Apoptosis Of Glomerular Cells Research Articles

Albumin-induced apoptosis of glomerular parietal epithelial cells is modulated by extracellular signal-regulated kinase 1/2

The biological role(s) of glomerular parietal epithelial cells (PECs) is not fully understood in health or disease. Given its location, PECs are constantly exposed to low levels of filtered albumin, which is increased in nephrotic states. We tested the hypothesis that PECs internalize albumin and increased uptake results in apoptosis. Confocal microscopy of immunofluorescent staining and immunohistochemistry were used to demonstrate albumin internalization in PECs and to quantitate albumin uptake in normal mice and rats as well as experimental models of membranous nephropathy, minimal change disease/focal segmental glomerulosclerosis and protein overload nephropathy. Fluorescence-activated cell sorting analysis was performed on immortalized cultured PECs exposed to fluorescein isothiocyanate (FITC)-labeled albumin in the presence of an endosomal inhibitor or vehicle. Apoptosis was measured by Hoechst staining in cultured PECs exposed to bovine serum albumin. Levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (p-ERK1/2) were restored by retroviral infection of mitogen-activated protein kinase (MEK) 1/2 and reduced by U0126 in PECs exposed to high albumin levels in culture and apoptosis measured by Hoechst staining. PECs internalized albumin normally, and this was markedly increased in all of the experimental disease models (P<0.05 versus controls). Cultured immortalized PECs also internalize FITC-labeled albumin, which was reduced by endosomal inhibition. A consequence of increased albumin internalization was PEC apoptosis in vitro and in vivo. Candidate signaling pathways underlying these events were examined. Data showed markedly reduced levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (ERK1/2) in PECs exposed to high albumin levels in nephropathy and in culture. A role for ERK1/2 in limiting albumin-induced apoptosis was shown by restoring p-ERK1/2 by retroviral infection, which reduced apoptosis in cultured PECs, while a forced decrease of p-ERK1/2 through inhibition of MEK 1/2 significantly increased albumin-induced PEC apoptosis. A normal role of PECs is to take up filtered albumin. However, this is increased in proteinuric glomerular diseases, leading to apoptosis through changes in ERK1/2.

Warfarin-Related Nephropathy Modeled by Nephron Reduction and Excessive Anticoagulation

An acute increase in international normalized ratio (INR) to >3.0 in patients with chronic kidney disease (CKD) can associate with an unexplained acute increase in serum creatinine and accelerated progression of CKD. A subset of these patients have renal tubular obstruction by casts of red blood cells, presumably the dominant mechanism of the acute kidney injury described as warfarin-related nephropathy. Here, we developed an animal model of this acute kidney injury that is based on the 5/6-nephrectomy model to aid future investigation of the pathogenesis of this condition. We found that acute excessive anticoagulation with brodifacoum ("superwarfarin") increased serum creatinine levels and hematuria in 5/6-nephrectomized rats but not in controls. In addition, morphologic findings in 5/6-nephrectomized rats included glomerular hemorrhage, occlusive red blood cell casts, and acute tubular injury, similar to the biopsy findings among affected patients. Furthermore, in the rat model, we observed an increase in apoptosis of glomerular endothelial cells. In summary, the 5/6-nephrectomy model combined with excessive anticoagulation may be a useful tool to study the pathogenesis of warfarin-related nephropathy.

Toxic effects of a high dose of non-ionic iodinated contrast media on renal glomerular and aortic endothelial cells in aged rats in vivo

Iodinated contrast media (CM) can induce apoptosis and necrosis of renal tubular cells. The injuries of endothelial cells induced by CM on the systemic condition have not been fully understood. To assess the toxic effects of non-ionic CM on the glomerular and aortic endothelial cells, iopromide and iodixanol, two kinds of representative non-ionic CM, were used for the in vivo study. Sixty aged rats were respectively received the agents or normal sodium intravascularly. No obvious apoptosis and morphological change was detected in the glomerular and aortic endothelial cells apart from renal tubules after CM administration. However, expressions of the nitric oxide synthase (eNOS) in glomerular endothelium were decreased at 12 h after CM injection. Furthermore, plasma creatinine and endothelin-1 were increased and plasma nitric oxide (NO) was decreased significantly after CM administration. However, we failed to observe the significant increase of plasma von Willebrand Factor. These results suggest that non-ionic iodinated CM do not induce apoptosis and necrosis of glomerular and aortic endothelial cells in vivo. Decreased eNOS expression and increased plasma endothelin-1 may be involved in non-ionic iodinated CM-induced endothelial dysfunction and kidney injury.

