Mesangial Cell Apoptosis Research Articles

Pattern recognition and renal defence in crescentic glomerulonephritis

Chavele et al. studied the role of the mannose receptor (MR) in crescentic nephritis. The accelerated nephrotoxic model of glomerulonephritis was induced on both wild-type (WT) and MR-deficient mice. The mice lacking the MR showed a markedly altered phenotype. They were largely protected against the development of glomerulonephritis with less affected glomeruli, less proteinuria and much better renal function when compared to the WT mice. Whilst more infiltrating macrophages were present in the WT animals, there was no difference in the deposition of sheep and mouse immunoglobulins. To elucidate the mechanism of MR-mediated damage, the authors additionally performed a series of in vitro experiments. They show that the Fab portion of sheep immunoglobulins binds to MR binding domains. Interestingly, the MR seems to interact with FcR-mediated cellular reactions. When MR-deficient macrophages and mesangial cells were treated with immune complexes, the macrophages showed a significantly poorer oxygen burst when compared with WT cells. In line with this possible interaction between Fc-receptors and MR, co-localization of Fcã-receptors and MR was demonstrated on macrophages. Additionally, the authors observed that MR-deficient mesangial cells in culture proliferated more abundantly and showed a markedly increased rate of spontaneous apoptosis compared with WT cells. These findings led the authors to test whether this increased apoptosis could play a role in the suppression of glomerular inflammation. Indeed, TNF-á production by LPS-stimulated macrophages was markedly reduced in the presence or apoptotic cells. The anti-inflammatory effect of apoptotic mesangial cells was increased when MR-deficient macrophages were used instead of WT cells (Figure 1). Fig. 1 Proposed role for mannose receptor in crescentic glomerulonephritis. (A) IgG may bind to the Fc receptor with the Fc domain and to the mannose receptor with the Fab portion leading to an increased oxygen burst (left). If the mannose receptor is absent, the oxygen burst is decreased (right). (B) Absence of the mannose receptor leads to an increased proliferation and apoptosis of mesangial cells. These apoptotic cells induce a non-inflammatory macrophage phenotype with IL-10 and TGF-beta production (right). If the mannose receptor is present, mesangial cells proliferate less and are less apoptotic, and a pro-inflammatory macrophage phenotype with TNF-alpha production prevails.

Oxidized high‐density lipoprotein enhances inflammatory activity in rat mesangial cells

Inflammation is a mechanism of glomerular damage in chronic glomerulopathies, in which dyslipidaemia plays an important role. Unlike native high-density lipoprotein (HDL), oxidized HDL is thought to be an adverse factor in chronic ischaemic disease and may increase the production of inflammatory cytokines in atheromatous plaques and plasma, but the effect of oxidized HDL on mesangial cells remains unclear. Intracellular reactive oxygen species level was measured. The inflammatory and proapoptotic effects of oxidized HDL were detected in rat mesangial cells by measuring levels of tumour necrosis factor-alpha, regulated upon activation, normal T-cell expressed and secreted, monocyte chemoattractant protein-1, CXC chemokine ligand-1 and early apoptosis. The expression of mitogen-activated protein kinase (MAPK) (p38/MAPK, extracellular-regulated kinase/MAPK and c-Jun N-terminal kinase/MAPK), nuclear factor-kappaB activity and lipoprotein scavenger receptors (CD36, low-density lipoprotein receptor-1 and scavenger receptor BI) were also detected. Oxidized HDL enhanced reactive oxygen species production and upregulated expression of proinflammatory factors, including tumour necrosis factor-alpha, regulated upon activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 and CXC chemokine ligand-1 by rat mesangial cells dose in a dependent fashion. Incubation with oxidized HDL also increased rat mesangial cells apoptosis in a dose-dependent manner. These effects partly depended on scavenger receptors CD36 and low-density lipoprotein receptor-1, but not scavenger receptor BI. In addition, co-culture with oxidized HDL activated P38/MAPK, extracellular-regulated kinase (ERK)/MAPK and nuclear factor-kappaB (NF-kappaB). The results of the present study suggest that oxidized HDL enhanced proinflammatory properties in mesangial cells partly via CD36 and low-density lipoprotein receptor-1. MAPK and nuclear factor-kappaB pathways were involved in the process. The ability of oxidized HDL to negatively influence mesangial cell biology may represent an important mechanism of chronic kidney disease.

