Cristina Zanchi

Protein Overload-Induced NF-κB Activation in Proximal Tubular Cells Requires H2O2 through a PKC-Dependent Pathway

Abnormal traffic of proteins through the glomerular capillary has an intrinsic toxicity that results in tubular dysfunction and interstitial inflammation. It has been previously shown that in porcine proximal tubular cells high concentrations of albumin activated NF-kappaB, which is responsible for the enhanced synthesis of the inflammatory chemokine RANTES. This study investigates whether reactive oxygen species (ROS) served as second messengers in protein overload-induced NF-kappaB activation. Human proximal tubular cells (HK-2) were incubated (5 to 60 min) with human albumin and IgG (1 to 30 mg/ml). Both proteins induced a rapid or significant increase in hydrogen peroxide (H(2)O(2)) production at 5 min and persisting at 60 min. This effect was dose-dependent. The contribution of H(2)O(2) in regulating NF-kappaB activation was evaluated by using the antioxidants dimethyl-thiourea and pyrrolidine dithiocarbamate in protein-overloaded HK-2 cells. Both agents, by preventing H(2)O(2) generation, induced human albumin or IgG inhibited NF-kappaB activation. Stimulation of HK-2 with exogenous H(2)O(2) resulted in the activation of a NF-kappaB subunit pattern similar to that obtained after protein challenge. Specific inhibitors of protein kinase C (PKC) activity significantly prevented H(2)O(2) production and consequent NF-kappaB activation, suggesting that ROS generation in HK-2 cells occurs downstream of PKC activation. Either antioxidants or PKC inhibitor almost completely abolished the upregulation of the monocyte chemoattractant protein-1 gene induced by excess albumin, as evaluated by real-time PCR, thus supporting a role for PKC and ROS as critical signals for the expression of NF-kappaB-dependent inflammatory genes. To identify the enzymatic sources responsible for the increased H(2)O(2) production, the effect of dyphenyleneiodonium, an inhibitor of the membrane NADP(H) oxidase, was studied, as was the effect of rotenone, which blocks complex I of the mitochondrial respiratory chain. It was found that both agents significantly reduced the exaggerated H(2)O(2) induced by protein overload. These data indicate that exposure to excess proteins in proximal tubular cells induces the formation of ROS, which are responsible for NF-kappaB activation and consequent induction of NF-kappaB-dependent inflammatory signals.

Add-On Anti–TGF-β Antibody to ACE Inhibitor Arrests Progressive Diabetic Nephropathy in the Rat

Renin-angiotensin system (RAS) inhibitors are effective in reducing renal disease progression in early diabetic nephropathy, but they provide imperfect protection at a later stage. Due to the pivotal role of transforming growth factor-beta (TGF-beta) in the pathogenesis of diabetic kidney disease, this study tested the effect of simultaneously interrupting TGF-beta and angiotensin II on disease progression in diabetic rats with overt nephropathy. Diabetes was induced by streptozotocin injection in uninephrectomized rats. Diabetic rats received murine (1D11) or human (CAT-192) anti-TGF-beta monoclonal antibodies alone or in combination with lisinopril, 13C4 irrelevant murine antibody, saline or lisinopril from month 4 (when animals had proteinuria) to month 8. Normal animals served as controls. Systolic BP increase was controlled by single treatments and even more by the combined therapies. 1D11 and lisinopril kept proteinuria at levels numerically lower than irrelevant antibody and saline, while CAT-192 was ineffective. The addition of either TGF-beta antibody to lisinopril normalized proteinuria. Consistent results were obtained for glomerulosclerosis and tubular damage, which were abrogated by the combined therapy. Interstitial volume expansion and infiltration of lymphocytes/macrophages were limited by 1D11 and lisinopril and further reduced by their combination. The increase of type III collagen in the renal interstitium was partially attenuated by 1D11 and lisinopril while normalized by their combination. It is concluded that anti-TGF-beta antibody when added to a background of chronic angiotensin-converting enzyme (ACE) inhibition fully arrests proteinuria and renal injury of overt diabetic nephropathy, providing a novel route to therapy and remission of disease for diabetic patients who do not respond to RAS inhibition.