Carnosine Prevents Apoptosis of Glomerular Cells and Podocyte Loss in STZ Diabetic Rats

Background/Aims: We identified carnosinase-1 (CN-1) as risk-factor for diabetic nephropathy (DN). Carnosine, the substrate for CN-1, supposedly is a protective factor regarding diabetic complications. In this study, we hypothesized that carnosine administration to diabetic rats might protect the kidneys from glomerular apoptosis and podocyte loss. Methods: We examined the effect of oral L-carnosine administration (1g/kg BW per day) on apoptosis, podocyte loss, oxidative stress, AGEs and hexosamine pathway in kidneys of streptozotocin-induced diabetic Wistar rats after 3 months of diabetes and treatment. Results: Hyperglycemia significantly reduced endogenous kidney carnosine levels. In parallel, podocyte numbers significantly decreased (-21% compared to non-diabetics, p<0.05), apoptotic glomerular cells numbers increased (32%, compared to non-diabetic, p<0.05) and protein levels of bax and cytochrome c increased (175% and 117%). Carnosine treatment restored carnosine kidney levels, prevented podocytes loss (+23% compared to diabetic, p<0.05), restrained glomerular apoptosis (-34% compared to diabetic; p<0.05) and reduced expression of bax and cytochrome c (-63% and -54% compared to diabetics, both p<0.05). In kidneys of all diabetic animals, levels of ROS, AGEs and GlcNAc-modified proteins were increased. Conclusion: By inhibition of pro-apoptotic signaling and independent of biochemical abnormalities, carnosine protects diabetic rat kidneys from apoptosis and podocyte loss.

AMP-activated Protein Kinase (AMPK) Negatively Regulates Nox4-dependent Activation of p53 and Epithelial Cell Apoptosis in Diabetes

Diabetes and high glucose (HG) increase the generation of NADPH oxidase-derived reactive oxygen species and induce apoptosis of glomerular epithelial cells (podocytes). Loss of podocytes contributes to albuminuria, a major risk factor for progression of kidney disease. Here, we show that HG inactivates AMP-activated protein kinase (AMPK), up-regulates Nox4, enhances NADPH oxidase activity, and induces podocyte apoptosis. Activation of AMPK blocked HG-induced expression of Nox4, NADPH oxidase activity, and apoptosis. We also identified the tumor suppressor protein p53 as a mediator of podocyte apoptosis in cells exposed to HG. Inactivation of AMPK by HG up-regulated the expression and phosphorylation of p53, and p53 acted downstream of Nox4. To investigate the mechanism of podocyte apoptosis in vivo, we used OVE26 mice, a model of type 1 diabetes. Glomeruli isolated from these mice showed decreased phosphorylation of AMPK and enhanced expression of Nox4 and p53. Pharmacologic activation of AMPK by 5-aminoimidazole-4-carboxamide-1-riboside in OVE26 mice attenuated Nox4 and p53 expression. Administration of 5-aminoimidazole-4-carboxamide-1-riboside also prevented renal hypertrophy, glomerular basement thickening, foot process effacement, and podocyte loss, resulting in marked reduction in albuminuria. Our results uncover a novel function of AMPK that integrates metabolic input to Nox4 and provide new insight for activation of p53 to induce podocyte apoptosis. The data indicate the potential therapeutic utility of AMPK activators to block Nox4 and reactive oxygen species generation and to reduce urinary albumin excretion in type 1 diabetes.