Advanced glycation end products‐induced apoptosis attenuated by PPARδ activation and epigallocatechin gallate through NF‐κB pathway in human embryonic kidney cells and human mesangial cells

Diabetic nephropathy has attracted many researchers' attention. Because of the emerging evidence about the effects of advanced glycation end products (AGEs) and receptor of AGE (RAGE) on the progression of diabetic nephropathy, a number of different therapies to inhibit AGE or RAGE are under investigation. The purpose of the present study was to examine whether peroxisome proliferator-activated receptor delta (PPARdelta) agonist (L-165041) or epigallocatechin gallate (EGCG) alters AGE-induced pro-inflammatory gene expression and apoptosis in human embryonic kidney cells (HEK293) and human mesangial cells (HMCs). The HEK cells and HMC were separated into the following groups: 100 microg/mL AGE alone for 18 h; AGE treated with 1 microM L-165041 or 10 microM EGCG, and untreated cells. Inflammatory cytokines, nuclear factor-kappaB pathway, RAGE expression, superoxide dismutase and cell apoptosis were determined. AGE significantly increased tumour necrosis factor-alpha (TNF-alpha), a major pro-inflammatory cytokine. The mRNA and protein expression of RAGE were up-regulated. These effects were significantly attenuated by pre-treatment with L-165041 or EGCG. AGE-induced nuclear factor-kappaB pathway activation and both cells apoptosis were also inhibited by L-165041 or EGCG. Furthermore, both L-165041 and EGCG increased superoxide dismutase levels in AGE-treated HEK cells and HMC. This study demonstrated that PPARdelta agonist and EGCG decreased the AGE-induced kidney cell inflammation and apoptosis. This study provides important insights into the molecular mechanisms of EGCG and PPARdelta agonist in attenuation of kidney cell inflammation and may serve as a therapeutic modality to treat patients with diabetic nephropathy.

Glucocorticoids protect renal mesangial cells from apoptosis by increasing cellular sphingosine-1-phosphate

Neutral ceramidase (NCDase) and sphingosine kinases (SphKs) are key enzymes regulating cellular sphingosine-1-phosphate (S1P) levels. In this study we found that stress factor-induced apoptosis of rat renal mesangial cells was significantly reduced by dexamethasone treatment. Concomitantly, dexamethasone increased cellular S1P levels, suggesting an activation of sphingolipid-metabolizing enzymes. The cell-protective effect of glucocorticoids was reversed by a SphK inhibitor, was completely absent in SphK1-deficient cells, and was associated with upregulated mRNA and protein expression of NCDase and SphK1. Additionally, in vivo experiments in mice showed that dexamethasone also upregulated SphK1 mRNA and activity, and NCDase protein expression in the kidney. Fragments (2285, 1724, and 1126 bp) of the rat NCDase promoter linked to a luciferase reporter were transfected into rat kidney fibroblasts and mesangial cells. There was enhanced NCDase promoter activity upon glucocorticoids treatment that was abolished by the glucocorticoid receptor antagonist RU-486. Single and double mutations of the two putative glucocorticoid response element sites within the promoter reduced the dexamethasone effect, suggesting that both glucocorticoid response elements are functionally active and required for induction. Our study shows that glucocorticoids exert a protective effect on stress-induced mesangial cell apoptosis in vitro and in vivo by upregulating NCDase and SphK1 expression and activity, resulting in enhanced levels of the protective lipid second messenger S1P.

Role of activating transcription factor 3 (ATF3) in sublytic C5b‐9‐induced glomerular mesangial cell apoptosis

Sublytic complement C5b-9 complexes can cause cell apoptosis, but the mechanism of glomerular mesangial cell (GMC) apoptosis mediated by these complexes has not been well defined. The activating transcription factor 3 (ATF3) gene is an immediate early gene for the cell to cope with a variety of stress signals and can promote apoptosis of some cells. In this study, ATF3 expression and cell apoptosis in GMCs induced by sublytic C5b-9 were measured, and then the effects of ATF3 gene over-expression or knockdown on GMC apoptosis induced by sublytic C5b-9 were examined at a fixed time. The results showed that both ATF3 expression and GMC apoptosis were markedly increased and ATF3 over-expression obviously increased sublytic C5b-9-induced GMC apoptosis, whereas ATF3 gene silencing had a significant opposite effect. Collectively, these findings indicate that upregulation of ATF3 gene expression is involved in regulating GMC apoptosis induced by sublytic C5b-9 complexes.