How To Fully Protect the Kidney in a Severe Model of Progressive Nephropathy: A Multidrug Approach

The current therapy for chronic proteinuric nephropathies is angiotensin-converting enzyme inhibitors (ACEi), which slow, but may not halt, the progression of disease, and which may be not effective to the same degree in all patients. In accelerated passive Heymann nephritis (PHN), this study assessed the effect of combining ACEi with angiotensin II receptor antagonist (AIIRA) and with statin that, besides lowering cholesterol, influences inflammatory and fibrogenic processes. Uninephrectomized PHN rats were divided into four groups (n = 10 each) and daily given oral doses of the following: vehicle; 40 mg/L lisinopril; 100 mg/L lisinopril plus L-158,809; 0.3 mg/kg lisinopril plus L-158,809 plus cerivastatin. Treatments started at 2 mo when rats had massive proteinuria and signs of renal injury and lasted until 10 mo. Increases in BP were equally lowered by treatments. ACEi kept proteinuria at levels comparable to pretreatment and numerically lower than vehicle. The addition of AIIRA to lisinopril was more effective, being proteinuria reduced below pretreatment values and significantly lower than vehicle. When cerivastatin was added on top of ACE inhibition and AIIR blockade, urinary protein regressed to normal values and renal failure was prevented. Renal ACE activity was increased threefold in PHN, it was inhibited by more than 60% after ACEi, and decreased below control values with triple therapy. Cerivastatin inhibited ACE activity by 30%. Glomerulosclerosis, tubular damage and interstitial inflammation were ameliorated by ACEi alone or combined with AIIRA, and prevented by addition of statin. TGF-beta(1) mRNA upregulation in PHN kidney was partially reduced after ACEi or combined with AIIRA and almost normalized after adding statin. Cerivastatin inhibited TGF-beta(1) gene upregulation by 25%. These data suggest a possible future strategy to induce remission of proteinuria, lessen renal injury, and protect from loss of function in those patients who do not fully respond to ACEi therapy.

Transforming Growth Factor-β1 Is Up-Regulated by Podocytes in Response to Excess Intraglomerular Passage of Proteins : A Central Pathway in Progressive Glomerulosclerosis

Chronic diseases of the kidney have a progressive course toward organ failure. Common pathway mechanisms of progressive injury, irrespectively of the etiology of the underlying diseases, include glomerular capillary hypertension and enhanced passage of plasma proteins across the glomerular capillary barrier because of impaired permselective function. These changes are associated with podocyte injury and glomerular sclerosis. Direct evidence for causal roles is lacking, particularly for the link between intraglomerular protein deposition and sclerosing reaction. Because transforming growth factor-beta1 (TGF-beta1) is the putative central mediator of scarring, we hypothesized that TGF-beta1 can be up-regulated by protein overload of podocytes thereby contributing to sclerosis. In rats with renal mass reduction, protein accumulation in podocytes as a consequence of enhanced transcapillary passage preceded podocyte dedifferentiation and injury, increase in TGF-beta1 expression in podocytes, and TGF-beta1-dependent activation of mesangial cells. Angiotensin-converting enzyme inhibitor prevented both accumulation of plasma proteins and TGF-beta1 overexpression in podocytes and sclerosis. Albumin load on podocytes in vitro caused loss of the synaptopodin differentiation marker and enhanced TGF-beta1 mRNA and protein. Conditioned medium of albumin-stimulated podocytes induced a sclerosing phenotype in mesangial cells, an effect mimicked by TGF-beta1 and blocked by anti-TGF-beta1 antibodies. Thus, the passage of excess plasma proteins across the glomerular capillary wall is the trigger of podocyte dysfunction and of a TGF-beta1-mediated mechanism underlying sclerosis. Agents to reduce TGF-beta1, possibly combined with angiotensin blockade, should have priority in novel approaches to treatment of progressive nephropathies.

In response to protein load podocytes reorganize cytoskeleton and modulate endothelin-1 gene: Implication for permselective dysfunction of chronic nephropathies

Effacement of podocyte foot processes occurs in many proteinuric nephropathies and is accompanied by rearrangement of the actin cytoskeleton. Here, we studied whether protein overload affects intracellular pathways, leading to cytoskeletal architecture changes and ultimately to podocyte dysfunction. Mouse podocytes bound and endocytosed both albumin and IgG via receptor-specific mechanisms. Protein overload caused redistribution of F-actin fibers instrumental to up-regulation of the prepro-endothelin (ET)-1 gene and production of the corresponding peptide. Increased DNA-binding activity for nuclear factor (NF)-kappaB and Ap-1 nuclear proteins was measured in nuclear extracts of podocytes exposed to excess proteins. Both Y27632, which inhibits Rho kinase-dependent stress fiber formation, and jasplakinolide, an F-actin stabilizer, decreased NF-kappaB and Ap-1 activity and reduced ET-1 expression. This suggested a role for the cytoskeleton, through activated Rho, in the regulation of the ET-1 peptide. Focal adhesion kinase (FAK), an integrin-associated nonreceptor tyrosine kinase, was phosphorylated by albumin treatment via Rho kinase-triggered actin reorganization. FAK activation led to NF-kappaB- and Ap-1-dependent ET-1 expression. These data suggest that reorganization of the actin cytoskeletal network in response to protein load is implicated in modulation of the ET-1 gene via Rho kinase-dependent FAK activation of NF-kappaB and Ap-1 in differentiated podocytes. Increased ET-1 generation might alter glomerular permselectivity and amplify the noxious effect of protein overload on dysfunctional podocytes.