Doksazosinin Kısmi Mesane Çıkış Obstrüksiyonu Sonrasında Renal Apopitozis Üzerine Yararlı Etkisi

Objective: It is not known whether inhibition of ?1-adrenoreceptor in a rat model of partial bladder outlet obstruction (BOO) causes decrease in tubular and glomerular cell death by apoptosis and prevention of subsequent inflammation and fibrosis. The aim of this study was to demonstrate the relationship between apoptosis and doxazosin ?1-adrenoceptor antagonist) in rat kidneys damaged by partial BOO. Material and Methods: A total of 45 adult female Wistar rats,with a mean weight of 250 g were randomly allocated to four experimental groups as: sham-operated rats (10 rats), sham-operated rats treated with doxazosin (10 rats), partial BOO group (14 rats), and partial BOO group treated with doxazosin (11 rats). Partial BOO was surgically induced. Detection of cell apoptosis was done by TUNEL assay. Results: This investigation revealed increased apoptosis in both tubular and glomerular cells of rats with partial BOO. Furthermore, the results of this study demonstrated that doxazosin treatment of rats reduced apoptosis in tubular and glomerular cells in both the controls and the rats with partial BOO. Conclusion: We believe that our observation of decreased apoptosis in both tubular and glomerular cells of rats with partial BOO might be useful to gain new insight into the therapeutic effect of doxazosin.

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.

Mechanisms of renal injury and progression of renal disease in congenital obstructive nephropathy

Congenital obstructive nephropathy accounts for the greatest fraction of chronic kidney disease in children. Genetic and nongenetic factors responsible for the lesions are largely unidentified, and attention has been focused on minimizing obstructive renal injury and optimizing long-term outcomes. The cellular and molecular events responsible for obstructive injury to the developing kidney have been elucidated from animal models. These have revealed nephron loss through cellular phenotypic transition and cell death, leading to the formation of atubular glomeruli and tubular atrophy. Altered renal expression of growth factors and cytokines, including angiotensin, transforming growth factor-beta, and adhesion molecules, modulate cell death by apoptosis or phenotypic transition of glomerular, tubular, and vascular cells. Mediators of cellular injury include hypoxia, ischemia, and reactive oxygen species, while fibroblasts undergo myofibroblast transformation with increased deposition of extracellular matrix. Progression of the lesions involves interstitial inflammation and interstitial fibrosis, both of which impair growth of the obstructed kidney and result in compensatory growth of the contralateral kidney. The long-term outcome depends on timing and severity of the obstruction and its relief, minimizing ongoing injury, and enhancing remodeling. Advances will depend on new biomarkers to evaluate the severity of obstruction, to determine therapy, and to follow the evolution of lesions.

Acute Renal Failure and Mechanical Ventilation: Reality or Myth?

Acute Renal Failure and Mechanical Ventilation: Reality or Myth?

SIRT1 Inhibits Transforming Growth Factor β-Induced Apoptosis in Glomerular Mesangial Cells via Smad7 Deacetylation

SIRT1, a class III histone deacetylase, is considered a key regulator of cell survival and apoptosis through its interaction with nuclear proteins. In this study, we have examined the likelihood and role of the interaction between SIRT1 and Smad7, which mediates transforming growth factor beta (TGFbeta)-induced apoptosis in renal glomerular mesangial cells. Immunoprecipitation analysis revealed that SIRT1 directly interacts with the N terminus of Smad7. Furthermore, SIRT1 reversed acetyl-transferase (p300)-mediated acetylation of two lysine residues (Lys-64 and -70) on Smad7. In mesangial cells, the Smad7 expression level was reduced by SIRT1 overexpression and increased by SIRT1 knockdown. SIRT1-mediated deacetylation of Smad7 enhanced Smad ubiquitination regulatory factor 1 (Smurf1)-mediated ubiquitin proteasome degradation, which contributed to the low expression of Smad7 in SIRT1-overexpressing mesangial cells. Stimulation by TGFbeta or overexpression of Smad7 induced mesangial cell apoptosis, as assessed by morphological apoptotic changes (nuclear condensation) and biological apoptotic markers (cleavages of caspase3 and poly(ADP-ribose) polymerase). However, TGFbeta failed to induce apoptosis in Smad7 knockdown mesangial cells, indicating that Smad7 mainly mediates TGFbeta-induced apoptosis of mesangial cells. Finally, SIRT1 overexpression attenuated both Smad7- and TGFbeta-induced mesangial cell apoptosis, whereas SIRT1 knockdown enhanced this apoptosis. We have concluded that Smad7 is a new target molecule for SIRT1 and SIRT1 attenuates TGFbeta-induced mesangial cell apoptosis through acceleration of Smad7 degradation. Our results suggest that up-regulation of SIRT1 deacetylase activity is a potentially useful therapeutic strategy for prevention of TGFbeta-related kidney disease through its effect on cell survival.

