Biomedical subjects

P C Singhal

Publications and source records attributed to P C Singhal.

At least 37 records · Page 2Linked to original sources

PubMed1999

Aging splenocyte and thymocyte apoptosis is associated with enhanced expression of p53, bax, and caspase-3.

Aging is associated with altered immune function. We previously reported that splenocytes and thymocytes undergo apoptosis with aging in rats. In the present study, we examined the expression of genes associated with apoptosis in splenocytes and thymus in aging rats. We evaluated the expression of bax, interleukin 1-beta-converting enzyme (ICE)/ced-3 protease family, caspase-3 and tumor suppressor gene p53. Rats in age groups of 6, 24, 48, and 96 weeks were sacrificed; thymocytes and splenocytes were isolated followed by lysis in a modified RIPA buffer containing protease inhibitors. Western blot analysis of proteins was performed by probing immunoblots with antibodies against p53, bax and PARP (poly ADP-ribose polymerase). Increased aging was associated with enhanced expression of bax, p53 and cleavage of PARP by Caspase-3. The expression of p53 and cleavage of PARP indicates the presence of damaged DNA; nevertheless, the cleavage of PARP or activation of caspase-3 may be playing an important role in the initiation of early events in apoptosis. These results suggest that aging of splenocytes and thymocytes is associated with the expression of cell death genes. The present study provides an insight into age-associated altered immune function.

Aging↗

PubMed1999

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.

Blotting, Northern↗

PubMed1999

Escherichia coli promotes macrophage apoptosis.

BACKGROUND: Escherichia coli is the bacterium most commonly isolated from the urine of patients with urinary tract infection (UTI). Recurrent episodes of UTI lead to renal interstitial scarring. In interstitial fibrosis and scarring, infiltration of mononuclear cells has been reported to play a key role. MATERIALS AND METHODS: We evaluated the effect of two strains of E. coli--the pathogenic BH-5 and the plasmidless, nonfimbriated HB-101-on human monocyte and murine macrophage apoptosis. RESULTS: E. coli BH-5 enhanced apoptosis in a time- and dose-dependent manner. It also promoted necrosis in a time- and dose-dependent manner. Strain HB-101 promoted monocyte apoptosis in a dose-dependent manner. However, the magnitude of HB-101-induced monocyte apoptosis was lower than BH-5-induced macrophage apoptosis. CONCLUSION: The ability of E. coli to induce apoptosis may contribute to its virulence and play a role in renal interstitial scarring.

Animals↗

PubMed1999

Absence of age-related increase in systolic blood pressure in ambulatory patients with HIV infection.

BACKGROUND: Systolic blood pressure is well known to increase significantly with age and is strongly correlated with stroke and coronary artery disease. We and other investigators have reported a low prevalence of hypertension in subgroups of patients with HIV infection. In the present study, we examined an ambulatory population of patients with HIV infection to determine whether in the outpatient setting they may lack an age-related increase in systolic blood pressure. METHODS: In an ambulatory outpatient practice, medical records of 178 consecutive patients with HIV infection and those of 200 control subjects were examined. Systolic and diastolic blood pressure and other clinical and laboratory variables were recorded. Scatter plots were generated to compare age with systolic blood pressure. Spearman rank correlation analysis was carried out to determine the relationship between systolic blood pressure and age and other variables. RESULTS: Patients ranged in age from 13 to 69 years. There was only a very slight increase (which did not achieve statistical significance) in systolic blood pressure with aging in the patients with HIV infection, in contrast to the control population, in which an age-related increase in systolic blood pressure was seen that was comparable to published Framingham data. Mean systolic blood pressure for the group as a whole was 118.2 +/- 1.1 mm Hg. Mean serum albumin was 4.2 +/- 0.04 g/dL and was only slightly diminished in older patients. Mean serum cholesterol was 176.8 +/- 3.4 mg/dL and this bore no relationship to aging. More advanced stages of HIV infection also did not correlate with the lack of age-associated systolic hypertension. CONCLUSION: The present population of ambulatory patients infected with HIV seem to lack an age-related increase in systolic blood pressure; this may be caused by such variables as autonomic dysfunction or factors that may attenuate the development of atherosclerosis.

