Overexpression of manganese superoxide dismutase suppresses increases in collagen accumulation induced by culture of mesangial cells in high-media glucose.

Superoxide has been implicated in the cellular signalling pathways, which regulate growth of mesangial cells (MC) and vascular smooth muscle cells. Manganese (Mn)(2+)-dependent superoxide dismutase (SOD-2) is primarily responsible for metabolism of superoxide produced in mitochondria by respiratory chain activity during aerobic metabolism of glucose and other substrates. In the current studies, we examined the role of superoxide in the stimulation of collagen accumulation induced in MC by culture in media containing a high concentration of glucose. Aconitase, an iron sulfur enzyme whose activity is inhibited by superoxide, was used as an index of cellular superoxide production and action. SV-40-transformed mouse MC were cultured in media containing 100 (low) or 500 (high) mg/dL D-glucose and infected with a recombinant adenoviral (Ad) vector encoding either human mitochondrial Mn(2+) SOD-2 or green fluorescent protein (GFP). In cells infected with SOD-2 (SOD-2-Ad) and cultured in low glucose, SOD-2 activity was 5-fold higher than in cells infected with GFP (GFP-Ad), whereas Cu(2+)/Zn(2+) cytoplasmic SOD (SOD-1) did not differ; culture in high-glucose media did not alter SOD-2 or SOD-1 activity in either GFD-Ad or SOD-2-Ad. In GFP-Ad, high glucose suppressed aconitase activity and increased collagen accumulation compared with corresponding values in low glucose. In SOD-2-Ad, high glucose failed to suppress aconitase activity or increase collagen accumulation. Addition of exogenous (presumably extracellular) SOD to GFP-Ad had no effect on the stimulation of collagen accumulation by high glucose. Analogous to the effects of SOD-2-Ad, diphenylene iodonium (DPI), a nonspecific inhibitor of the production of superoxide by mitochondrial respiration and other nicotinamide adenine dinucleotide (phosphate) (NAD)(P)H oxidase activities, reduced collagen accumulation in GFP-Ad cultured in low glucose and blocked stimulation of collagen accumulation induced by culture in high glucose. These results support a role for increased cellular superoxide production derived from NAD(P)H oxidase activity in the stimulation of collagen accumulation induced in MC by high glucose and demonstrate that an increase in mitochondrial SOD-2 activity suppresses this response.

Overexpression of Cu2+/Zn2+ superoxide dismutase protects against early diabetic glomerular injury in transgenic mice.

Ex vivo and in vitro observations implicate superoxide as a mediator of cell injury in diabetes, but in vivo evidence is lacking. In the current studies, parameters of glomerular injury were examined in hemizygous nondiabetic transgenic mice (SOD) and streptozotocin-diabetic (D) transgenic mice (D-SOD), which overexpress human cytoplasmic Cu2+/Zn2+ superoxide dismutase (SOD-1), and in corresponding wild-type littermates (WT, D-WT) after 4 months of diabetes. In both SOD and D-SOD mice, renal cortical SOD-1 activity was twofold higher than values in the WT mice; blood glucose and glycosylated hemoglobin (GHb) levels did not differ in the two diabetic groups. Urinary albumin excretion, fractional albumin clearance, urinary transforming growth factor-beta (TGF-beta) excretion, glomerular volume, glomerular content of immunoreactive TGF-beta, and collagen alpha1 (IV) and renal cortical malondialdehyde (MDA) levels were significantly higher in D-WT mice compared with corresponding values in D-SOD mice. Glomerular volume, glomerular content of TGF-beta and collagen IV, renal cortical MDA, and urinary excretion of TGF-beta in D-SOD mice did not differ significantly from corresponding values in either the nondiabetic SOD or WT mice. In separate groups of mice studied after 8 months of diabetes, mesangial matrix area, calculated as a fraction of total glomerular tuft area, and plasma creatinine were significantly higher in D-WT but not in D-SOD mice, compared with corresponding values in the nondiabetic mice. In vitro infection of mesangial cells (MC) with a recombinant adenovirus encoding human SOD-1 increased SOD-1 activity threefold over control cells and prevented the reduction of aconitase activity, an index of cellular superoxide, and the increase in collagen synthesis that otherwise occurred in control MC in response to culture with 300 or 500 mg/dl glucose. Thus, increases in cellular SOD-1 activity attenuate diabetic renal injury in vivo and also prevent stimulation of MC matrix protein synthesis induced in vitro by high glucose.

