Lymphopenia in dialysis patients: a preliminary study indicating a possible role of apoptosis.

Lymphopenia is a common finding in dialysis patients. Since infection rate and mortality associated with infection are high in dialysis patients, lymphopenia may be one of the contributing factors. In the present study, we evaluated the mechanism responsible for lymphopenia in these patients. Lymphocytes isolated from dialysis patients showed increased apoptosis (p < 0.001) when compared to lymphocytes isolated from healthy subjects (healthy subjects, 0.5 +/- 0.2% vs. dialysis patients, 8.8 +/- 0.7% apoptotic cells/field). Sera from dialysis patients promoted lymphocyte apoptosis in a time- and dose-dependent manner. These sera also enhanced lymphocyte DNA fragmentation into multiple integers of 180 base pairs in the form of a ladder pattern. Cellulose acetate membranes promoted T cell apoptosis when compared to polysulfone membranes and to control. Cellulose acetate dialysis membranes also appear to promote lymphocyte FasL expression. Similarly, dialysis sera enhanced T cell Fas as well as FasL expression. Neither the cellulose acetate nor polysulfone membranes could induce FasL expression on B cells. Similarly, dialysis sera failed to induce FasL expression on B cells. On the other hand, anti-FasL antibodies attenuated dialysis sera-induced apoptosis in T as well as B cells. Interestingly, dialysis serum showed a 5-fold increase in FasL content when compared with control serum. These results suggest that dialysis-associated factors can induce autocrine death in T cells but the help of activated T cells is required to induce death in B cells.

Opiates promote T cell apoptosis through JNK and caspase pathway.

Opiate addicts are prone to recurrent infections. In the present study we evaluated the molecular mechanism of opiate-induced T cell apoptosis. Both morphine and DAGO ([D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin) enhanced T cell apoptosis. Morphine as well as DAGO activated c-Jun NH2-terminal kinase (JNK) in T cells. Moreover, opiates increased the expression of ATF-2. a specific substrate for JNK and P38 mitogen activated kinases (MAPK). Furthermore, opiates attenuated extracellular signal related kinase (ERK) in T cells. Both morphine and DAGO cleaved pro-caspases 8, 9, and 10 and generated caspases 8, 9 and 10 (active products). Morphine as well as DAGO also cleaved poly-(ADP-ribose) polymerase (PARP) into 116 and 85 kD proteins indicating the activation of caspase-3. These results suggest that opiate-induced T cell apoptosis may be mediated through the JNK cascade and activation of caspases 8 and 3.

Tubular cell senescence and expression of TGF-beta1 and p21(WAF1/CIP1) in tubulointerstitial fibrosis of aging rats.

Kidney aging has been recognized as a chronic process of compromised renal function and structural changes in the tubulointerstitium and glomerulus. Cell senescence is associated with alterations in cell structure and function, including expression of cytokines and structural and regulatory components of extracellular matrix proteins. In this investigation, we tested the hypothesis that senescent renal cells may accumulate in vivo with advancing age. We also evaluated the expression of transforming growth factor (TGF)-beta1 and p21WAF1/CIP1 in aging kidneys. Sprague-Dawley rats at the ages of 3, 12, and 24 months were used for this study. Renal tissues were processed for morphometric and senescence analysis. Expression of TGF-beta1 and p21WAF1/CIP1 was evaluated by Northern or Western blot analysis and immunohistochemistry. Substantial tubulointerstitial injury occurred at the age of 12 months, but significant glomerular structure alteration was observed at the age of 24 months. Tubular cells developed senescence, which was detected by beta-galactosidase staining. This staining increased in frequency and intensity with age. Renal cortices showed a significant increase in the mRNA expression for TGF-beta1 and protein level for p21WAF1/CIP1. The enhanced expression of TGF-beta1 and p21WAF1/CIP1 was localized in the tubulointersititial cells. These data suggest that tubular cells undergo senescence and express increased TGF-beta1 and p21WAF1/CIP1 with advancing age. These age-related cellular and molecular alterations may play an important role in the initiation and/or progression of tubulointerstitial fibrosis and glomerulosclerosis in aging.

