ANG II has been shown to modulate kidney cell growth and contribute to the pathobiology of glomerulosclerosis. Glomerular visceral epithelial cell (GEC) injury or loss is considered to play a pivotal role in the initiation and progression of glomerulosclerosis. In the present study, we investigated the effect of ANG II on GEC apoptosis. Rat GECs were incubated with increasing doses of ANG II for variable time periods. Apoptosis was evaluated by cell nucleus staining and DNA fragmentation assay. ANG II induced GEC apoptosis in a dose- and time-dependent manner. The proapoptotic effect was attenuated by the ANG II receptor type 1 antagonist losartan or the ANG II receptor type 2 antagonist PD-123319 and was completely blocked by incubation with the combined antagonists. Moreover, ANG II stimulated transforming growth factor (TGF)-beta1 production as measured by ELISA. GECs exposed to TGF-beta1 demonstrated a dose- and time-dependent increase in apoptosis. ANG II-induced apoptosis was significantly inhibited by addition of anti-TGF-beta1 antibody. ANG II also upregulated the expression of Fas, FasL, and Bax and downregulated the expression of Bcl-2 in GECs. These studies suggest that ANG II induces GEC apoptosis by a mechanism involving TGF-beta1 expression that may, importantly, contribute to the pathogenesis of glomerulosclerosis.

II induces apoptosis in renal proximal tubular cells. Am J Physiol Renal Physiol 284: F955-F965, 2003; 10.1152/ajprenal.00246.2002-ANG II has been demonstrated to play a role in the progression of tubulointerstial injury. We studied the direct effect of ANG II on apoptosis of cultured rat renal proximal tubular epithelial cells (RPTECs). ANG II promoted RPTEC apoptosis in a dose-and time-dependent manner. This effect of ANG II was attenuated by anti-transforming growth factor (TGF)-␤ antibody. Moreover, TGF-␤ triggered RPTEC apoptosis in a dose-dependent manner. ANG II also enhanced RPTEC expression of Fas and Fas ligand (FasL); furthermore, anti-FasL antibody attenuated ANG II-induced RPTEC apoptosis. In addition, ANG II increased RPTEC expression of Bax, a cell death protein. Both ANG II type 1 (AT1) and type 2 (AT2) receptor blockers inhibited ANG II-induced RPTEC apoptosis. SB-202190, an inhibitor of p38 MAPK phosphorylation, and caspase-3 inhibitor also attenuated ANG II-induced RPTEC apoptosis. ANG II enhanced RPTEC heme oxygenase (HO)-1 expression. Interestingly, pretreatment with hemin as well as curcumin (inducers of HO-1) inhibited the ANG II-induced tubular cell apoptosis; conversely, pretreatment with zinc protoporphyrin, an inhibitor of HO-1 expression, promoted the effect of ANG II. These results suggest that ANG II-induced apoptosis is mediated via both AT1 and AT2 receptors through the generation of TGF-␤, followed by the transcription of cell death genes such as Fas, FasL, and Bax. Modulation of tubular cell expression of HO-1 has an inverse relationship with the ANG II-induced tubular cell apoptosis.Bax; Bcl-2; Fas; Fas ligand; proximal tubular epithelial cells; heme oxygenase-1 ANG II HAS BEEN DEMONSTRATED to contribute to the progression of renal injury through its hemodynamic effects (35, 36). These effects are confirmed by blocking its production and receptor sites (15,16,20). However, apart from its hemodynamic effects, the direct effects of ANG II on kidney cells are being increasingly recognized (2,6,9,25,27). It has been demonstrated that in addition to circulating ANG II, tissue (intrarenal) generation of ANG II is also important for its net effect (35).Tubulointerstitial lesions have been demonstrated to correlate with the progression of renal failure (5,11,18,21,26), thus suggesting their contribution to the progression of renal failure (11). Transforming growth factor (TGF)-␤, a fibrogenic cytokine, has been shown to play a role in the inception and progression of renal lesions in both human renal diseases and experimental animal models of human immunodeficiency virus-associated nephropathy, renal ablation, and ureteric obstruction (16,33,36). Interestingly, in these conditions, elevated blood ANG II levels have been reported. Moreover, modalities, which inhibit the production of ANG II, have been demonstrated to slow the progression of renal lesions (15,16,20).The effect of ANG II on the growth of proximal tubular cells has been evaluated in both in vivo and in vitro studies (4, 3...