Wnt/β-Catenin Signaling Modulates Survival of High Glucose–Stressed Mesangial Cells

Glomerulosclerosis and diabetic nephropathy are attributable to high glucose induction of mesangial cell apoptosis. Whereas Wnt signaling has been found to regulate renal morphogenesis and pathogenesis, the biologic role of Wnt/beta-catenin signaling in controlling high glucose-induced mesangial cell apoptosis is not well defined. Herein is reported that Wnt/beta-catenin signaling is required for protecting glomerular mesangial cells from high glucose-mediated cell apoptosis. High glucose downregulated Wnt4 and Wnt5a expression and the subsequent nuclear translocation of beta-catenin, whereas it increased glycogen synthase kinase-3beta (GSK-3beta) and caspase-3 activities and apoptosis of glomerular mesangial cells. Suppression of GSK-3beta activation or increase in nuclear beta-catenin by transfection of Wnt4 or Wnt5a or stable beta-catenin (S33Y) reversed Akt activation and reduced the high glucose-mediated caspase-3 cleavage and cell apoptosis. Pharmacologic inhibition of GSK-3beta by recombinant Wnt5a or bromoindirubin-3'-oxime or LiCl increased Akt phosphorylation and beta-catenin translocation and abrogated high glucose-mediated proapoptotic activities. Exogenous bromoindirubin-3'-oxime treatment reduced phospho-Ser(9)-GSK-3beta and beta-catenin expression and apoptosis of cells adjacent to glomeruli in diabetic kidneys and attenuated urinary protein secretion in diabetic rats. Taken together, mesangial cells responded to high glucose by impairing that canonical Wnt pathway to increase proapoptotic activities. Sustaining Wnt/beta-catenin signaling is beneficial for promoting survival of mesangial cells that are exposed to high glucose stress.

Roles of TGF-b1 and apoptosis in the progression of glomerulosclerosis in human IgA nephropathy

Apoptotic glomerular cells have been detected in the severely damaged glomeruli that are a consequence of human IgA nephropathy. Transforming growth factor-(TGF) beta1 is known to induce apoptosis in cultured mesangial cells. To clarify whether TGF-beta1 contributes to the progression of IgA nephropathy by activating apoptosis in glomerular cells, we examined the expression of TGF-beta1 gene and apoptotic changes in kidney biopsy samples, and assessed those relations to the severity of nephropathy. 32 patients with IgA nephropathy, showing proteinuria (> 1 g/day) and serum creatinine less than 1.5 mg/dl were classified according to glomerular sclerosis index (GSI) into 3 groups (Group I: GSI < 0.3,Group 11: 0.3 < or = GSI < 1.0, Group: III GSI > or = 1.0). Computer-aided morphometry of glomeruli and arteries, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of fragmented DNA (TUNEL) staining were performed. Expression of TGF-beta1 and caspase-3 mRNAs in renal biopsy samples was analyzed by real-time PCR (Taq Man method). Increased glomerular area, interstitial fibrosis, lymphocytic infiltration, and tubulointerstitial changes were observed to accompany increased severity of GSI. TUNEL index was higher in Group III. The levels of TGF-beta1 and caspase-3 mRNAs were significantly increased in Group III (183 and 190%, respectively). Furthermore, caspase-3 mRNA levels were tightly associated with TGF-beta1 mRNA expression (r = 0.677, p < 0.0001). The present study suggests that the activation of TGF-beta1 plays a role in the progression of IgA nephropathy even in the moderate degree of glomerular injury, in part via activation of apoptosis of glomerular cells.

Silent information regulator 2 (SIRT1) attenuates oxidative stress-induced mesangial cell apoptosis via p53 deacetylation