Puromycin aminonucleoside induces glomerular epithelial cell apoptosis.

Glomerular epithelial cell (GEC) injury has been considered to play an important role in puromycin aminonucleoside (PAN)-induced nephrosis. We studied the effect of PAN on rat as well as human GEC apoptosis. Morphogic evaluation of GEC apoptosis and necrosis was carried out by staining with H-33342 and propidium iodide. GEC apoptosis was further confirmed by DNA fragmentation assay (by both agarose gel electrophoresis and end-labeling). To determine the dose- and time-response effect of PAN, GECs were treated with variable concentrations of PAN (10 to 500 microg/ml) for variable time periods (6 to 48 h). To determine the role of gene synthesis, we studied the effect of actinomycin D (a transcriptional inhibitor) on PAN-induced GEC apoptosis. To determine the role of free radicals, we evaluated the effect of superoxide dismutase (SOD), dimethylthiourea (DMTU), and catalase on PAN-induced GEC apoptosis. PAN induced GEC apoptosis in a dose- and time-dependent manner. PAN at a high concentration (PAN, 100 microg/ml) also induced a moderate degree of GEC necrosis. In DNA fragmentation assays PAN-treated GECs showed the classic ladder pattern. PAN-induced GEC apoptosis was partly attenuated with free radical scavengers, such as SOD, DMTU, and catalase. In addition, actinomycin D attenuated PAN-induced GEC apoptosis. PAN induces GEC apoptosis, which may be mediated through the generation of reactive oxygen species.

Transforming growth factor beta induces mesangial cell apoptosis through NO- and p53-dependent and -independent pathways.

BACKGROUND: Because transforming growth factor beta (TGF-beta) has been shown to have a bimodal effect on mesangial cell (MC) proliferation, we studied its effect on MC apoptosis. METHODS: Cultured mouse MCs were used to evaluate the effect of TGF-beta. Morphologic evaluation of MC apoptosis was performed by staining cells with H-33342 and propidium iodide. To confirm the effect of TGF-beta on MC apoptosis, DNA was extracted from control and TGF-beta-treated MCs and run on gel electrophoresis. We evaluated the effect of NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, on TGF-beta-induced MC apoptosis to determine the role of NO and studied the effect of sodium nitroprusside (SNP) and SNAP (S-nitroso-N-acetyl-penicillamine) on MC apoptosis to confirm the effect of NO. We examined the role of p53 by studying the effect of TGF-beta on MCs derived from p53 knockout mice (p53KO-MC) as well as a normogenic strain (N-MC). We also examined the effect of TGF-beta, SNP, and SNAP on apoptosis of p53 mutant (MDAMB-231) and wild-type p53 (MCF-7) breast cancer cell lines. In addition, Western blots were generated from control, TGF-beta-treated, and SNAP-treated MCs and probed for the expression of p53. RESULTS: TGF-beta promoted MC apoptosis. Moreover, TGF-beta-treated MCs displayed integer multiples of 180 base pairs (ladder pattern). L-NAME inhibited TGF-beta-induced MC apoptosis. Furthermore, SNP and SNAP, NO donors, promoted MC apoptosis. TGF-beta also enhanced the MC expression of p53. TGF-beta induced only a moderate degree of apoptosis in MCs derived from p53KO-MC when compared with N-MCs. Similarly, the TGF-beta-induced apoptosis of MDAMB-231 was of a moderate degree when compared with MCF-7 cells. CONCLUSIONS: We hypothesize that TGF-beta promotes MC apoptosis through NO generation and p53-dependent and -independent pathways.

Morphine-induced macrophage apoptosis: the role of transforming growth factor-beta.