Adolescent↗

PubMed1999

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.

Animals↗

PubMed1998

Metal-catalyzed oxidation of immunoglobulin G impairs Fc receptor-mediated binding to macrophages.

Enhanced oxidative stress is a feature of inflammatory and infectious conditions. Proteins may be important targets of oxidation and this may alter their function. We evaluated whether metal-catalyzed oxidation of IgG could alter its ability to bind to Fc receptors on macrophages. Human IgG incubated with an FeCl3/EDTA/ascorbate metal-catalyzed oxidation system resulted in a significant increase in carbonyl content, a measure of protein oxidation, compared to IgG treated with EDTA alone (control). Western blot analysis using an antibody to oxidized protein revealed an increase in antibody binding to both the heavy (Fc portion-containing) and light chains of IgG treated with the oxidizing system. Western blot analysis of papain-digested IgG confirmed oxidative modification of the Fc portion. Binding studies carried out with J774.16 macrophages demonstrated significantly diminished ability of the oxidized IgG to bind to macrophage Fc receptors compared to control IgG. These data demonstrate that IgG is susceptible to metal-catalyzed oxidation and that this impairs its ability to bind to macrophage Fc receptors. Oxidation of IgG might play a role in modulating immune function in infection and disorders associated with immune complex formation by diminishing IgG binding to phagocytic cells.

Ascorbic Acid↗

PubMed1998

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.

Animals↗

PubMed1998

Outcome of stroke in patients undergoing hemodialysis.

BACKGROUND: While elevated levels of serum creatinine have been shown to be a risk factor for diminished survival after stroke, it is unknown how renal replacement therapy may affect the outcome. METHODS: Strokes occurring in 26 consecutive patients undergoing hemodialysis at our institution were reviewed and clinical and laboratory variables and outcome were compared with those of patients who had a stroke but had normal renal function. RESULTS: Twenty-four strokes in the patients undergoing hemodialysis were ischemic while only 2 were hemorrhagic. Virtually all the patients had hypertension, half had diabetes mellitus, and most had some prior evidence of cardiovascular disease at the time of their stroke. Fifty percent of the patients undergoing hemodialysis had a good outcome (defined as being discharged home) while the remainder had a poor outcome (defined as dying or being discharged to a nursing facility). The combined presence of hypertension and coronary artery disease had a sensitivity of 91.2% for identifying patients with a poor outcome, while male sex, the presence of coronary artery disease, and the combined presence of hypertension, coronary artery disease, and/or congestive heart failure had sensitivities greater than 80% but low specificity. The outcome of patients undergoing hemodialysis was comparable with that of a control group of patients who had a stroke but had normal renal function, although the length of hospital stay was greater (mean [+/-SEM] 29.8+/-6.4 days vs 12.7+/-1.1 days, respectively; P<.01). CONCLUSIONS: Hospitalized patients undergoing hemodialysis in whom stroke occurs appear to have as good an outcome as that of patients with normal renal function, although they are hospitalized longer. In addition, certain clinical variables seem to be associated with a worse outcome. Aggressive measures to prevent and treat stroke seem as warranted for patients undergoing hemodialysis as for patients with normal renal function, although interventions to reduce the length of hospital stay are needed.

Adult↗

PubMed1998

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.

Animals↗

PubMed1998

Extracellular matrix modulates mesangial cell apoptosis and mRNA expression of cathepsin-B and tissue transglutaminase.