In this study, we evaluated the molecular mechanisms involved in morphine-induced macrophage apoptosis. Both morphine and TGF-β promoted P38 mitogen-activated protein kinase (MAPK) phosphorylation, and this phosphorylation was inhibited by SB 202190 as well as by SB 203580. Anti-TGF-β Ab as well as naltrexone (an opiate receptor antagonist) inhibited morphine-induced macrophage P38 MAPK phosphorylation. Anti-TGF-β Ab also attenuated morphine-induced p53 as well as inducible NO synthase expression; in contrast, NG-nitro-l-arginine methyl ester, an inhibitor of NO synthase, inhibited morphine-induced P38 MAPK phosphorylation and Bax expression. Morphine also enhanced the expression of both Fas and Fas ligand (FasL), whereas anti-FasL Ab prevented morphine-induced macrophage apoptosis. Moreover, naltrexone inhibited morphine-induced FasL expression. In addition, macrophages either deficient in FasL or lacking p53 showed resistance to the effect of morphine. Inhibitors of both caspase-8 and caspase-9 partially prevented the apoptotic effect of morphine on macrophages. In addition, caspase-3 inhibitor prevented morphine-induced macrophage apoptosis. These findings suggest that morphine-induced macrophage apoptosis proceeds through opiate receptors via P38 MAPK phosphorylation. Both TGF-β and inducible NO synthase play an important role in morphine-induced downstream signaling, which seems to activate proteins involved in both extrinsic (Fas and FasL) and intrinsic (p53 and Bax) cell death pathways.

Summary Laboratory and clinical reports indicate that opiate addicts are prone to infections. This effect of opiates is partly attributed to opiate‐induced macrophage (Mφ) apoptosis. In the present study, we evaluated the role of transforming growth factor‐β (TGF‐β) in morphine‐induced apoptosis of murine J774 cells and peritoneal Mφ. Mφ harvested from morphine‐treated mice showed greater (P < 0·0001) apoptosis when compared with control Mφ. Morphine also enhanced apoptosis of J774 cells and peritoneal Mφ. Anti‐TGF‐β antibody inhibited (P < 0·001) the morphine‐induced apoptosis in J774 cells (control 0·7 ± 0·4%; 10−6 m morphine 23·5 ± 0·7%; anti‐TGF‐β antibody (Ab) + 10−6 m morphine 8·1 ± 0·7%; apoptotic cells/field) and peritoneal Mφ (control 1·5 ± 0·9%; 10−6 m morphine 29·1 ± 1·4%; 10−6 m morphine + anti‐TGF‐β Ab 19·1 ± 1·8%; apoptotic cells/field). TGF‐β enhanced (P < 0·001) apoptosis of J774 cells and peritoneal Mφ. TGF‐β also promoted Mφ DNA fragmentation into integer multiples of 180 bp (ladder pattern). Immunocytochemical studies revealed that morphine enhanced the Mφ cytoplasmic content of TGF‐β. In addition, Western blotting showed increased production of TGF‐β by morphine‐treated J774 cells when compared with control cells. Morphine increased J774 cell expression of bax. Interestingly, morphine‐induced bax expression was inhibited by anti‐TGF‐β Ab. As both morphine‐induced J774 cell apoptosis and bax expression were inhibited by anti‐TGF‐β Ab, it appears that morphine‐induced J774 cell apoptosis may be mediated through the generation of TGF‐β.