Laboratory and clinical reports indicate that opiate addicts are prone to infections. This effect of opiates is partly attributed to opiate-induced macrophage (Mphi) apoptosis. In the present study, we evaluated the role of transforming growth factor-beta (TGF-beta) in morphine-induced apoptosis of murine J774 cells and peritoneal Mphi. Mphi harvested from morphine-treated mice showed greater (P < 0. 0001) apoptosis when compared with control Mphi. Morphine also enhanced apoptosis of J774 cells and peritoneal Mphi. Anti-TGF-beta 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-beta antibody (Ab) + 10-6 M morphine 8.1 +/- 0.7%; apoptotic cells/field) and peritoneal Mphi (control 1.5 +/- 0.9%; 10-6 M morphine 29.1 +/- 1.4%; 10-6 M morphine + anti-TGF-beta Ab 19. 1 +/- 1.8%; apoptotic cells/field). TGF-beta enhanced (P < 0.001) apoptosis of J774 cells and peritoneal Mphi. TGF-beta also promoted Mphi DNA fragmentation into integer multiples of 180 bp (ladder pattern). Immunocytochemical studies revealed that morphine enhanced the Mphi cytoplasmic content of TGF-beta. In addition, Western blotting showed increased production of TGF-beta 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-beta Ab. As both morphine-induced J774 cell apoptosis and bax expression were inhibited by anti-TGF-beta Ab, it appears that morphine-induced J774 cell apoptosis may be mediated through the generation of TGF-beta.

Ethanol-induced neutrophil apoptosis is mediated through nitric oxide.

Clinical reports indicate that acute ethanol intoxication in chronic ethanol abusers is associated with neutropenia. We hypothesize that ethanol accelerates the apoptosis of neutrophils thus decreasing the peripheral blood count of neutrophils. We studied the effect of ethanol on neutrophil apoptosis in vivo as well as in vitro. Human neutrophils harvested from healthy subjects after an alcohol drinking binge showed enhanced apoptosis (before, 0.5+/-0.25 vs. after, 26.1+/-2.6% apoptotic neutrophils/field). Peritoneal neutrophils isolated from ethanol-treated rats also showed increased (P < 0.0001) apoptosis when compared with neutrophils isolated from control rats (control, 0.8+/-0.2% vs. ethanol, 11.8+/-0.7% apoptotic neutrophils/field). In in vitro studies, ethanol in concentrations of 50 mM and higher accelerated the apoptosis of human and rat neutrophils. This effect of ethanol on human neutrophils was time dependent. DNA isolated from ethanol-treated human neutrophils displayed integer multiples of 180 base pairs (ladder pattern), further confirming the effect of ethanol on neutrophil apoptosis. N(G)-monomethyl-L-arginine monoacetate and N(G)-nitro-L-arginine methyl ester, inhibitors of nitric oxide (NO) synthase, attenuated the ethanol-induced neutrophil apoptosis. Sodium nitroprusside, a NO donor, also promoted neutrophil apoptosis. Moreover, ethanol enhanced neutrophil expression of inducible NO synthase. In addition, ethanol stimulated neutrophil NO generation. These results suggest that ethanol accelerates neutrophil apoptosis. This effect of ethanol on neutrophil apoptosis seems to be mediated through the generation of NO.

HIV-1 gp120 envelope protein modulates proliferation of human glomerular epithelial cells.

Glomerular epithelial cells (GEC) have been demonstrated to undergo morphological alterations in human immunodeficiency virus (HIV)-associated focal glomerulosclerosis. In the present study, we evaluated the effect of HIV-1 gp120 envelope protein on the growth of cultured human (H) GEC. gp120 protein enhanced (P < 0.001) the proliferation of HGEC at lower concentrations. The mitogenic effect of gp120 protein on HGEC was further confirmed by enhanced accumulation of proliferating nuclear cell antigen (PCNA) by gp120 protein-treated cells, as compared with control cells. On the contrary, gp120 protein at higher concentrations suppressed (P < 0. 001) the growth of HGEC. To evaluate the mechanism of gp120 protein-induced HGEC growth suppression, we examined the effect of gp120 protein on HGEC apoptosis. gp120 protein at higher concentrations promoted the apoptosis of HGEC. At higher concentrations, gp120 protein also enhanced DNA fragmentation of HGEC. Anti-gp120 antibody attenuated the proliferative as well as the apoptotic effects of gp120 protein on HGEC. Because protein kinase C as well as tyrosine kinase inhibitors partially inhibited gp120-induced proliferation, gp120 appears to be activating both the protein kinase C and tyrosine kinase pathways. In addition, gp120 protein at lower concentrations enhanced mRNA expression of c-fos and at higher concentrations promoted mRNA expression of c-jun. We conclude that gp120 has a bimodal effect on proliferation of HGEC. This effect may be mediated through the activation of early growth genes.