Mesangial matrix is a dynamic structure which modulates mesangial cell function. Since accumulation of matrix precedes the development of focal glomerulosclerosis, we studied the effect of different matrices on mesangial cell (MC) apoptosis. Suspended mesangial cells became apoptotic in a time dependent manner. Collagen type III did not modulate MC apoptosis when compared to cells grown on plastic. MCs grown on Matrigel, collagen type I and IV showed an increased number of apoptotic cells when compared to MCs grown on plastic. DNA end-labeling further confirmed these observations. MCs grown on Matrigel showed enhanced (P < 0.05) mRNA expression for tissue transglutaminase (TTG) and cathepsin-B. Mesangial cells grown on Matrigel also showed enhanced expression of superoxide dismutase (SOD). We conclude that mesangial cells require attachment to the matrix for their survival and alteration of the quality of matrix modulates mesangial cell apoptosis.

Animals↗

PubMed1998

Tubular cell and HIV-1 gp120 interaction products promote migration of monocytes.

Renal interstitial accumulation of monocytes is an important feature of HIV-associated nephropathy. We studied the effects of proximal tubular cell products (TCP) and proximal tubular cell-gp120 interaction products (TC-120IP) on the migration of monocytes across a modified Boyden chamber. TC-120IP promoted (P < 0.001) the migration of monocytes when compared with TCP (TCP, 45.0 +/- 5.9 vs. TC-120IP, 192.3 +/- 39.5 migrated monocytes/field). This effect of TC-120IP on monocyte migration was dose dependent. Anti-MCP-1 (TCP, 24.7 +/- 2.6; TC-120IP, 82.3 +/- 5.5; TC120-IP + anti-MCP-1 antibody, 46.5 +/- 3.5 migrated monocytes/field) as well as anti-TGF-beta antibodies (TCP, 25.8 +/- 3.4; TC120-IP, 80.3 +/- 6.9; TC-120IP + anti-TGF-beta antibody, 43.8 +/- 5.6 migrated monocytes/field) partly attenuated TC-120IP-induced migration of monocytes across a filter. Moreover, anti-MCP-1 and anti-TGF antibodies showed an additive inhibitory effect on TC-120IP-induced migration of monocytes across a filter. These results suggest that TC-120IP-induced migration of monocytes may be mediated through the generation of MCP-1 and TGF-beta by tubular cells. The present study provides the basis for a hypothesis that HIV-1 gp120 protein may be contributing to the infiltration of monocytes in the renal interstitium of patients with HIV-associated nephropathy.

AIDS-Associated Nephropathy↗

PubMed1998

Oxidation of the mesangial matrix metalloproteinase-2 impairs gelatinolytic activity.

Glomerulosclerosis is characterized by an accumulation of mesangial extracellular matrix. Oxygen radicals are strongly implicated in glomerular injury but it is unclear by what mechanism they could modulate matrix turnover dynamics. We evaluated whether oxidation of the 72 kD mesangial matrix metalloproteinase-2 (MMP-2), the major mesangial matrix-degrading enzyme, could alter its gelatinolytic activity. Oxidation of the MMP-2 using a FeCl3/ascorbate system resulted in impaired ability to degrade [3H]gelatin compared to control. Samples were also subjected to SDS-PAGE gelatin substrate zymography. At the 72 kD position a significant impairment of gelatinolytic activity of oxidized samples was observed, a decrease attenuated by coincubation of samples with the FeCl3/ascorbate system plus the radical spin trap N-tert-butyl-alpha-phenylnitrone suggesting specificity of oxidative changes in the decrease in enzymatic activity. These data represent the first report demonstrating that oxidation of the MMP-2 diminishes its activity and suggest a previously undescribed mechanism by which oxygen radicals may contribute to altered turnover of extracellular matrix.

Animals↗

PubMed1998

Peroxynitrite formation and apoptosis in transgenic sickle cell mouse kidneys.