Upregulation of Hic-5 in glomerulosclerosis and its regulation of mesangial cell apoptosis

Glomerulosclerosis is characterized by the loss of glomerular cells by apoptosis and deposition of collagen type I into the normal collagen IV-containing mesangial matrix. We sought to determine the alterations that might contribute to these changes by performing proteomic analysis of rat mesangial cell lysates comparing cells cultured on normal collagen type IV to those grown on abnormal collagen type I surfaces. Subculture on collagen type I was associated with changed expression of several proteins, including a significant upregulation of the paxillin-like LIM protein, hydrogen-peroxide-induced clone 5 (Hic-5), and increased the susceptibility of the cells to apoptosis in response to physiological triggers. When we knocked down Hic-5 (using siRNA), we found mesangial cells grown on collagen type I were protected from apoptosis to the same degree as untreated cells grown on collagen type IV. Further we found that the level of Hic-5 in vivo was almost undetectable in control rats but increased dramatically in the glomerular mesangium of remnant kidneys 90 and 120 days after subtotal nephrectomy. This induction of Hic-5 paralleled the upregulation of mesangial collagen type I expression and glomerular cell apoptosis. Our results suggest that Hic-5 is pivotal in mediating the response of mesangial cells to attachment on abnormal extracellular matrix during glomerular scarring.

Influence of glucosamine on glomerular mesangial cell turnover: implications for hyperglycemia and hexosamine pathway flux

Cells exposed to high glucose may undergo hypertrophy, proliferation, and apoptosis, but the role of hexosamine flux in mediating these effects has not been fully elucidated. Accordingly, we studied the effects of glucose and glucosamine on rat glomerular mesangial cells (MC) turnover. Compared with physiological glucose (5.6 mM), treatment with high glucose (25 mM) for 24 h stimulated MC proliferation, an effect that was mimicked by exposure to low concentrations of glucosamine (0.05 mM). The percentage of cells in G(0)/G(1) phase of the cell cycle was reduced with a concomitant increase of the number of cells in G(2)/M phase. Proliferating cell nuclear antigen, phosphorylated mammalian target of rapamycin [phospho-mTOR (Ser(2448))], and total regulatory-associated protein of mTOR were increased by high glucose and glucosamine treatment. Inhibition of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme for hexosamine flux, with 6-diazo-5-oxonorleucine (10 muM) and of mTOR with rapamycin both attenuated glucose-mediated MC proliferation. Higher glucosamine concentrations (0.25-10 mM) caused MC apoptosis after 48 h, and, in addition, GFAT overexpression also increased MC apoptosis (TdT-dUTP nick end-labeling-positive cells: 3.8 +/- 0.3 vs. 1.1 +/- 0.2% for empty vector; P < 0.05). Hence, hexosamine flux is an important determinant of MC proliferation and apoptosis. The proliferative response to high glucose and hexosamine flux is rapamycin-sensitive, suggesting that this effect is associated with signaling through rapamycin-sensitive mTOR complex 1 (mTORC1).

Apoptosis of glomerular mesangial cells induced by sublytic C5b‐9 complexes in rats with Thy‐1 nephritis is dependent on Gadd45γ upregulation

The complement C5b-9 complexes can result in cell apoptosis, but the mechanism of sublytic C5b-9-mediated glomerular mesangial cell (GMC) apoptosis in Thy-1 nephritis (Thy-1N) remains largely unclear. The Gadd45 gene is involved in the cellular response to DNA damage and can promote cell apoptosis. In this study, both Gadd45 gamma expression patterns and pathologic changes of renal tissue were examined in rat Thy-1N. Both Gadd45 gamma expression and GMC apoptosis were significantly decreased in Thy-1N rats upon the depletion of complement with cobra venom factor. Our in vitro studies showed that Gadd45 gamma over-expression increased sublytic C5b-9-induced GMC apoptosis, while Gadd45 gamma gene knockdown by siRNA greatly reduced GMC apoptosis. Moreover, Gadd45 gamma gene silencing in vivo markedly inhibited the pathologic changes in the renal tissue of Thy-1N rats. These data suggest that Gadd45 gamma gene expression is involved in regulating GMC apoptosis mediated by sublytic C5b-9 in Thy-1N.