Human glomerular epithelial cell express CD4 and interaction with gp120 protein promotes PYK2 tyrosine phosphorylation.

Focal segmental glomerulosclerosis (FSGS) is the predominant glomerular lesion in patients with HIV infection. Visceral glomerular epithelial cell (vGEC) injury is a key feature of this glomerular lesion. However, the exact mechanism of HIV-1-induced vGEC injury is not clear. We studied the presence of CD4 (HIV-1 receptor) in vGECs. vGECs were cultured from human kidneys and used during the 5th to 10th passages. Immunocytochemical studies were carried out to visualize CD4 receptors in these cells. Protein and RNA were extracted from vGECs and renal cortical tissues. Western and Northern blots were generated and probed for the expression of CD4. To determine the downstream effect of ligand receptor interaction, vGECs were treated either with variable concentrations of HIV-1 gp120 protein (0.001 to 0.1 microg/ml) for 1 min or with a fixed dose of gp120 protein (0.01 microg/ml) for variable time periods (0 to 10 min), and at the end of the incubation period, tyrosine phosphorylation of pyk2 was studied. Immunocytochemical studies showed the presence of CD4 receptors in vGECs. Western and Northern blot studies confirmed the presence of CD4 expression in these cells. gp120 protein promoted vGEC tyrosine phosphorylation of pyk2 in a dose- and time-dependent manner. The present study provides a mechanistical insight for the role of HIV-1 in the development of glomerular injury in patients with HIV infection.

Morphine promotes apoptosis in Jurkat cells.

Patients with intravenous heroin addiction are prone to recurrent infections and at times these infections are fatal. We evaluated the effect of morphine on the apoptosis of Jurkat cells and freshly isolated human T lymphocytes. Morphine promoted apoptosis of both the Jurkat cells and the freshly isolated T lymphocytes in a dose-dependent manner. DAGO, a specific mu receptor agonist, also promoted Jurkat cell apoptosis. DNA isolated from morphine-treated Jurkat cells and T lymphocytes also showed integer multiples of 200 base pairs. Superoxide dismutase (SOD) enhanced lymphocyte apoptosis; whereas catalase attenuated the morphine-induced apoptosis of Jurkat cells as well as of T lymphocytes. Morphine-treated Jurkat cells also showed a decreased expression of bcl-2 and an enhanced expression of bax. In addition, morphine-treated Jurkat cells showed activation of caspase-3. These results indicate that morphine-induced T lymphocyte apoptosis may be mediated through the generation of reactive oxygen species. The change in ratio of bax and bcl-2 seems to tilt the balance toward apoptosis, leading to the activation of caspase-3. This study provides further support for the hypothesis that morphine may be directly compromising immune function by enhancing apoptosis of T lymphocytes in patients with heroin addiction.

Circulating factor in patients with recurrent focal segmental glomerulosclerosis postrenal transplantation inhibits expression of inducible nitric oxide synthase and nitric oxide production by cultured rat mesangial cells.