Regulation of the laminin C1 promoter in cultured mesangial cells.

Expression of the genes encoding several matrix proteins, including the laminin gamma1 and beta1 subunits, is increased in glomeruli or renal cortex from diabetic animals or in mesangial cells cultured in high concentrations of glucose. Transforming growth factor (TGF)-beta1 and IGF-1 have been implicated as mediators of this response. In the present study, we assessed the influence of high glucose concentrations and the roles of TGF-beta1 and IGF-1 in the regulation of laminin C1 gene expression in cultured mesangial cells. Culture of normal rat mesangial cells (RMC) or SV40-transformed mouse mesangial (MES-13) cells in 500 mg/dl D-glucose for 2 days to 3 weeks significantly increased laminin C1 mRNA abundance compared with cells cultured in 100 mg/dl D-glucose. IGF-1 also increased laminin C1 mRNA abundance in RMC or MES-13 cells, whereas TGF-beta1 was without effect. The influence of raising the medium glucose concentration on laminin C1 promoter activity was further studied in MES-13 cells that had been stably transfected with a reporter gene containing the promoter linked to luciferase. Culture in 500 mg/dl D-glucose for 4 h to at least 1 week increased laminin C1 promoter activity compared with cells maintained in 100 mg/dl glucose. In contrast, culture of cells in medium that contained 400 mg/dl mannitol or 400 mg/dl L-glucose in addition to 100 mg/dl D-glucose did not increase laminin C1 promoter activity. The ability of high glucose to increase laminin C1 promoter activity was absolutely dependent on the presence of serum. Consistent with results obtained with mRNA, TGF-beta1 had no influence on promoter activity in stable integrants. Whereas IGF-1 transiently increased promoter activity in stable integrants, the increase was not sustained (6 h). Moreover, neutralizing antibody to TGF-beta or to IGF-1 receptor did not suppress increases in laminin C1 promoter activity induced by culture of stable integrants in high glucose. Several inhibitors of protein kinase C, including bisindolylmaleimide (GFX), myristoylated PKC inhibitor peptide, and LY333531, were also without effect on increases in laminin C1 promoter activity induced by culture in high glucose. Exposure to the NO donor (+/-)-s-nitroso-n-acetylpenicillamine (SNAP) blocked increases in laminin C1 promoter activity induced by serum and by culture in high glucose without influencing promoter activity in cells cultured in the absence of serum and in 100 mg/dl glucose. The ability of high glucose concentrations and IGF-1 to increase laminin C1 promoter activity in cultured mesangial cells, and the suppression of glucose actions by the NO donor SNAP, provide potential mechanisms whereby the synthesis of the laminin gamma1 chain may be regulated in the glomerulus in diabetes. Of note, the mechanism by which high glucose increases laminin C1 promoter activity appears to differ from mechanisms previously described for some other glucose actions on matrix protein synthesis. In this regard, TGF-beta and protein kinase C were not implicated as mediators of the effect of high glucose on laminin C1 promoter activity.

Effects of supplementation with vitamin C or E on albuminuria, glomerular TGF-beta, and glomerular size in diabetes.