The role of endoplasmic reticulum in cadmium-induced mesangial cell apoptosis

Cd is an industrial and environmental pollutant that affects many organs in humans and other mammals. However, the molecular mechanisms of Cd-induced nephrotoxicity are unclear. In this study, we show that endoplasmic reticula (ER) played a pivotal role in Cd-induced apoptosis in mesangial cells. Using Fluo-3 AM, the intracellular concentration of calcium ([Ca 2+] i) was detected as being elevated as time elapsed after Cd treatment. Co-treatment with BAPTA-AM, a calcium chelator, was able to significantly suppress Cd-induced apoptosis. Calcineurin is a cytosolic phosphatase, which was able to dephosphorylate the inositol-1,4,5-triphosphate receptor (IP 3R) calcium channel to prevent the release of calcium from ER. Cyclosporine A, a calcineurin inhibitor, increased both [Ca 2+] i and the percentage of Cd-induced apoptosis. However, EGTA and the IP 3R inhibitor, 2-APB, were able to partially modulate Cd cytotoxicity. These results led us to suggest that the extracellular and ER-released calcium plays a crucial role in Cd-induced apoptosis in mesangial cells. Following this line, we further detected the ER stress after Cd treatment since ER is one of the major calcium storage organelles. After Cd exposure, GADD153, a hallmark of ER stress, was upregulated (at 4 h of exposure), followed by activation of ER-specific caspase-12 and its downstream molecule caspase-3 (at 16 h of exposure). The pan caspase inhibitor, Z-VAD, and BAPTA-AM were able to reverse the Cd-induced cell death and ER stress, respectively. Furthermore, the mitochondrial membrane potential (Δ Ψ m) was depolarized significantly and cytochrome c was released after 24 h of exposure to Cd and followed by mild activation of caspase-9 at the 36-h time point, indicating that mitochondria stress is a late event. Therefore, we concluded that ER is the major killer organelle in Cd-induced mesangial cell apoptosis and that calcium oscillation plays a pivotal role.

Statin Pleiotropy Against Renal Injury

Statins may exhibit significant renoprotective effects beyond their lipid-lowering capacity. Herein, the authors review data from human and animal models of renal disease as well as from studies in cultured renal cells with regard to extralipid renoprotective properties of statins. Statins may exert lipid-independent benefits against renal injury in experimental states of chronic or acute renal function impairment. These include diabetic and hypertensive glomerulosclerosis, autoimmune glomerulonephritis, ischemia/reperfusion-induced renal damage, and unilateral ureteral obstructive nephropathy. Also, statins, by reducing the synthesis of mevalonate products, inhibit the activation of Rho and Ras guanosine triphosphatases that may influence various signaling pathways involving renal inflammatory, fibrogenic, proliferative, and cell-death responses. Therefore, statins exert anti-inflammatory actions in renal tissue, prevent renal scarring, and diminish mesangial or other kidney cell-type proliferation while promoting mesangial cell apoptosis. Renal antioxidant effects with consequent endothelial function regulation of renal vasculature following statin treatment may also account for pleiotropic protection against renal injury.

Candesartan attenuates Angiotensin II-induced mesangial cell apoptosis via TLR4/MyD88 pathway

Angiotensin II (Ang II) can stimulate Toll-like receptor 4 (TLR4) expression in mesangial cells (MCs), but the role of TLR4 in the Ang II-induced apoptosis and the effect of candesartan on TLR4 expression remain unclear. Here, we report that Ang II-induced MC apoptosis in a time-dependent manner and up-regulated TLR4/MyD88 expression, and that the intracellular ROS was subsequently increased. We also show that candesartan attenuated the Ang II-induced MC apoptosis, and that this protective effect was dependent on decreased TLR4/MyD88 expression as well as reduced intracellular ROS formation. Furthermore, Ang II increased the apoptosis inducing factor protein level, while candesartan markedly reduced this increase. These results demonstrate that TLR4/MyD88 pathway was involved in the Ang II promoted MC apoptosis, which was related to TLR4/MyD88 mediated oxidative stress. These data also suggest that candesartan exerted anti-apoptotic effect as an antioxidant by modulating this pathway.