Oxidative stress-induced apoptosis of renal glomerular cells is an important factor for the development of various kidney diseases. Identification of molecules that modulate this process could lead to the development of new strategies for preventing kidney diseases. In this study, we evaluated whether mammalian silent information regulator 2 (SIRT1), which has been recently identified as a cell survival factor countering various stressors, is a key regulator of oxidative stress-induced mesangial cell apoptosis. Morphological features of apoptotic cell death (nuclear condensation) and the expression of biochemical proapoptotic markers [cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP)] were assessed in murine mesangial cells (MMCs) exposed to hydrogen peroxide (H 2O 2). H 2O 2 increased mesangial cell apoptosis, predominantly through p53 activation by acetylation, which is a posttranscriptional modification for p53 activation. H 2O 2-induced apoptosis was significantly attenuated in SIRT1-overexpressing MMCs, but enhanced in SIRT1-knockdown MMCs. Although SIRT1 did not affect H 2O 2-mediated phosphorylation of mitogen-activated protein (MAP) kinase, it interacted with p53 and inhibited H 2O 2-mediated p53 acetylation but not phosphorylation in MMCs. Our results indicate that SIRT1 can prevent oxidative stress-induced apoptosis through p53 deacetylation in mesangial cells. Upregulation of SIRT1 may provide a new strategy for preventing kidney glomerular diseases.

Inhibition of Fas/Fas ligand interaction reduces apoptosis of glomerular endothelial cells induced by ischemia and reperfusion in mouse kidney

Ischemia and reperfusion (I/R) injury of renal graft is one of the major problems for successful transplantation of kidney. Apoptosis plays a crucial role in the renal injury induced by I/R and the apoptosis of renal tubular epithelial cells has been predominantly investigated. However, it is not clear whether I/R provokes endothelial cell injury within glomeruli. Here, we focused on the apoptotic change of glomerular endothelial cells (GECs) during I/R injury. DNA ladder formation, morphological changes and TUNEL staining in accordance with Fas and FasL expressions in the kidney were examined. Apoptotic cell death in kidney was observed as early as 3 hours after reperfusion. Early apoptotic change was mainly occurred in vascular endothelial cells, including GECs. The double staining with TUNEL and anti-vascular endothelial cadherin (VE-cadherin) Ab demonstrated that I/R induced apoptosis in GECs. Electron microscopic examination supports the findings. In addition, laser microdissection and RT-PCR demonstrated that enhanced mRNA expression of FasL in glomeruli was observed after ischemia, suggesting involvement of Fas/FasL system in apoptotic death of GECs after I/R. Furthermore, the apoptosis of GECs was predominantly inhibited by the intravenous injection of Fas-Fc fusion protein before ischemia. These findings suggest that I/R induces apoptotic cell death not only in tubular epithelial cells but also in endothelial cells in glomeruli and the Fas/FasL system is importantly involved in I/R-induced endothelial cell apoptosis. Thus, the pretreatment with Fas-Fc fusion protein may help to reduce the injury of endothelial cells in glomeruli and effectively improve renal function after renal transplantation.

Conditional Ablation of Macrophages Halts Progression of Crescentic Glomerulonephritis

The presence of macrophages in inflamed glomeruli of rat kidney correlates with proliferation and apoptosis of resident glomerular mesangial cells. We assessed the contribution of inflammatory macrophages to progressive renal injury in murine crescentic glomerulonephritis (GN). Using a novel transgenic mouse (CD11b-DTR) in which tissue macrophages can be specifically and selectively ablated by minute injections of diphtheria toxin, we depleted renal inflammatory macrophages through days 15 and 20 of progressive crescentic GN. Macrophage depletion reduced the number of glomerular crescents, improved renal function, and reduced proteinuria. Morphometric analysis of renal tubules and interstitium revealed a marked attenuation of tubular injury that was associated with reduced proliferation and apoptosis of tubular cells. The population of interstitial myofibroblasts decreased after macrophage depletion and interstitial fibrosis also decreased. In the presence of macrophages, interstitial myofibroblasts exhibited increased levels of both proliferation and apoptosis, suggesting that macrophages act to support a population of renal myofibroblasts in a high turnover state and in matrix deposition. Finally, deletion of macrophages reduced CD4 T cells in the diseased kidney. This study demonstrates that macrophages are key effectors of disease progression in crescentic GN, acting to regulate parenchymal cell populations by modulating both cell proliferation and apoptosis.

