Sophie Doublier

Nephrin Redistribution on Podocytes Is a Potential Mechanism for Proteinuria in Patients with Primary Acquired Nephrotic Syndrome

We investigated the distribution of nephrin by immunofluorescence microscopy in renal biopsies of patients with nephrotic syndrome: 13 with membranous glomerulonephritis (GN), 10 with minimal change GN, and seven with focal segmental glomerulosclerosis. As control, six patients with IgA GN without nephrotic syndrome and 10 normal controls were studied. We found an extensive loss of staining for nephrin and a shift from a podocyte-staining pattern to a granular pattern in patients with nephrotic syndrome, irrespective of the primary disease. In membranous GN, nephrin was co-localized with IgG immune deposits. In the attempt to explain these results, we investigated in vitro whether stimuli acting on the cell cytoskeleton, known to be involved in the pathogenesis of GN, may induce redistribution of nephrin on the surface of human cultured podocytes. Aggregated but not disaggregated human IgG(4), plasmalemmal insertion of membrane attack complex of complement, tumor necrosis factor-alpha, and puromycin, induced the shedding of nephrin with a loss of surface expression. This phenomenon was abrogated by cytochalasin and sodium azide. These results suggest that the activation of cell cytoskeleton may modify surface expression of nephrin allowing a dislocation from plasma membrane to an extracellular site.

Statins Prevent Oxidized LDL-Induced Injury of Glomerular Podocytes by Activating the Phosphatidylinositol 3-Kinase/AKT-Signaling Pathway

The injury of podocytes is associated with alterations of the glomerular size-selective barrier to proteins. In this study, oxidized LDL (oxLDL) but not native LDL induced apoptosis in human cultured podocytes and reduced Akt activity and P-Akt/Akt ratio. Moreover, oxLDL-induced redistribution and loss of nephrin, an adhesion molecule specific for the glomerular slit diaphragm. Nephrin reduction was preceded by inhibition of nephrin tyrosine phosphorylation and of its association with p85 phosphatidylinositol 3-kinase (PI3K). Moreover, three different statins, mevastatin, pravastatin, and simvastatin, inhibited in a dose-dependent manner apoptosis and loss of nephrin induced by oxLDL by stimulating Akt activity. In addition, simvastatin significantly increased the expression of nephrin protein and mRNA by podocytes. The protective effects of statins were blocked by treatment of podocytes with two unrelated pharmacologic inhibitors of PI3K, LY294002 and wortmannin, suggesting a role for PI3K, and by mevalonate, indicating dependency on HMG-CoA reductase activity. Statins directly stimulated Akt phosphorylation ad activity. Finally, oxLDL induced a retraction of cultured podocytes and an increase in the albumin diffusion across their monolayer that was inhibited by treatment with statins. In conclusion, statins reduced the oxLDL-induced apoptosis and loss of nephrin in glomerular podocytes. The statin-induced Akt activation may protect from the loss of nephrin by an inhibition of its redistribution and shedding and by a stimulation of its synthesis. These data provide a rationale for the anti-proteinuric effect of statins.

Artemisinin induces doxorubicin resistance in human colon cancer cells via calcium-dependent activation of HIF-1α and P-glycoprotein overexpression

Background and purpose:  Artemisinin is an antimalarial drug exerting pleiotropic effects, such as the inhibition of the transcription factor nuclear factor‐kappa B and of the sarcoplasmic/endoplasmic reticulum Ca++‐ATPase (SERCA) of P. falciparum. As the sesquiterpene lactone thapsigargin, a known inhibitor of mammalian SERCA, enhances the expression of P‐glycoprotein (Pgp) by increasing the intracellular Ca++ ([Ca++]i) level, we investigated whether artemisinin and its structural homologue parthenolide could inhibit SERCA in human colon carcinoma HT29 cells and induce a resistance to doxorubicin.

Calpain Activation and Secretion Promote Glomerular Injury in Experimental Glomerulonephritis: Evidence from Calpastatin-Transgenic Mice

Glomerular injury and albuminuria in acute glomerulonephritis are related to the severity of inflammatory process. Calpain, a calcium-activated cysteine protease, has been shown to participate in the development of the inflammatory process. Therefore, for determination of the role of calpain in the pathophysiology of acute glomerulonephritis, transgenic mice that constitutively express high levels of calpastatin, a calpain-specific inhibitor protein, were generated. Wild-type mice that were subjected to anti-glomerular basement membrane nephritis exhibited elevated levels of calpain activity in kidney cortex at the heterologous phase of the disease. This was associated with the appearance in urine of calpain activity, which originated potentially from inflammatory cells, abnormal transglomerular passage of plasma proteins, and tubular secretion. In comparison with nephritic wild-type mice, nephritic calpastatin-transgenic mice exhibited limited activation of calpain in kidney cortex and limited secretion of calpain activity in urine. This was associated with less severe glomerular injury (including capillary thrombi and neutrophil activity) and proteinuria. There was a reduction in NF-kappaB activation, suggesting that calpain may participate in inflammatory lesions through NF-kappaB activation. There also was a reduction in nephrin disappearance from the surface of podocytes, indicating that calpain activity would enhance proteinuria by affecting nephrin expression. Exposure of cultured podocytes to calpain decreased nephrin expression, and, conversely, exposure of these cells to calpastatin prevented TNF-alpha from decreasing nephrin expression, demonstrating a role for the secreted form of calpain. Thus, both activation and secretion of calpains participate in the development of immune glomerular injury.