Effect of tissue factor pathway inhibitor on apoptosis of rat mesangial cells and Fas and bcl-2 expression

To study the biological impact and mechanism of recombinant tissue factor pathway inhibitor (rTFPI) on apoptosis of rat kidney mesangial cells (MsC). TFPI expression in human glomerular minor lesion (GML), mesangial proliferative glomerulonephritis (MPGN) and cultured rat MsC was detected using immunohistochemistry and immunofluorescence, respectively. Rat MsC were incubated with rTFPI and its variant peptides. Morphological changes of apoptosis were investigated by Hoechst 33258 and the apoptotic rate was assessed by flow cytometry. DNA fragmentation and effect of rTFPI on expression of caspase-3, Fas and bcl-2 were studied using gel electrophoresis and Western blot respectively. The expression of TFPI in MPGN was higher than that in GML. TFPI was expressed in cultured rat mesangial cells. Apoptosis of MsC was induced by rTFPI, especially by its C-termianl, in a dose- and time-dependent manner. Apoptosis ratios of MsC treated with rTFPI were 2.1, 3.0 and 4.9 times more than control, respectively. Expression of gene caspase-3 and Fas was up-regulated in a dose-dependent manner wherease bcl-2 expression did not show any changes. rTFPI induces apoptosis in cultured rat mesangial cells by its C-terminal possibly via Fas/FasL pathway.

Overexpression of decorin induces apoptosis and cell growth arrest in cultured rat mesangial cells in vitro

Decorin (DCN) is a small leucine-rich proteoglycan that plays an important role in the regulation of intercellular contact, cell migration and proliferation. DCN suppresses cell growth and induces apoptosis in various tumour cells. The aim of this study was to investigate whether overexpression of DCN could induce apoptosis and cell growth arrest in mesangial cells (MsCs) in vitro. PcDNA3.1A-DCN plasmid was transfected into cultured rat MsCs, and positive clones stably expressing DCN (MsC/DCN) were selected. SiRNA was used for blocking DCN expression in MsC/DCN. Apoptosis and cell growth of MsCs were assayed by flow cytometry. Hoechst staining was used for observing apoptotic cells. Expressions of active Caspase-3, epidermal growth factor receptor (EGFR), P21 and transforming growth factor-beta (TGF-beta1) were analyzed using Western blot. Overexpression of DCN in MsCs induced apoptosis and arrested cells in the G(0)/G(1) phase. The protein level of active Caspase-3 was significantly elevated in MsC/DCN (P < 0.01). DCN transfection induced downregulation of EGFR and up-expression of P21. In addition, the expression of TGF-beta1 was significantly inhibited. DCN-siRNA transfection remarkably blocked the expression of DCN and reversed the downregulatory effects of DCN on MsC's proliferation. Overexpression of DCN could inhibit MsCs proliferation by inducing apoptosis and cell growth arrest in vitro and it also downregulates expression of TGF-beta1. These results suggest novel strategies for regulating the proliferation of MsC in glomerular diseases.

Urokinase induces survival or pro-apoptotic signals in human mesangial cells depending on the apoptotic stimulus

Deregulated apoptosis of MCs (mesangial cells) is associated with a number of kidney diseases including end-stage diabetic nephropathy. Cell death by apoptosis is a tightly orchestrated event, whose mechanisms are not completely defined. In the present study we show that the uPA (urokinase-type plasminogen activator)/uPAR (uPA receptor) system can initiate both cell survival and pro-apoptotic signals in human MCs in response to different apoptotic stimuli. uPA abrogated MC apoptosis induced by serum withdrawal conditions and enhanced apoptosis initiated in MCs by high glucose. Effects of uPA were independent of its proteolytic activity and required uPAR for both pro- and anti-apoptotic effects. Studies on the uPAR interactome provide evidence that the opposing effects of uPA were directed via different uPAR-interacting transmembrane partners. Exposure of MCs to RGD (Arg-Gly-Asp) peptide led to abrogation of the anti-apoptotic effect of uPA, which implies involvement of integrins in this process. A pro-apoptotic effect of uPA under high-glucose conditions was mediated via association of uPAR and the cation-independent M6P (mannose-6-phosphate)/IGF2R (insulin-like growth factor 2 receptor). Both receptors were co-precipitated and co-localized in MCs. Studies on the underlying signalling indicate that the ERK1/2 (extracellular-signal-regulated kinase 1/2), Akt and BAD (Bcl-2/Bcl-X(L)-antagonist, causing cell death) protein were involved in regulation of apoptosis by uPA in MCs. M6P/IGF2R mediated BAD perinuclear localization during apoptosis initiated by uPA and high glucose. In conclusion, we provide evidence that, in MCs, the uPA/uPAR system regulates survival/apoptosis processes in a stimulus-specific fashion via a mitochondria-dependent mechanism and that BAD protein serves as a downstream molecule.