Oxidant stress and a reduction in antioxidant status, including reduced plasma and tissue ascorbic acid content, occur in diabetic patients and experimental models of diabetes. In this study, the effects of treatment of streptozotocin diabetic rats for 2 mo with vitamin C (10 g/kg body wt/d) or dietary vitamin E (200 mg/kg body wt/d) in the drinking water on urinary albumin excretion, glomerular transforming growth factor (TGF)-beta content, and glomerular size were examined. Treatment of diabetic rats with vitamin C or E had no effect on blood glucose levels compared with that in untreated diabetics (453 +/- 28 g/dl +/- SEM). Body weight, BP, and creatinine clearance rates were not significantly different among the study groups. Kidney weight was significantly higher in all of the diabetic groups compared with age-matched control rats. Treatment with vitamin C, but not vitamin E, significantly reduced kidney weight compared with that in untreated diabetic rats. Immunohistochemical staining for TGF-beta was 2.5-fold higher in glomeruli of cortical sections from untreated diabetic rats versus control rats. Treatment with vitamin C or E prevented the increase in glomerular TGF-beta immunoreactivity. Glomerular volume was also significantly increased (twofold) in kidneys of untreated diabetic rats compared with control rats, as assessed by light microscopy. Treatment with vitamin C prevented and treatment with vitamin E reduced the increase in glomerular volume. Treatment with vitamin C also prevented the sevenfold increase in albumin clearance otherwise seen in untreated diabetic rats. By contrast, treatment with vitamin E had no effect on albumin clearance despite reductions in glomerular size and TGF-beta. Renal cortical vitamin E and plasma, but not renal cortical vitamin C, were reduced in diabetic rats versus control rats. Supplementation of diabetic rats with vitamin C markedly increased plasma and renal cortical vitamin C content to values greater than those in control rats. Supplementation with vitamin E increased renal cortical vitamin E content by 50% compared with values in control rats and also increased plasma and renal cortical vitamin C. These results support the potential utility of antioxidant treatment for the prevention of renal injury in diabetes.

Antioxidant inhibition of protein kinase C-signaled increases in transforming growth factor-beta in mesangial cells.

Protein kinase C (PKC)-signaled increases in transforming growth factor beta (TGF beta) have been implicated in the stimulation of matrix protein synthesis induced by high concentrations of glucose, thromboxane, angiotension II (AII), and other stimuli in cultured glomerular mesangial cells. In the present study, the effects of several antioxidants on mesangial cell responses to high glucose, thromboxane, and AII were examined. alpha-Tocopherol blocked increases in PKC, TGF beta bioactivity, collagen, and/or fibronectin synthesis induced in mesangial cells by high glucose, the thromboxane analog U46619, and AII. By contrast, alpha-tocopherol did not alter increases in matrix protein synthesis in mesangial cells in response to exogenous TGF beta, a cytokine that does not activate PKC in mesangial cells and whose actions to stimulate matrix protein synthesis in these cells are not blocked by PKC inhibition or downregulation. Taurine and N-acetylcystein similarly inhibited activation of PKC and increases in TGF beta in response to high glucose, U46619, and AII. alpha-Tocopherol but not taurine or N-acetylcysteine partially blocked increases in PKC activity in mesangial cells in response to the diacylglycerol (DAG) analog, phorbol dibutyrate (PDBu). Thus, alpha-tocopherol may have direct effects on interaction of the PKC system of mesangial cells with DAG that are not shared by N-acetylcysteine or taurine. Increases in TGF beta have been implicated in the pathogenesis of glomerulosclerosis in diabetes and other nephropathies. The capacity of antioxidants to block increases in TGF beta in mesangial cells in response to high glucose, thromboxane, and All suggests their potential therapeutic utility to attenuate glomerulosclerosis.

Nitric oxide inhibition of transforming growth factor-beta and collagen synthesis in mesangial cells.