Activation of Nuclear Factor-κB Pathway by Simvastatin and RhoA Silencing Increases Doxorubicin Cytotoxicity in Human Colon Cancer HT29 Cells

Doxorubicin efficacy in cancer therapy is hampered by the dose-dependent side effects, which may be overcome by reducing the drugs dose and increasing its efficacy. In the present work, we suggest that the activation of the nuclear factor-κB (NF-κB) pathway and of nitric-oxide (NO) synthase increases the doxorubicin efficacy in human colon cancer HT29 cells. To induce NF-κB, we took into account the effect of doxorubicin itself and of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin; as NF-κB inhibitors, we chose the sesquiterpene lactones parthenolide and artemisinin. Simvastatin increased the NF-κB activity and NO synthesis, elicited the tyrosine nitration of the multidrug resistance-related protein 3, and enhanced the doxorubicin intracellular accumulation and cytotoxicity. Simvastatin potentiated the effect of doxorubicin on the NF-κB pathway and the inducible NO synthase expression. The effects of simvastatin were due to the inhibition of the small G-protein RhoA and of its effector Rho kinase. Parthenolide and artemisinin prevented all of the statin effects by inducing RhoA/Rho kinase activation. On the other hand, they did not reduce the NF-κB translocation and doxorubicin intracellular content when RhoA was silenced by small interfering RNA (siRNA). It is interesting that RhoA siRNA was sufficient to increase NF-κB translocation, NO synthase activity, doxorubicin accumulation, and cytotoxicity also in non-stimulated cells. Our results suggest that artemisinin, a widely used antimalarial drug, may impair the response to doxorubicin in colon cancer cells; on the contrary, simvastatin and RhoA siRNA may represent future therapeutic approaches to improve doxorubicin efficacy, reducing the risk of doxorubicin-dependent adverse effects.

The NADPH oxidase inhibitor apocynin induces nitric oxide synthesis via oxidative stress.

We have recently shown that apocynin elicits an oxidative stress in N11 mouse glial cells and other cell types. Here we report that apocynin increased the accumulation of nitrite, the stable derivative of nitric oxide (NO), in the extracellular medium of N11 cell cultures, and the NO synthase (NOS) activity in cell lysates. The increased synthesis of NO was associated with increased expression of inducible NOS (iNOS) mRNA, increased nuclear translocation of the redox-sensitive transcription factor NF-kappa B and decreased intracellular level of its inhibitor IkB alpha. These effects, accompanied by increased production of H2O2, were very similar to those observed after incubation with bacterial lipopolysaccharide (LPS) and were inhibited by catalase. These results suggest that apocynin, similarly to LPS, induces increased NO synthesis by eliciting a generation of reactive oxygen species (ROS), which in turn causes NF-kappa B activation and increased expression of iNOS. Therefore, the increased bioavailability of NO reported in the literature after in vivo or in vitro treatments with apocynin might depend, at least partly, on the drug-elicited induction of iNOS, and not only on the inhibition of NADPH oxidase and the subsequent decreased scavenging of NO by oxidase-derived ROS, as it is often supposed.

Overexpression of Human Insulin-Like Growth Factor Binding Protein-1 in the Mouse Leads to Nephron Deficit

IGFs and their binding proteins are important regulators of fetal development. We have previously reported that overexpression of the human IGF binding protein-1 in mice is associated with glomerulosclerosis. The aim of this study was to investigate whether, in that model, decreased bioavailability of IGFs also affected nephrogenesis. When the mothers expressed human IGF binding protein-1, pups were growth retarded and had a reduced number of nephrons. Even nontransgenic pups born to heterozygous mothers had a nephron reduction, indicating that renal hypoplasia was secondary to fetal growth retardation. When the transgene was expressed only in the fetus, pups had a normal birth weight and the kidney was normal at birth, as indicated by histologic studies. However, a significant reduction in the nephron number was observed at 3 mo of age. Because nephrogenesis continues for a few days after birth in the mouse, this indicated that human IGF binding protein-1 overexpression altered postnatal nephrogenesis. In addition, exogenously added IGF-II, but not IGF-I, was effective in stimulating in vitro nephrogenesis. Together these elements suggest that reduced amounts of circulating IGFs, presumably IGF-II, impair kidney development.