BACKGROUND: Primary focal segmental glomerulosclerosis (FSGS) recurs in nearly 30% of patients who progress to end-stage renal disease and then receive a kidney transplant. A circulating plasma factor has been isolated from these patients that increases glomerular permeability to albumin in vitro. Because of the pivotal role of the mesangial cell in the accumulation of extracellular matrix (ECM) material within the glomerulus and the modulation of matrix protein synthesis by nitric oxide (NO), we examined the effect of the FSGS factor on inducible nitric oxide synthase (iNOS) expression and NO production by cultured rat mesangial cells (RMC). METHODS: RMC were incubated with the supernatant following 70% ammonium sulfate precipitation of serum from patients with recurrent FSGS. RESULTS: Addition of the FSGS factor to cultured RMC led to a significant inhibition of nitrite accumulation, an index of NO synthesis. There was a parallel decline in iNOS gene and protein expression. Sera obtained from control patients or those with minimal change nephrotic syndrome or diabetic nephropathy that was processed in the same manner as FSGS samples had no effect NO synthesis or iNOS activity. The inhibitory effect of the FSGS factor on NO production persisted despite addition of indomethacin (0.1-1 mumol/L) or cyclosporine (25 micrograms/mL) to test media. CONCLUSIONS: These data indicate that the FSGS factor independently alters two aspects of glomerular function--permselectivity and matrix protein synthesis--by distinct mechanisms. FSGS factor-induced disturbances in iNOS gene and protein expression and NO production by mesangial cells may antagonize the antifibrotic effect of NO within the mesangium and contribute to progressive glomerulosclerosis in patients with primary FSGS.

Ethanol-induced macrophage apoptosis: the role of TGF-beta.

Both clinical and laboratory reports indicate that ethanol addicts are prone to recurrent infections. We hypothesize that ethanol promotes macrophage apoptosis, thus compromising the efficiency of the mononuclear phagocyte system in dealing with infection. We studied the effect of ethanol on macrophage apoptosis. Human monocytes isolated from healthy subjects after an alcohol drinking binge showed enhanced apoptosis (before, 1.2 +/- 0.3% vs after, 28.4 +/- 3.7% apoptotic cells/field). Peritoneal macrophages harvested from ethanol-treated rats also showed increased (p < 0.0001) apoptosis. DNA isolated from peritoneal macrophages of ethanol-treated rats displayed integer multiples of 200 base pairs (ladder pattern). Furthermore, macrophages harvested from ethanol-treated rats had an enhanced expression as well as accumulation of TGF-beta. In in vitro studies, ethanol promoted apoptosis of human monocytes as well as rat peritoneal macrophages. In addition, ethanol enhanced apoptosis of murine macrophages (J774) in a time-dependent manner. The ethanol-induced apoptosis was amplified by LPS and partly attenuated (p < 0.001) by anti-TGF-beta Ab. TGF-beta also promoted macrophage apoptosis in a dose-dependent manner. Moreover, ethanol enhanced TGF-beta protein production by macrophages. These results indicate that ethanol promotes macrophage apoptosis. This effect of ethanol seems to be partly mediated through the generation of TGF-beta by macrophages.

Matrix-mesangial cell interaction modulates migration of macrophages.

BACKGROUND: Macrophages (Møs) have been demonstrated to play an important role in immune-mediated renal injury. Accumulation of macrophages in the mesangium has been reported to be a key event in the development of focal glomerulosclerosis. We hypothesized that mesangial cells (MCs) and matrix interaction may be a determinant for the migration of Møs into the mesangium. Therefore, we studied the effect of the interaction between matrix and MCs on the migration of Møs. METHODS: Mouse MCs were plated on Petri dishes coated either with buffer, collagen type I, III, IV, or Matrigel in media containing 1% fetal calf serum for 48 hours. Subsequently, supernatants were collected and stored. The effect of these supernatants (conditioned media) was evaluated on the migration of Møs across a filter in a modified Boyden chamber. RESULTS: Conditioned media from MCs grown on Matrigel (MC-Matrigel interaction products, MC-MGP) enhanced the migration of macrophages across a filter in a modified Boyden chamber when compared with conditioned media from MCs grown on plastic, collagen type I, type III, or type IV (MC-PP, MC-CI, MC-CIII, and MC-CIV). MC-MGP enhanced the migration of Møs in a dose dependent manner. Anti-MCP-1 antibodies attenuated (P < 0.05) the MC-MGP-induced Mø migration (MC-MGP, 16.8 +/- 2.5 vs MC-MGP + anti-MCP-1 antibody, 6.5 +/- 1.2 migrated macrophages/field, n = 12). Anti-TGF-beta antibodies did not attenuate MC-MGP-induced Mø migration. MCs grown on Matrigel showed a 5-fold increase of MCP-1 mRNA when compared with cells grown on plastic or collagen type IV. CONCLUSIONS: The present study suggests that matrix components may modulate the migration of Møs. This effect of MC-matrix interaction on macrophage migration may be mediated through the generation of MCP-1.