N-Acetylcysteine-Mediated Antioxidation Prevents Hyperglycemia-Induced Apoptosis and Collagen Synthesis in Rat Mesangial Cells

Background/Aims: High-glucose (HG)-induced mesangial apoptosis and fibrogenesis possibly involves reactive oxygen species (ROS) formation and activated mitochondrial stress. We investigated the therapeutic effect of the antioxidant N-acetylcysteine (NAC) on cellular apoptosis and matrix accumulation in HG-treated rat mesangial cells (RMCs). Methods: RMCs were cultured in media containing 5 (control) or 35 mM (HG) glucose. Cellular apoptosis was assayed by TdT-mediated dUTP nick-end labeling staining. Collagen and transforming growth factor-1 gene expression were measured by reverse transcriptase-polymerase chain reaction or Northern blotting. Mitochondrial capacity and intracellular ROS generation was assayed by fluorescence microscopy and flow cytometry, respectively. Cellular ATP production and malondialdehyde (MDA) formation were determined by a luciferin-luciferase reaction and high-performance liquid chromatography, respectively. Cytochrome c release, caspase activation and poly(ADP)ribose polymerase cleavage were assayed by Western blotting. Results: HG-treated RMCs displayed enhanced cellular apoptosis (65%) and collagen gene expression (1.8-fold increase); these reactions could be significantly suppressed by 1 mM NAC (p < 0.05). Intracellular ROS generation, production of ATP and MDA, and caspase-3, -8 and -9 activities were significantly increased in HG-treated RMCs, and were effectively attenuated by addition of NAC. Conclusion: It is concluded that NAC prevents HG-induced mesangial apoptosis and fibrogenesis pathways by the reduction of oxidative stress.

Aldosterone induces mesangial cell apoptosis both in vivo and in vitro.

Both clinical and experimental reports indicate that aldosterone contributes to the progression of renal failure independent of its hemodynamic effects. In the present study, we evaluated effect of aldosterone on human mesangial cell (MC) growth. Aldosterone induced apoptotic and mitogenic effects on MCs. Aldosterone promoted MC apoptosis in a dose- and time-dependent manner. Spironolactone, a mineralocorticoid receptor antagonist, inhibited aldosterone-induced MC apoptosis. Similarly, antioxidants and free radical scavengers partially attenuated proapoaptotic effects of aldosterone. Aldosterone also enhanced dephosphorylation of phospho-Bad and accumulation of cytosolic cytochrome c in MCs. In in vivo studies, rats were randomly assigned to receive normal saline, aldosterone, or eplerenone + aldosterone for 28 days. Systolic blood pressure, urinary albumin excretion rate, serum creatinine, and aldosterone were measured. Aldosterone-infused rats developed elevated systolic blood pressure and albuminuria when compared with control rats. Aldosterone-treated rats also showed greater numbers of apoptosed MCs. This proapoptotic effect of aldosterone was inhibited by eplerenone, a selective aldosterone antagonist. These findings suggest that aldosterone, besides its hemodynamic effects, may also directly contribute to the occurrence of MC apoptosis.

Superoxide Destabilization of β-Catenin Augments Apoptosis of High-Glucose-Stressed Mesangial Cells

Intense mesangial cell apoptosis contributes to the pathogenesis of diabetic nephropathy. Although reactive oxygen radicals and Wnt signaling components are potent regulators that modulate renal tissue remodeling and morphogenesis, cross-talk between oxidative stress and Wnt/beta-catenin signaling in controlling high-glucose-impaired mesangial cell survival and renal function have not been tested. In this study, high glucose induced Ras and Rac1 activation, superoxide burst, and Wnt5a/beta-catenin destabilization and subsequently promoted caspase-3 and poly (ADP-ribose) polymerase cleavage and apoptosis in mesangial cell cultures. The pharmacological and genetic suppression of superoxide synthesis by superoxide dismutase and diphenyloniodium, dominant-negative Ras (S17N), and dominant-negative Rac1 (T17N) abrogated high-glucose-induced glycogen synthase kinase (GSK-3beta) activation and caspase-3 and poly (ADP-ribose) polymerase degradation. Inactivation of Ras and Racl also reversed Wnt/beta-catenin expression and survival of mesangial cells. Stabilization of beta-catenin by the transfection of stable beta-catenin (Delta45) and kinase-inactive GSK-3beta attenuated high-glucose-mediated mesangial cell apoptosis. Exogenous superoxide dismutase administration attenuated urinary protein secretion in diabetic rats and abrogated diabetes-mediated reactive oxygen radical synthesis in renal glomeruli. Immunohistological observation revealed that superoxide dismutase treatment abrogated diabetes-induced caspase-3 cleavage and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) and increased Wnt5a/beta-catenin expression in renal glomeruli. Taken together, high glucose induced oxidative stress and apoptosis in mesangial cells. The Ras and Rac1 regulation of superoxide appeared to raise apoptotic activity by activating GSK-3beta and inhibiting Wnt5a/beta-catenin signaling. Controlling oxidative stress and Wnt/beta-catenin signaling has potential for protecting renal tissue against the deleterious effect of high glucose.