A neutralizing VEGF antibody prevents glomerular hypertrophy in a model of obese type 2 diabetes, the Zucker diabetic fatty rat

Antagonism of vascular endothelial growth factor (VEGF) has improved the outcome in experimental nephropathies of various origins, including diabetic nephropathy in a type 1 diabetic rat model and a type 2 diabetic mouse model. Neutralizing VEGF antibodies prevented glomerular hypertrophy in these models. We examined the renal effects of VEGF blockade in an obese rat model of type 2 diabetic nephropathy and investigated the mechanism underlying the inhibition of glomerular hypertrophy. Twenty female Zucker diabetic fatty (ZDF) rats, fed a high-fat diet and aged 10 weeks, were treated with VEGF antibodies or an irrelevant isotype-matched IgG. Ten heterozygous (fa/+) littermates served as additional non-diabetic, lean controls. Urinary albumin excretion (UAE) and creatinine clearance (CrCl) were assessed at baseline, and at 3 and 5 weeks. Kidney weight and glomerular volume were determined at the end of the study. Glomerular apoptosis was examined with anti-active caspase-3 immunohistochemistry. All obese animals had established diabetes, hyperlipidaemia and normal blood pressure, which were not influenced by VEGF antibody treatment. ZDF control rats had increased UAE, CrCl, kidney weights and glomerular volumes compared with non-diabetic, lean control rats. VEGF antibody treatment prevented the glomerular hypertrophy, but did not affect UAE, CrCl and kidney weight. Glomerular anti-active caspase-3 immunostaining was not different between the groups. Inhibition of VEGF prevented early glomerular hypertrophy in ZDF rats with established diabetes. Increased apoptosis of glomerular endothelial cells does not appear to underly the inhibition of glomerular growth.

Sterile vesicoureteral reflux decreases tubular cell apoptosis in rat kidney

Objectives To assess the possible role of apoptosis in reflux-related renal scarring by investigating how sterile vesicoureteral reflux affects apoptosis of glomerular and tubular cells in the rat kidney. Methods Twenty-nine rats were assigned to one of three groups: group 1, reflux (n = 10); group 2, sham surgery (n = 9); and group 3, controls (n = 10). All rats underwent bilateral nephrectomy 42 days later. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling was used to detect the apoptotic cells, and separate apoptotic index values for glomerular cells (GCAI) and tubular cells (TCAI) were calculated in each kidney (left and right from each animal). Results The mean TCAI value in the left kidney from groups 1, 2, and 3 was 0.86% ± 0.26%, 1.20% ± 0.18%, and 1.19% ± 0.14%, respectively. The corresponding TCAI values for the right kidneys were 0.77% ± 0.21%, 1.10% ± 0.34%, and 1.06% ± 0.15%. The mean GCAI value in the left kidney from groups 1, 2, and 3 was 0.26% ± 0.14%, 0.28% ± 0.08%, and 0.20% ± 0.10%, respectively. The corresponding GCAI values for the right kidneys were 0.23% ± 0.06%, 0.26% ± 0.06%, and 0.22% ± 0.12%. Analysis revealed no significant differences among the groups with respect to GCAI in the left kidneys ( P = 0.258) or right kidneys ( P = 0.618). The mean TCAI values in group 1 were significantly lower than the corresponding values in groups 2 and 3 for the left ( P = 0.001) and right ( P = 0.012) kidneys. Conclusions This is the first study to have shown that sterile vesicoureteral reflux decreases apoptosis in tubular cells in rat kidneys with vesicoureteral reflux.

High glucose evokes an intrinsic proapoptotic signaling pathway in mesangial cells

In response to chronic hyperglycemia, microvascular cells undergo stress and injury, which can lead to cell death. We characterized a proapoptotic signaling pathway whereby high glucose evokes an intrinsic, caspase-9-dependent mechanism of cell death in human mesangial cells. Biochemical (caspase activity, cytochrome-c release, etc.) and morphologic (chromatin condensation and nuclear segmentation) features of apoptotic cell death were assessed in cultured human mesangial cells exposed to high glucose, a risk factor for mesangial cell injury and diabetic glomerulosclerosis. Proapoptotic signaling was also analyzed in the db/db murine model of kidney injury in diabetes. Incubation in high glucose caused cytotoxicity and apoptosis in mesangial cells. High glucose stimulated mitochondrial release of cytochrome-c, cleavage of procaspase-9, and caspase-9 enzyme activity, suggesting an intrinsic pathway of proapoptotic signaling. In contrast, caspase-8 was unaffected by high glucose. A cell-permeable, caspase-9-selective inhibitor blocked caspase-3 activation and prevented chromatin condensation and nuclear segmentation in cells treated with high glucose. To determine whether an intrinsic signaling pathway occurs in the diabetic kidney in vivo, apoptosis was investigated in diabetic 8- and 16-week db/db murine kidneys. Effector caspases-3 and -7 were activated in diabetic db/db kidneys but not in age-matched nondiabetic db/m controls. At 16 weeks, apoptotic cells in db/db glomeruli were identified on the basis of nuclear segmentation and DNA fragmentation. Apoptosis of glomerular cells correlated with expansion of the mesangial matrix and with worsening of albuminuria. Consistent with an intrinsic signaling pathway, caspase-9 cleavage was elevated only in db/db kidneys, whereas activation of caspase-8 and caspase-12 was undetectable. These findings support the hypothesis that hyperglycemia evokes an intrinsic pathway of proapoptotic signaling in mesangial cells. In addition, these results point to an important role for the intrinsic pathway in microvascular injury in the diabetic kidney in vivo.