Enhanced proliferation, apoptosis, and matrix accumulation by mesangial cells derived from HIV-1 transgenic mice.

BACKGROUND: Mice, transgenic for HIV-1 genes, have been demonstrated to develop renal lesions mimicking HIV-associated nephropathy. Focal glomerulosclerosis (FGS) has been reported to be the predominant glomerular lesion in these animals. In the other models of FGS, the accumulation of mesangial matrix and mesangial cell proliferation have been shown to be the preceding abnormalities. We evaluated the proliferation, apoptosis, and matrix accumulation by mesangial cells derived from mice transgenic for HIV-1 genes as well as from nontransgenic mice. METHODS: Mesangial cells were cultured from mice transgenic for HIV-1 genes (HTrMC) and nontransgenic mice (NTrMC) of the same age and sex. The growth rate of HTrMC and NTrMC was determined under identical conditions. Morphologic evaluation of apoptosis was performed by staining cells with Hoechst (H)-33342 and propidium iodide. Accumulation of mesangial cell collagen type IV, laminin, and fibronectin was measured by the dot blot assay. Total RNA was extracted from HTrMC and NTrMC and Northern blots were generated. These blots were probed with specific probes for TGF-beta, proteoglycan (P16), and GAPDH. RESULTS: Mesangial cells (HTrMC) derived from transgenic mice had greater (P < 0.004) proliferation when compared to mesangial cells (NTrMCs) from nontransgenic mice (HTrMCs, 4.2 +/- 0.3 vs NTrMCs, 3.0 +/- 0.2 x 10(4) cells/well). HTrMCs also showed enhanced (P < 0.0001) apoptosis compared to NTrMCs (HTrMCs, 13.2 +/- 1.5% vs NTrMCs, 3.1 +/- 0.5% apoptotic cells/field). HTrMCs accumulated an increased (P < 0.02) amount of collagen type IV (HTrMCs, 5659.7 +/- 472.8 vs NTrMCs, 3882.2 +/- 339.7 ng/well); whereas NTrMCs accumulated a greater amount of laminin when compared to HTrMCs (HTrMCs, 12.8 vs NTrMCs, 29.6 +/- 2.9 ng/well). HTrMCs also showed an enhanced mRNA expression of TGF-beta and an attenuated expression of proteoglycan (P16). CONCLUSIONS: These results suggest that mesangial cells derived from mice transgenic for HIV-1 genes have enhanced proliferation and collagen accumulation. The enhanced expression of TGF-beta may have contributed to enhanced HTrMC proliferation and the accumulation of collagen. The present study provides the basis for a hypothesis that mesangial cells may be contributing to the development of focal glomerulosclerosis in mice transgenic for HIV-1 genes.

HIV-1 gp120 envelope protein and morphine-tubular cell interaction products modulate kidney fibroblast proliferation.