Pro-apoptotic effect of decorin on rat mesangial cells in vitro

To investigate the effects of over-expression of decorin (DCN) gene on apoptosis of cultured rat mesangial cells (MsC). PcDNA3.1A-DCN plasmid was transfected into cultured rat MsC by the induction of liposome and positive clones were selected by treating the cells with G418. The MsC clones stably expressing DCN (MsC/DCN) were confirmed by cellular immunofluorescence, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. The DCN-siRNA was used for blocking DCN expression in MsC/DCN, and was confirmed by Western blot. The apoptosis of MsC was assayed by flow cytometry and Hoechst staining. Expression of Caspase-3 was assayed by Western blot. Positive clones with DCN over-expression were established. The apoptotic rate in MsC/DCN was (20.40 +/- 8.01)% and was much higher than the (2.07 +/- 0.99)% in MsC (P < 0.01). Some of the MsC/DCN cells showed typical morphologic changes of apoptosis. The protein expression of active Caspase-3 was also significantly increased in MsC/DCN compared to MsC (P < 0.01). DCN-siRNA transfection not only significantly blocked the expression of DCN and reduced the rate of apoptotic cells, but also down-regulated the expression of active Caspase-3. Over-expression of DCN induces apoptosis of cultured rat MsC in vitro. This effect of DCN inducing apoptosis suggests a novel strategy for regulating the proliferation of MsC in glomerular diseases.

TWEAKing renal injury

TWEAK is a recently identified cytokine of the TNF superfamiliy. Through activation of the Fn14 receptor, TWEAK regulates cell proliferation, cell death and inflammation. Recent studies show increased TWEAK and Fn14 expression in tubular cells during acute kidney injury as well as elevated urinary TWEAK levels in patients with active lupus nephritis. Furthermore, glomerular mesangial cells and renal tubular epithelial cells express the Fn14 receptor under the regulation of proinflammatory cytokines. TWEAK weakly increases cell death and promotes secretion of inflammatory mediators in non-stimulated mesangial cells. In addition, in a proinflammatory milieu, TWEAK induces apoptosis of mesangial and tubular cells. The available data suggest that TWEAK is a new player in kidney injury both at the glomerular and tubulointerstitial levels and might be a target for therapeutic intervention.

Glomerular Regeneration Is Delayed in Nephritic α8-Integrin-Deficient Mice: Contribution of α8-Integrin to the Regulation of Mesangial Cell Apoptosis

Background/Aims: α₈β₁-Integrin is expressed in mesangial cells. In vitro studies suggest a role for α₈-integrin in the regulation of cell proliferation and apoptosis. We tested the hypothesis that α₈-integrin is essential for the healing process after mesangioproliferative glomerulonephritis. Methods: Mice homozygous for a deletion of the α₈-integrin chain were compared with wild-type mice. To study glomerular healing, we used the habu toxin model of reversible mesangioproliferative glomerulonephritis. Animals received 6 mg/kg habu toxin intravenously; controls received saline only. Results: Early mesangiolysis occurred in wild-type and α₈-integrin-deficient mice. However, mesangiolysis was no longer detectable after 7 days in wild types but persisted after 14 days in α₈-integrin-deficient animals. Mesangial activation marker α-smooth muscle actin was detectable only at day 7 in wild-type mice but persisted until day 14 in α₈-integrin-deficient mice. In wild types, glomerular cell proliferation and apoptosis peaked at day 7 and decreased thereafter but remained elevated in α₈-integrin-deficient mice until day 28. In cultivated mesangial cells, α₈-integrin expression was associated with increased cell survival. Conclusion: Interactions between α₈-integrin and the mesangial matrix may contribute to healing of glomerular injury by influencing cell proliferation and apoptosis.