Background: Angiotensin II (ANG II) has been shown to play a role in the induction of glomerular injury. In the present study, we evaluated the effects of ANG II on mesangial cell apoptosis and the involved molecular mechanism. Materials and Methods: The effect of ANG II on apoptosis of mouse mesangial cells (MC) was evaluated by morphologic, DNA fragmentation and TUNEL assays. To evaluate the role of oxidative stress and involved mechanisms, we studied the effect of antioxidants, anti-TGF-␤ antibody, inhibitors of nitric oxide synthase and modulators of cytosolic calcium/heme oxygenase (HO) activity. In addition, we studied the effect of ANG II on the generation of reactive oxygen species (ROS) by MCs. Results: ANG II promoted apoptosis of MCs in a dose dependent manner. This effect of ANG II was not only associated with ROS production, but also inhibited by

Renal interstitial scarring is an important component of heroin-associated nephropathy. Kidney fibroblasts have been demonstrated to play a role in the development of renal scarring in a variety of renal diseases. We studied the effect of morphine, an active metabolite of heroin, on the proliferation of kidney fibroblasts. Morphine at a concentration of 10(-12) M enhanced (P < 0.001) the proliferation of kidney fibroblasts (control, 67.5 +/- 2.0 vs. morphine, 112.2 +/- 10.1 x 10(4) cells/well). [3H]thymidine incorporation studies further confirmed these results. Morphine at concentrations of 10(-12) M to 10(-10) M also modulated mRNA expression of early growth related genes (c-fos, c-jun and c-myc). Morphine at concentrations of 10(-8) to 10(-4) M promoted apoptosis of kidney fibroblasts and also enhanced the synthesis of p53 by kidney fibroblasts. We speculate that morphine-induced kidney fibroblast proliferation may be mediated through the activation of early growth related genes, whereas morphine induced kidney fibroblast apoptosis may be mediated through the generation of p53. The present in vitro study provides a hypothetical basis for the role of morphine in the development of renal interstitial scarring in patients with heroin-associated nephropathy.

Stromal cell-derived Factor-1a (SDF-1a) stimulates the migration of bone marrow (BM) cells, similar to vascular endothelial growth factor (VEGF). We previously demonstrated that inhibition of VEGF 165 by small interfering RNA inhibited Ewing's sarcoma tumor growth, tumor vessel formation and recruitment of BM cells to the tumor. To determine the importance of BM cells in tumor vessel development, we investigated the effects of SDF-1a on VEGF-inhibited TC/siVEGF 7-1 Ewing's tumor neovasculature formation and growth. The effect of SDF-1a on CD34 1 progenitor cell chemotaxis was determined in vivo. Using a BM transplantation model with GFP 1 transgenic mice as BM donors and nude mice as recipients, we evaluated the effect of SDF-1a on the recruitment of BM-derived cells to VEGF 165 -inhibited TC/ siVEGF 7-1 tumors, as well as its effect on neovasculature development, vessel morphology and tumor growth. SDF-1a stimulated the migration of CD34 1 progenitor cells to Matrigel plugs in vivo and promoted the retainment of BM-derived pericytes in close association with perfused, functional tumor vessels. Intratumor inoculation of Ad-SDF-1a into TC/siVEGF 7-1 tumors resulted in increased SDF-1 and PDGF-BB expression, augmented tumor growth, an increase in the number of large, lumen-bearing vascular structures, and enhanced vessel pericyte coverage, with no change in VEGF 165 . SDF-1a stimulates BM cell chemotaxis and the association of these cells with functional tumor vessels. Furthermore, SDF-1a enhances tumor neovascularization and growth with no alteration in VEGF 165 . Our work suggests that SDF-1-mediated vasculogenesis may represent an alternate pathway that could potentially be utilized by tumors to sustain growth and neovasculature expansion after anti-VEGF therapy. '

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