BACKGROUND: Renal interstitial scarring is an important feature of HIV-associated nephropathy. Intravenous drug abuse has been demonstrated to be a risk factor for the development of HIV-associated nephropathy in patients with HIV infection. We studied the effect of tubular cell-morphine and/or HIV-1 gp120 envelope protein interaction products on kidney fibroblast (KF) proliferation and apoptosis. METHODS: Tubular cell-morphine and/or gp120 interaction products were prepared by incubating confluent human proximal tubular cells with buffer (TCP), morphine (10(-3) mol/L) (TCM-IP), gp120 (0.01 microgram/mL)(TC-120IP), or morphine (10(-3) mol/L) + gpl20 (0.01 microgram/mL) (TCM-120IP). To evaluate the effect of tubular cell interaction products (TCIP) on KF proliferation, growth arrested kidney fibroblasts were treated with variable concentrations (5%, 10%, 20%, 30%, and 50%) of TCP, TCM-IP, TC-120IP, or TCM-120IP for 48 hours. To evaluate the role of cytokines in TCIP-induced KF proliferation, cells were incubated with TCIP with or without cytokine neutralizing antibodies to TGF-beta, TNF-alpha, FGF, or IL-6 for 48 hours. Subsequently, cells were counted in a hemocytometer (n = 3). To evaluate the effect of TCIP on KF apoptosis, cells were treated with 50% TCP, 50% TCM-IP, 50% TC-120IP, or 50% TCM-120IP for 24 hours and stained with H-33342 and propidium iodide. In parallel experiments KFs were harvested under identical conditions, DNA was isolated and run on gel electrophoresis. To evaluate the role of early growth genes in TCM-120-induced KF proliferation, TCM-120IP-treated cells were probed with cDNA for c-fos and c-jun. RESULTS: TC-120IP at a lower concentration (20%) enhanced (P < 0.001) proliferation of KF when compared with TCP. TCM-IP did not stimulate KF proliferation. On the contrary, TCM-120IP at a lower concentration (20%) promoted (P < 0.001) KF proliferation when compared with TCP, TCM-IP and TC-120IP. TCM-120IP at a lower concentration (20%) also enhanced KF mRNA expression of c-fos and c-jun. TCM-120IP enhanced KF proliferation in a dose-dependent manner. All tubular cell interaction products at a higher concentration (50%) promoted apoptosis of KF. CONCLUSIONS: Tubular cell-gp120 interaction products stimulated KF proliferation. Morphine amplified the effect of tubular cell-gp120 interaction on the proliferation of KF. TCM-120IP-induced KF proliferation may be mediated through the expression of early growth genes; whereas TCM-120IP-induced KF growth suppression may be mediated through the induction of apoptosis.

High glucose enhances growth factor-stimulated nitric oxide production by cultured rat mesangial cells.

Nitric oxide (NO) contributes to the alterations in glomerular hemodynamics and extracellular matrix accumulation observed in diabetic nephropathy. High glucose concentrations directly inhibit NO production by rat mesangial cells (RMC). However, the role of peptide growth factors and chemokines in regulating NO synthesis by RMC under normal and high glucose conditions has not been studied. Therefore, we examined the effect of IGF-I, EGF, TGF-beta and RANTES on NO production by RMC maintained in normal (5.6 mM) or high glucose (33.3 mM) for 48 h. No synthesis was determined by measuring nitrite accumulation in conditioned media with the Greiss reaction. In normal glucose media, IGF-I, EGF, and RANTES had no effect on nitrite accumulation while TGF-beta inhibited NO synthesis. In high glucose conditions, IGF-I and EGF significantly enhanced NO production. The effects of RANTES and TGF-beta were unchanged by an elevated glucose concentration. EGF-induced stimulation of NO production in high glucose media was associated with parallel alterations in iNOS gene and protein expression. The modest enhancement in nitrite accumulation provoked by IGF-I in high glucose conditions was not accompanied by demonstrable increases in iNOS mRNA abundance or protein content. In conclusion, peptide growth factors modulate the direct inhibitory effect of high glucose on NO production by cultured mesangial cells. These actions in vivo may limit the adverse consequences of reduced NO production in promoting diabetic nephropathy.

Effect of vascular endothelial growth factor on nitric oxide production by cultured rat mesangial cells.

Vascular endothelial growth factor (VEGF) stimulates nitric oxide (NO) production by endothelial cells in vitro and in vivo. However, the impact of VEGF on inducible nitric oxide synthase (iNOS) activity and NO synthesis in cultured mesangial cells is not known. Therefore, we measured nitrite accumulation in cytokine-stimulated, rat mesangial cells (RMC) in response to graded concentrations of VEGF. Addition of VEGF (10-50 ng/ml) did not alter RMC viability or NO production in either normal (5.6 mM) or high (33.3 mM) glucose conditions. Exposure of RMC to VEGF did not modify the effects of L-arginine (20 mM) or L-NAME (1 mM) on nitrite accumulation in normal or high glucose media. The steady state abundance of iNOS mRNA and the cytosolic content of iNOS protein were unaffected by addition of VEGF. Cultured RMC expressed the high-affinity tyrosine kinase VEGF receptors, flt and flk/KDR, and the levels were not modulated by incubation in normal or high glucose media. We conclude that VEGF does not regulate proliferation or NO production in cultured RMC. These findings suggest that disturbances in the normal interaction between VEGF and NO are not involved in the pathogenesis of abnormal mesangial cell structure or function in diabetic nephropathy.

