtration barrier possessing a unique phenotype maintained by proteins including podocalyxin and nephrin, the expression of which is suppressed in pathological conditions. We used an in vitro model of human glomerular epithelial cells (HGEC) to investigate the role of high glucose in dysregulating the podocytic epithelial phenotype and determined the time needed for this change to occur. RESULTS: In our in vitro podocyte system changes indicating podocyte dedifferentiation in the prolonged presence of high glucose included loss of podocalyxin, nephrin and CD10/CALLA concomitant with upregulation of mesenchymal vimentin. Our study demonstrates for the first time that podocyte-specific markers undergo changes of expression at different time intervals, since glucose-mediated podocalyxin downregulation is a progressive process that precedes downregulation of nephrin expression. Finally we demonstrate that high glucose permanently impaired WT1 binding to the podocalyxin gene promoter region but did not affect WT1 binding on the nephrin gene promoter region. CONCLUSION: The presence of high glucose induced a phenotypic conversion of podocytes resembling partial dedifferentiation. Our study demonstrates that dysregulation of the normal podocytic phenotype is an event differentially affecting the expression of function-specific podocytic markers, exhibiting downregulation of the epithelial marker CD10/CALLA and PC first, followed by stably downregulated nephrin. Furthermore, it is herein suggested that WT1 may not be directly involved with upregulation of previously reduced PC and nephrin expression.

Synthesis of a folate functionalized PEGylated poly(propylene imine) dendrimer as prospective targeted drug delivery system.

Based on fourth generation diaminobutane poly(propylene imine) dendrimer, a novel targeted drug nanocarrier was prepared, bearing protective PEG chains and a folate targeting ligand. As a control a PEGylated derivative without folate was also synthesized. The encapsulation and release properties of these PEGylated derivatives were investigated employing etoposide, an anticancer hydrophobic drug. Enhanced solubility of etoposide was achieved inside the dendrimeric scaffold which was subsequently released in a controlled manner. These properties coupled with specificity towards the folate receptor and the low toxicity render folate functionalized PEGylated poly(propylene imine) dendrimer promising candidate for targeted drug delivery.

Rapid magnetic heating treatment by highly charged maghemite nanoparticles on Wistar rats exocranial glioma tumors at microliter volume.

One of the most significant challenges implementing colloidal magnetic nanoparticles in medicine is the efficient heating of microliter quantities by applying a low frequency alternating magnetic field. The ultimate goal is to accomplish nonsurgically the treatment of millimeter size tumors. Here, we demonstrate the synthesis, characterization, and the in vitro as well as in vivo efficiency of a dextran coated maghemite (gamma-Fe(2)O(3)) ferrofluid with an exceptional response to magnetic heating. The difference to previous synthetic attempts is the high charge of the dextran coating, which according to our study maintains the colloidal stability and good dispersion of the ferrofluid during the magnetic heating stage. Specifically, in vitro 2 mul of the ferrofluid gives an outstanding temperature rise of 33 degrees C within 10 min, while in vivo treatment, by infusing 150 mul of the ferrofluid in animal model (rat) glioma tumors, causes an impressive cancer tissue dissolution.

High glucose induces suppression of insulin signalling and apoptosis via upregulation of endogenous IL-1beta and suppressor of cytokine signalling-1 in mouse pancreatic beta cells.

Chronic hyperglycemia and inflammatory cytokines disrupt and/or attenuate signal transduction pathways that promote normal beta-cell survival, leading to the destruction of endocrine pancreas in type 2 diabetes. There is convincing evidence that autocrine insulin signalling exerts protective anti-apoptotic effects on beta cells. Suppressors of cytokine signalling (SOCS) were induced by several cytokines and inhibit insulin-initiated signal transduction. The aim of this study was to investigate whether high glucose can influence endogenous interleukin-1beta (IL-1beta) and SOCS expression thus affecting insulin signalling and survival in insulin-producing mouse pancreatic beta cells (betaTC-6). Results showed that prolonged exposure of betaTC-6 cells to increased glucose concentrations resulted in significant inhibition of insulin-induced tyrosine phosphorylation of the insulin receptor (IR), and insulin receptor substrate-2 (IRS-2) as well as PI3-kinase activation. These changes were accompanied by impaired activation of the anti-apoptotic signalling protein Akt and annulment of Akt-mediated suppression of the Forkhead family of transcription factors (FoxO) activation. Glucose-induced attenuation of IRS-2/Akt-mediated signalling was associated with increased IL-1beta expression. Enhanced endogenous IL-1beta specifically induced mRNA and protein expression of SOCS-1 in betaTC-6 cells. Inhibition of SOCS-1 expression by SOCS-1-specific small interfering RNA restored IRS-2/PI3K-mediated Akt phosphorylation suppressed by high glucose. The upregulation of endogenous cytokine signalling and FoxO activation were accompanied by enhanced caspase-3 activation and increased susceptibility of cells to apoptosis. These results indicated that glucose-induced endogenous IL-1beta expression increased betaTC-6 cells apoptosis by inhibiting, at least in part, IRS-2/Akt-mediated signalling through SOCS-1 upregulation.

Impaired transcription factor interplay in addition to advanced glycation end products suppress podocalyxin expression in high glucose-treated human podocytes.

Podocalyxin represents a Wilms' tumor suppressor protein (WT1)-regulated differentiation marker for glomerular epithelium. We provide evidence concerning mechanisms involved in the regulation of podocalyxin expression following long-term exposure to increased (25 mM) glucose levels. Prolonged culture of conditionally immortalized human podocytes in 25 mM glucose induced suppression of podocalyxin expression both at the protein and mRNA levels, whereas WT1 protein levels remained unaltered. WT1 interacted with another transcription factor, CRE-binding protein (CBP). This association was decreased by 40% in the presence of 25 mM glucose. Chromatin immunoprecipitation assays on chromatin from podocytes cultured in 25 mM glucose revealed reduced WT1 binding to podocalyxin promoter sequences, probably resulting from impaired WT1-CBP interactions. We explored the possible role of glucose-induced adducts (advanced glycation end products; AGEs) in impairing interactions between WT1 and CBP, with the use of aminoguanindine, an inhibitor of AGE formation. Podocytes were cultured in the simultaneous presence of 20 mM aminoguanidine and 25 mM glucose, and podocalyxin protein levels were examined. Aminoguanidine effectively prevented downregulation of podocalyxin protein levels but could not restore podocalyxin levels once expression was suppressed. Thus increased glucose apparently impaired the ability of WT1 to initiate transcription in part by decreased association of WT1 with CBP. Administration of aminoguanidine concomitant with increasing glucose levels in our in vitro model system protected from glucose-induced "silencing" of the podocalyxin gene, suggesting that AGEs play an important role in suppressing its expression in diabetic conditions.