Role of p38 MAP kinase pathway in a toxin-induced model of hemolytic uremic syndrome.

The role of proinflammatory cytokines in a rat model of toxin-induced hemolytic uremic syndrome (HUS) was studied. Male Sprague-Dawley rats underwent continuous saline infusion (6 ml/h) via a tail vein and received a bolus injection of saline (control), lipopolysaccharide (LPS, 10 microg/100 g body weight), ricin (6.7 microg/100 g body weight), or ricin with LPS (ricin+LPS). They were then observed for 8 h. Blood samples and kidney tissues were obtained at the end of the experiment. The effects of FR 167653, a potent inhibitor of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) production, were also examined in ricin+LPS-treated rats. Only ricin+LPS-treated rats developed significant thrombocytopenia, hemolysis, and oliguric acute renal failure with extensive glomerular thrombotic microangiopathy, which was characterized by glomerular microthrombi and apoptosis of glomerular endothelial cells. Thrombotic microangiopathy was not detected in other organs, including the brain, liver, spleen, pancreas, lung, colon, and intestine. Significantly elevated levels of serum IL-1beta and TNF-alpha were detected only in ricin+LPS-treated rats. Treatment of ricin+LPS-treated rats with FR 167653 significantly reduced the serum levels of IL-1beta and TNF-alpha, accompanied by improvement of the oliguric renal failure and glomerular thrombotic microangiopathy. These findings indicate that the increased serum levels of IL-1beta and TNF-alpha, which probably result in the apoptosis of glomerular endothelial cells, play a pivotal role in the development of this rat model of toxin-induced HUS. The findings also suggest that inhibition of these proinflammatory cytokines may prevent the development of HUS.

Shedding of growth-suppressive gangliosides from glomerular mesangial cells undergoing apoptosis

Apoptosis of glomerular mesangial cells is a common feature in several types of glomerular diseases. However, its pathophysiologic significance is not known. We recently identified gangliosides as a major growth-inhibitory substance in the conditioned medium of mesangial cells. In this report, we tested whether biologically distinct forms of cell fate, apoptosis and necrosis, could modulate ganglioside shedding from mesangial cells. Mesangial cells were exposed to low (10 to 40 mJ/cm2) and high (400 mJ/cm2) doses of ultraviolet light to induce apoptosis and necrosis, respectively. Conditioned media were collected and examined for its growth-inhibitory activity for mesangial cells. Ganglioside shedding was analyzed using metabolic labeling and thin-layer chromatography (TLC). Shedding of gangliosides as well as growth-inhibitory activity in the conditioned medium predominantly increased when mesangial cells were undergoing apoptosis in contrast to that of viable or necrotic mesangial cells. The inhibitory substance in the conditioned medium from apoptotic mesangial cells completely fulfilled the characteristic criteria of gangliosides. This substance was less than 3 kD and was sensitive to neuraminidase digestion. Shedding of gangliosides from mesangial cells reduced significantly when apoptosis was inhibited by overexpression of antiapoptotic gene, Bcl-XL. In addition, ganglioside shedding also increased when mesangial cells were exposed to other inducers of apoptosis for mesangial cells (i.e., H2O2 and staurosporin). These results provide the novel link between masangial cell apoptosis and increased release of gangliosides that potentially suppress mesangial cell proliferation and thus indicate a mechanism for the negative regulation of mesangial cell growth by apoptosis.