Culture of mesangial cells (MCs) in 5.6 vs. 30.0 mmol/l glucose for 3 weeks induced a sustained increase in protein kinase C (PKC) activity, transforming growth factor (TGF)-beta1 mRNA, bioactive TGF-beta, and collagen synthesis. Nitric oxide (NO), generated exogenously by the NO donor S-nitroso-N-acetyl, D,L-penicillamine (SNAP) or endogenously after the exposure of MC to interleukin-1beta (IL-1beta), suppressed bioactive TGF-beta in MCs cultured in 5.6 or 30.0 mmol/l glucose and suppressed or abolished increases in TGF-beta1 mRNA and collagen synthesis induced by high concentrations of glucose or phorbol 12,13-dibutyrate without altering values obtained with normal glucose concentrations. SNAP had a transient suppressive effect on PKC activity, which may explain at least in part some of the actions of SNAP. The selective inhibitor of PKC, bisindolylmaleimide (GFX), mimicked NO action. The ability of SNAP and IL-1beta to suppress TGF-beta and collagen synthesis was not mediated by cGMP, since the cGMP analog, 8-Br-PET-cGMP, did not mimic NO action and an antagonist of cGMP-dependent protein kinase, Rp-8-pCPT-cGMPs, did not prevent the inhibitory actions of SNAP. N-omega-L-arginine methyl ester (NMMA) increased TGF-beta in glomerular capillary endothelial cells (GCECs) and stimulated collagen synthesis by MC in a co-culture with GCECs. Captopril inhibited TGF-beta and collagen synthesis and increased cGMP in co-cultures of GCECs and MCs. These effects of captopril were abolished by NMMA, implying mediation by NO. Thus, endogenous NO produced by GCECs may modulate TGF-beta production by both GCECs and MCs and act to suppress matrix protein synthesis by MCs.

Thromboxane/prostaglandin endoperoxide-induced hypertrophy of rat vascular smooth muscle cells is signaled by protein kinase C-dependent increases in transforming growth factor-beta.

In the present study, we examined the effect of the thromboxane/prostaglandin endoperoxide analogue U46619 on proliferation and hypertrophy in cultured rat vascular smooth muscle cells and the roles of protein kinase C and transforming growth factor-beta (TGF-beta) in the mediation of the hypertrophic response to U46619. Since an increase in basic fibroblast growth factor (bFGF) was previously shown to mediate the hypertrophic response to U46619, we also assessed the relationship between bFGF and TGF-beta in the expression of U46619 actions. U46619 increased [35S]methionine incorporation into protein and protein content of vascular smooth muscle cells but had no effect on cell number. A role for TGF-beta was supported by the following observations: (1) exogenous human TGF-beta 1 increased protein synthesis; (2) antibody to TGF-beta blocked both TGF-beta- and U46619-induced increases in protein content; (3) U46619 increased active and total TGF-beta bioactivities; and (4) the actions of U46619 on protein content and TGF-beta bioactivity were blocked by the thromboxane/prostaglandin endoperoxide receptor antagonist SQ 29,548. Previous observations had demonstrated a role for bFGF in the expression of U46619 actions on protein synthesis. Results of the present study suggest that TGF-beta and bFGF interact in mediating the protein synthetic response to U46619. First, the concentration of exogenous TGF-beta (10 pmol/L) alone required to produce a protein synthetic response equivalent to that induced by U46619 was much higher than the concentration of endogenous active TGF-beta that accumulated in the media in response to U46619 (0.7 pmol/L). Second, bFGF (20 ng/mL) increased total TGF-beta bioactivity and stimulated protein synthesis. The hyper-trophic response to bFGF was blocked by anti-TGF-beta. The ability of U46619 and bFGF to increase protein synthesis and protein content in vascular smooth muscle cells was associated with TGF-beta-induced suppression of proliferation, as evidenced by the ability of antibody to TGF-beta to enhance U46619- and bFGF-induced increases in [3H]thymidine incorporation into DNA. Results of the present study also supported a role for protein kinase C in the expression of U46619 and bFGF actions. U46619 increased protein kinase C activity in the particulate fraction of vascular smooth muscle cells. Moreover, the protein kinase C inhibitors GF109203X and staurosporine blocked U46619- and bFGF-induced increases in protein synthesis as well as active and total TGF-beta bioactivities. By contrast, the protein kinase C inhibitors did not prevent the increases in protein synthesis induced by exogenous TGF-beta. The results demonstrate that thromboxane/prostaglandin endoperoxide signals increased TGF-beta bioactivity via protein kinase C. Increases in both bFGF and TGF-beta are required for an optimal hypertrophic response to U46619. The hypertrophic response to TGF-beta occurs through a protein kinase C-independent pathway.