Protein Overload Induces Fractalkine Upregulation in Proximal Tubular Cells through Nuclear Factor κB– and p38 Mitogen-Activated Protein Kinase–Dependent Pathways

Investigated was the effect of high albumin concentrations on proximal tubular cell expression of fractalkine. Human proximal tubular cells (HK-2) were incubated with human serum albumin (HSA), which induced a dose-dependent increase in fractalkine mRNA associated with increased levels of both membrane-bound and soluble forms of the protein. To evaluate the role of nuclear factor kappaB (NF-kappaB) activation in HSA-induced fractalkine mRNA, HK-2 cells were infected with a recombinant adenovirus encoding the natural inhibitor of NF-kappaB, IkBalpha; a 43% reduction of fractalkine mRNA levels resulted. Similarly, when cells were infected with the recombinant adenovirus expressing dominant negative mutant of the IkB kinase 2, a 55% inhibition of fractalkine mRNA was achieved. p38 mitogen-activated protein kinase was activated by HSA and was involved in NF-kappaB-dependent transcription of fractalkine. In kidneys of mice with bovine serum albumin overload proteinuria, fractalkine mRNA levels were 2.3-fold greater than those of controls. Fractalkine expression was also induced in tubular epithelial cells in this model. Anti-CXCR1 antibody treatment limited interstitial accumulation of mononuclear cells. Protein overload is a promoter of fractalkine gene induction mediated by NF-kappaB and p38 activation in proximal tubular cells. Fractalkine might contribute to direct mononuclear cells into peritubular interstitium and enhance their adhesion property, which in turn would favor inflammation and disease progression.

Transcriptional Regulation of Nephrin Gene by Peroxisome Proliferator–Activated Receptor-γ Agonist: Molecular Mechanism of the Antiproteinuric Effect of Pioglitazone

The renoprotective potential of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist pioglitazone was explored in an immune model of progressive nephropathy, passive Heymann nephritis (PHN), compared with that of an angiotensin II receptor antagonist, taken as standard therapy for renoprotection. PHN rats received orally vehicle, pioglitazone (10 mg/kg twice daily), or candesartan (1 mg/kg twice daily) from months 2 to 8. Pioglitazone reduced proteinuria as effectively as candesartan and limited renal functional and structural changes. Kidneys from untreated PHN rats showed lower nephrin mRNA and protein than controls, both restored by pioglitazone. The effect was seen both early and late during the course of the disease. Whether the antiproteinuric effect of pioglitazone could be due to its effect on nephrin gene transcription also was investigated. HK-2 cells were transfected with plasmids that harbor the luciferase gene under portions (2-kb or 325-bp) of human nephrin gene promoter that contain putative peroxisome proliferator-responsive elements (PPRE) and incubated with pioglitazone (10 muM). Transcriptional activity of luciferase gene was highly increased by pioglitazone, with the strongest expression achieved with the 325-bp fragment. Increase in luciferase activity was prevented by bisphenol A diglycidyl ether, a PPAR-gamma synthetic antagonist. Electrophoretic mobility shift assay experiments showed a direct interaction of PPAR/retinoid X receptor heterodimers to PPRE present in the enhancer region of the nephrin promoter. In conclusion, pioglitazone exerts an antiproteinuric effect in immune-mediated glomerulonephritis as angiotensin II receptor antagonist does. Enhancement of nephrin gene transcription through specific PPRE in its promoter discloses a novel mechanism of renoprotection for PPAR-gamma agonists.

Renal expression of FGF23 in progressive renal disease of diabetes and the effect of ACE inhibitor.

Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone mainly produced by bone that acts in the kidney through FGF receptors and Klotho. Here we investigated whether the kidney was an additional source of FGF23 during renal disease using a model of type 2 diabetic nephropathy. Renal expression of FGF23 and Klotho was assessed in Zucker diabetic fatty (ZDF) and control lean rats at 2, 4, 6, 8 months of age. To evaluate whether the renoprotective effect of angiotensin converting enzyme (ACE) inhibitor in this model was associated with changes in FGF23 and Klotho, ZDF rats received ramipril from 4, when proteinuric, to 8 months of age. FGF23 mRNA was not detectable in the kidney of lean rats, nor of ZDF rats at 2 months of age. FGF23 became measurable in the kidney of diabetic rats at 4 months and significantly increased thereafter. FGF23 protein localized in proximal and distal tubules. Renal Klotho mRNA and protein decreased during time in ZDF rats. As renal disease progressed, serum phosphate levels increased in parallel with decline of fractional phosphorus excretion. Ramipril limited proteinuria and renal injury, attenuated renal FGF23 upregulation and ameliorated Klotho expression. Ramipril normalized serum phosphate levels and tended to increase fractional phosphorus excretion. These data indicate that during progressive renal disease the kidney is a site of FGF23 production which is limited by ACE inhibition. Interfering pharmacologically with the delicate balance of FGF23 and phosphorus in diabetes may have implications in clinics.

Mesenchymal stem cell therapy promotes renal repair by limiting glomerular podocyte and progenitor cell dysfunction in adriamycin-induced nephropathy

We previously reported that in a model of spontaneously progressive glomerular injury with early podocyte loss, abnormal migration, and proliferation of glomerular parietal epithelial progenitor cells contributed to the formation of synechiae and crescentic lesions. Here we first investigated whether a similar sequence of events could be extended to rats with adriamycin (ADR)-induced nephropathy. As a second aim, the regenerative potential of therapy with bone marrow-derived mesenchymal stem cells (MSCs) on glomerular resident cells was evaluated. In ADR-treated rats, decrease of WT1(+) podocyte number due to apoptosis was associated with reduced glomerular expression of nephrin and CD2AP. As a consequence of podocyte injury, glomerular adhesions of the capillary tuft to the Bowmans capsule were observed, followed by crescent-like lesions and glomerulosclerosis. Cellular components of synechiae were either NCAM(+) parietal progenitor cells or nestin(+) podocytes. In ADR rats, repeated injections of MSCs limited podocyte loss and apoptosis and partially preserved nephrin and CD2AP. MSCs attenuated the formation of glomerular podocyte-parietal epithelial cell bridges and normalized the distribution of NCAM(+) progenitor cells along the Bowmans capsule, thereby reducing glomerulosclerosis. Finding that MSCs increased glomerular VEGF expression and limited microvascular rarefaction may explain the prosurvival effect by stem cell therapy. MSCs also displayed anti-inflammatory activity. Coculture of MSCs with ADR-damaged podocytes showed a functional role of stem cell-derived VEGF on prosurvival pathways. These data suggest that MSCs by virtue of their tropism for damaged kidney and ability to provide a local prosurvival environment may represent a useful strategy to preserve podocyte viability and reduce glomerular inflammation and sclerosis.

Complement-Mediated Dysfunction of Glomerular Filtration Barrier Accelerates Progressive Renal Injury

Intrarenal complement activation leads to chronic tubulointerstitial injury in animal models of proteinuric nephropathies, making this process a potential target for therapy. This study investigated whether a C3-mediated pathway promotes renal injury in the protein overload model and whether the abnormal exposure of proximal tubular cells to filtered complement could trigger the resulting inflammatory response. Mice with C3 deficiency were protected to a significant degree against the protein overload-induced interstitial inflammatory response and tissue damage, and they had less severe podocyte injury and less proteinuria. When the same injury was induced in wild-type (WT) mice, antiproteinuric treatment with the angiotensin-converting enzyme inhibitor lisinopril reduced the amount of plasma protein filtered, decreased the accumulation of C3 by proximal tubular cells, and protected against interstitial inflammation and damage. For determination of the injurious role of plasma-derived C3, as opposed to tubular cell-derived C3, C3-deficient kidneys were transplanted into WT mice. Protein overload led to the development of glomerular injury, accumulation of C3 in podocytes and proximal tubules, and tubulointerstitial changes. Conversely, when WT kidneys were transplanted into C3-deficient mice, protein overload led to a more mild disease and abnormal C3 deposition was not observed. These data suggest that the presence of C3 increases the glomerular filtration barriers susceptibility to injury, ultrafiltered C3 contributes more to tubulointerstitial damage induced by protein overload than locally synthesized C3, and local C3 synthesis is irrelevant to the development of proteinuria. It is speculated that therapies targeting complement combined with interventions to minimize proteinuria would more effectively prevent the progression of renal disease.