Simulated glomerular hypertension promotes mesangial cell apoptosis and expression of cathepsin-B and SGP-2.

BACKGROUND: Focal glomerulosclerosis (FGS) is characterized by the accumulation of mesangial matrix and lack of mesangial cells (MC). We studied the role of glomerular hypertension (GH) in the development of MC hypoplasia. METHODS: We used an in vitro model of GH to study the effect of GH on MC apoptosis. Cultured rat endothelial cells were grown in the intracapillary space and MCs were grown in the extracapillary space. MC proliferation as well as apoptosis was evaluated under simulated normal glomerular pressure (SNGP) as well as simulated glomerular hypertension (SGH). Apoptosis was determined morphologically by DNA fragmentation using Hoechst staining as well as with the use of DNA gel electrophoresis. MCs grown under SNGP and SGH were also evaluated for the expression of genes associated with active cell death. In addition, we evaluated the effect of direct applied pressure on MC apoptosis. RESULTS: MCs grown under SGH were less elliptical and had a tendency to develop a dome in the center. Direct applied pressure promoted MC apoptosis in a dose and time dependent manner. DNA gel electrophoresis from MCs grown under SGH also showed integer multiples of 180 base pairs (ladder pattern); whereas cells grown under SNGP showed no DNA fragmentation. SGH increased mRNA expression of cathepsin-B (SNGP, 0.31 +/- 0.04 vs SGH, 0.57 +/- 0.03, P < 0.01, n = 3) and SGP-2 (SNGP, 0.55 +/- 0.05 vs SHG, 1.08 +/- 0.12, P < 0.01, n = 3) in MCs when compared to cells grown under SNGP. CONCLUSIONS: The present in vitro study suggests that GH has the potential to convert a hypercellular mesangium into a hypocellular one. This effect of GH is associated with expression of genes associated with active cell death.

Morphine enhances macrophage apoptosis.

Laboratory data indicate that morphine decreases the numbier of peritoneal and alveolar macrophages (Mphi) and compromises their phagocytic capability for immune complexes and bacteria. We hypothesize that morphine decreases the number of, as well as compromises the phagocytic capability of, Mphi by programming their death. We studied the effect of morphine on Mphi apoptosis in vivo as well as in vitro. Peritoneal Mphi harvested from morphine-treated rats showed DNA fragmentation. Morphine enhanced murine Mphi (J 774.16) apoptosis in a dose-dependent manner. Human monocytes treated with morphine showed a classic ladder pattern in gel electrophoretic and end-labeling studies. Morphine promoted nitric oxide (NO) production both under basal and LPS-activated states. N(G)-nitro-L-arginine methyl ester (L-NAME) and N(G)-monomethyl-L-arginine monoacetate (L-NMMA), inhibitors of NO synthase, attenuated the morphine-induced generation of NO by Mphi. Morphine also enhanced Mphi mRNA expression of inducible NO synthase (iNOS). Since morphine-induced Mphi apoptosis was inhibited by L-NAME and L-NMMA, it appears that morphine-induced Mphi apoptosis may be mediated through the generation of NO. Morphine promoted the synthesis of Bax and p53 proteins by Mphi. Moreover, IL-converting enzyme (ICE)-1 inhibitor attenuated morphine-induced Mphi apoptosis. These studies suggest that morphine activates the induction phase of the apoptotic pathway through accumulation of p53. The effector phase of morphine-induced apoptosis appears to proceed through the accumulation of Bax and activation of ICE-1. The present study provides a basis for a hypothesis that morphine may be directly compromising immune function by promoting Mphi apoptosis in patients with opiate addiction.

Morphine modulates proliferation of kidney fibroblasts.

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.