Nitric oxide suppresses increases in mesangial cell protein kinase C, transforming growth factor beta, and fibronectin synthesis induced by thromboxane.

Thromboxane (TX) stimulation of fibronectin (FN) synthesis in mesangial cells (MC) is dependent on protein kinase C (PKC)-mediated increases in transforming growth factor beta (TGF beta), and is suppressed by increases in cellular cGMP. The current studies evaluate the role of cGMP-dependent and -independent actions of nitric oxide (NO) in modulating the responses of MC to the TX analogue U46619. TX-stimulated increases in PKC activity, TGF beta, and FN synthesis in MC were suppressed by either 8-Br-PET-cGMP or the NO donor S-nitroso-N-acetylpenicillamine (SNAP). By contrast, NO, but not cGMP, inhibited basal PKC activity, TGF beta bioactivity and FN synthesis. The cGMP-dependent protein kinase 1-alpha inhibitor 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphorothioate (Rp) restored the PKC, TGF beta, and the FN synthetic responses to TX when added to MC before exposure of the cells to either cGMP or SNAP. However, neither Rp nor the guanylate cyclase inhibitor Ly83583 significantly altered SNAP inhibition of basal PKC. In addition, Rp failed to alter the decreases in basal TGF beta bioactivity and FN synthesis seen in the presence of SNAP. In contrast to the FN response to U46619, cGMP and SNAP did not affect the stimulation of FN synthesis by exogenous TGF beta. The later findings are consistent with inhibitory actions of NO and cGMP at, or proximal to, U46619-induced increases in TGF beta in the suppression of TX-signaled increases in FN synthesis. Thus, NO depresses basal PKC and TGF beta bioactivity in MC by mechanisms that are largely independent of cGMP, whereas NO inhibition of these MC responses to TX is mediated primarily by increases in cGMP and activation of protein kinase 1-alpha.

Protein kinase C in diabetic nephropathy.

Protein kinase C is activated in numerous tissues obtained from diabetic animals and in several cultured cell systems exposed to high media glucose in vitro including glomerular mesangial cells. Several activators of protein kinase C, such as high media glucose, angiotensin II, phorbol ester, low density lipoprotein, and the thromboxane analogue U-46619, increase TGF beta bioactivity or mRNA expression and increase the synthesis of extracellular matrix proteins by mesangial cells in culture. The studies described in the present report support the hypothesis that activation of protein kinase C by thromboxane, an eicosanoid whose production is known to be elevated in diabetes, increases TGF beta production by mesangial cells in culture. TGF beta then acts to increase extracellular matrix protein synthesis through a mechanism that does not require active protein kinase C. Thus, activation of protein kinase C in the glomerulus in diabetes could contribute to mesangial expansion by stimulating active TGF beta production.

Protein kinase C signals thromboxane induced increases in fibronectin synthesis and TGF-beta bioactivity in mesangial cells.