HIV-1 Tat reduces nephrin in human podocytes : a potential mechanism for enhanced glomerular permeability in HIV-associated nephropathy

Objective:To determine whether HIV-1 Tat may directly alter glomerular permeability in HIV-associated nephropathy (HIVAN). Design:Heavy proteinuria is a hallmark of HIVAN. The slit diaphragm is the ultimate glomerular filtration barrier critical for maintaining the efficiency of the ultrafiltration unit of the kidney. In this study, we evaluated the direct effect of Tat protein on the permeability of isolated glomeruli and on the expression of nephrin, the main slit diaphragm component, by human cultured podocytes. Methods:Permeability was studied by measuring the permeability to albumin in isolated rat glomeruli. We also evaluated the expression of nephrin in human cultured podocytes by using immunofluorescence and Western blot. Results:We found that Tat increased albumin permeability in isolated glomeruli, and rapidly induced the redistribution and loss of nephrin in cultured podocytes. Pretreatment of glomeruli and podocytes with blocking antibodies showed that Tat reduced nephrin expression by engaging vascular endothelial growth factor receptors types 2 and 3 and the integrin αvβ3. Pre-incubation of podocytes with two platelet-activating factor (PAF) receptor antagonists prevented the loss and redistribution of nephrin induced by Tat, suggesting that PAF is an intracellular mediator of Tat action. Tat induced a rapid PAF synthesis by podocytes. When podocytes transfected to overexpress PAF-acetylhydrolase, the main catabolic enzyme of PAF, were stimulated with Tat, the redistribution and loss of nephrin was abrogated. Conclusion:The present results define a mechanism by which Tat may reduce nephrin expression in podocytes, thus increasing glomerular permeability. This provides new insights in the understanding of HIVAN pathogenesis.

RhoA Silencing Reverts the Resistance to Doxorubicin in Human Colon Cancer Cells

The efficacy of doxorubicin in the treatment of cancer is limited by its side effects and by the onset of drug resistance. Reverting such resistance could allow the decrease of the dose necessary to eradicate the tumor, thus diminishing the toxicity of the drug. We transfected doxorubicin-sensitive (HT29) and doxorubicin-resistant (HT29-dx) human colon cancer cells with RhoA small interfering RNA. The subsequent decrease of RhoA protein was associated with the increased sensitivity to doxorubicin in HT29 cells and the complete reversion of doxorubicin resistance in HT29-dx cells. RhoA silencing increased the activation of the nuclear factor-κB pathway, inducing the transcription and the activity of nitric oxide synthase. This led to the tyrosine nitration of the multidrug resistance protein 3 transporter (MRP3) and contributed to a reduced doxorubicin efflux. Moreover, RhoA silencing decreased the ATPase activity of P-glycoprotein (Pgp) in HT29 and HT29-dx cells as a consequence of the reduced expression of Pgp. RhoA silencing, by acting as an upstream controller of both MRP3 nitration and Pgp expression, was effective to revert the toxicity and accumulation of doxorubicin in both HT29 and HT29-dx cells. Therefore, we suggest that inactivating RhoA has potential clinical applications and might in the future become part of a gene therapy protocol. (Mol Cancer Res 2008;6(10):1607–20)

HIV type 1 Tat protein is a survival factor for Kaposi's sarcoma and endothelial cells.

The HIV-1 Tat protein has been directly implicated in the pathogenesis of AIDS-related Kaposis sarcoma (KS); however, its effects on KS spindle-shaped and endothelial cell apoptosis are largely unexplored. Since susceptibility to apoptosis is relevant for tumor development and response to therapy, we investigated the effects of Tat on KS and endothelial cell survival from apoptosis. The effect of Tat was evaluated in three KS cell lines (KS-imm, KS-C1, and KS-L3) exposed to the chemotherapy agent vincristine, currently used for the treatment of this tumor, and in human umbilical vein-derived endothelial cells (HUVECs) induced to undergo apoptosis by serum withdrawal. Apoptosis was assessed by enzymatic assays, microscopic examination of chromatin and cytoskeleton, evaluation of plasma membrane integrity and subdiploid DNA content, TUNEL assays, and measurement of caspase-3 activity. Tat, in a dose-dependent manner, protected the three KS cell lines and HUVECs from apoptosis induced by vincristine or serum starvation, respectively. This effect appeared to be independent of modulation of Fas, Bcl-2, or Bax expression. In contrast, Tat upregulated Bcl-X(L) expression and induced a relevant decrease in caspase-3 activity in vincristine-treated KS cells. Taken together, these results suggest that the HIV-1 Tat protein may factor KS development and progression by sustaining endothelial and transformed cell survival.