BACKGROUND: In a previous study, nitric oxide synthases (NOS) were found to be strongly expressed in the tubular epithelium of kidneys of a transgenic mouse model of sickle cell disease (alphaHbetaS[betaMDD]). Because NOS activity is often associated with peroxynitrite formation when superoxide radical (.O-2) is present in abundance, we examined the kidneys of sickle cell mice for nitrotyrosine, considered to be a footprint of ONOO-. METHODS: Western blot and immunohistochemistry for nitrotyrosine was carried out. Since peroxynitrite and other reactive oxygen radicals are capable of causing apoptosis, we also performed agarose gel electrophoresis of kidney DNA and TUNEL staining of nuclei, indicators of apoptosis. RESULTS: Nitration of tyrosine residues of three proteins (kD 66, 57 and 22) was found on Western blot of kidney protein extracts of the sickle cell mice. The degree of tyrosine nitration of the 66 kD protein was not significantly different in the control versus transgenic mice, whereas tyrosine nitration of the 57 and 22 kD proteins was clearly increased in transgenic mice. Strong immunostaining for nitrotyrosine was seen in tubular epithelial cells of the sickle cell mice, in close proximity to positive immunostaining of iNOS. Neither iNOS nor nitrotyrosine was expressed in the control mice. DNA "laddering" was found localized to the same zones of the kidney as nitrotyrosine and iNOS immunostaining. TUNEL assay on mouse kidney tissue sections showed minimal tubular cell apoptosis in normal mouse with hypoxia, mild tubular cell apoptosis in sickle cell mouse in room air, and moderate tubular cell apoptosis in sickle cell mouse with hypoxia. CONCLUSIONS: The observations suggest that ONOO- and perhaps other reactive oxygen species are being produced in the sickle cell kidney. The mechanism may be ischemia/reperfusion due to intermittent vascular occlusion by sickle cells. The resulting hypoxia could result in iNOS activation, superoxide radical and peroxynitrite formation. Two consequences of these reactions appear to be nitration of tyrosine residues of some renal proteins and enhanced apoptosis.

Anemia, Sickle Cell↗

PubMed1998

Metal-catalyzed oxidation of extracellular matrix increases macrophage nitric oxide generation.

BACKGROUND: Oxygen radicals are believed to play a significant role in glomerular disease. In part this may be due to oxidation of lipids, but protein oxidation may play a contributory role as well. We have demonstrated that the mesangial extracellular matrix is susceptible to metal-catalyzed oxidation and that this increases scavenger receptor-mediated adhesion of macrophages, cells which appear to be important participants in glomerular injury via their secretory products. As other scavenger receptor ligands can increase macrophage nitric oxide generation, we examined whether oxidation of matrix could increase the activity of macrophage inducible nitric oxide synthase (iNOS). METHODS: Extracellular matrix was oxidized using a metal-catalyzed oxidation system. Matrix oxidation was measured using carbonyl analysis, and iNOS activity in macrophages seeded onto the matrix was measured by nitrite determination and Western and Northern analyses for iNOS. RESULTS: Macrophages exposed to oxidized matrix demonstrated a significant enhancement of iNOS activity. This enhancement could be antagonized by cotreatment of matrix with the radical spin trap N-tert-butyl-a-phenylnitrone, resulting in a corresponding decrease in protein carbonyl content, a measure of protein oxidation. Seeding macrophages onto oxidized matrix and adding the scavenger receptor ligand polyinosinic acid further augmented iNOS activity, suggesting that additional scavenger receptors were available to bind ligand and that further augmentation of iNOS activity did not require an additional change in cell shape. Western blot analysis revealed an increase in iNOS protein expression as a consequence of interaction with the oxidized matrix, but there was no difference in iNOS mRNA expression by Northern analysis suggesting a post-transcriptional mechanism for enhanced iNOS activity. CONCLUSION: These data demonstrate that oxidation of extracellular matrix enhances macrophage nitric oxide generation, and suggest a previously undescribed role for extracellular matrix modification in the regulation of cellular function and possibly the mediation of glomerular injury.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Acquired Immunodeficiency Syndrome↗