Previous studies have demonstrated that thromboxane (TX) stimulates matrix protein synthesis in mesangial cells (MC), and that this action is signalled by receptor mediated activation of protein kinase C (PKC). In the present study, we examined the hypothesis that activation of PKC by TX signals increases in transforming growth factor beta (TGF-beta) bioactivity, which in turn induces enhanced matrix protein synthesis. In cultured rat MC, the TXA2/prostaglandin endoperoxide analogue U-46619, but not exogenous human platelet TGF-beta 1, activated PKC as reflected by enhanced in situ phosphorylation of MARCKS protein, an endogenous substrate of PKC. U-46619 and TGF-beta 1 stimulated fibronectin (Fn) synthesis in MC, as shown by [35S]methionine incorporation into immunoprecipitable Fn. Pan-specific rabbit anti-TGF-beta antibody blocked the increases in Fn synthesis induced by exogenous TGF-beta and those induced by U-46619 at 24 to 72 hours after addition. Anti-TGF-beta antibody did not block the small increases in FN synthesis observed six hours after addition of U-46619, suggesting that this acute response was not dependent on TGF-beta. Anti-TGF-beta antibody also failed to block activation of PKC by U-46619. U-46619 and 50 nM of the PKC agonist phorbol dibutyrate (PDBu) significantly increased both the active fraction and total (latent plus active) TGF-beta in MC culture media, as assayed with the mink lung epithelial cell bioassay system.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired nitric oxide release by glomeruli from diabetic rats.

Basal nitric oxide (NO) production and NO responses to carbamylcholine (CCh) and the Ca2+ ionophore A23187 were measured with a NO electrode in glomeruli isolated from 2 to 3-month-diabetic versus age-matched control rats. In the presence of CCh or A23187, NO production was markedly reduced in glomeruli from diabetic versus control rats. Spontaneous generation of NO by s-nitrosopenicillamine (SNAP) was also reduced in the presence of glomeruli from diabetic rats as compared with values either in control glomeruli or in buffer alone. The results demonstrate an impairment of NO generation and/or stability in glomeruli from 2 to 3-month-diabetic rats, which correlates with the previously observed suppression of NO-dependent glomerular cyclic guanosine 3',5'-monophosphate (cGMP) induced by diabetes.

Role for transforming growth factor beta in thromboxane-induced increases in mesangial cell fibronectin synthesis.

Thromboxane A2 (TXA2) has been implicated in the pathogenesis of progressive glomerulosclerosis and stimulates the synthesis of matrix protein by mesangial cells (MCs). This study examined the role of transforming growth factor beta (TGF-beta) in the mediation of the action of the stable TXA2/prostaglandin (PG) endoperoxide analog U-46619 to stimulate fibronectin (Fn) synthesis in cultured rat MC. Exogenous TGF-beta increased Fn synthesis by MC in a concentration- and time-dependent fashion, as reflected by incorporation of [35S]methionine into immunoprecipitable Fn. Submaximal concentrations of TGF-beta (1-2.5 pmol/l) increased Fn synthesis two- to threefold, a response comparable in magnitude to that observed with a maximal stimulatory concentration (1 mumol/l) of U-46619. Anti-TGF-beta antibody, but not isotypic IgG, blocked the increases in Fn synthesis induced by both U-46619 and exogenous TGF-beta. Endogenous TGF-beta bioactivity in MC culture media, assessed by the mink lung epithelial cell system, was significantly increased by 1 mumol/l U-46619 (1.7 +/- 0.3 pmol/l) compared with that of control media (0.6 +/- 0.1 pmol/l, P < 0.05). Total (active plus latent) TGF-beta bioactivity, assayed after heat activation of latent TGF-beta, was also significantly higher in media of MCs cultured with U-46619 (45 +/- 4 pmol/l) compared with control (24 +/- 4 pmol/l). Thus, U-46619 increased endogenous TGF-beta bioactivity to a level sufficient to account for the enhancement of Fn synthesis observed with U-46619, as reflected by the Fn synthetic response to exogenous TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Low-density lipoprotein stimulation of mesangial cell fibronectin synthesis: role of protein kinase C and transforming growth factor-beta.

Low-density lipoprotein (LDL) cholesterol has been implicated in the pathogenesis of glomerulosclerosis in diabetes and other forms of glomerular injury. In the present study we evaluated the effect of LDL on fibronectin synthesis in cultured rat mesangial cells (MCs) and the roles of protein kinase C (PKC) and transforming growth factor-beta (TGF-beta) in mediating this LDL action. In MCs, 25 micrograms to 100 micrograms/ml LDL increased PKC activity within 15 minutes, as reflected by enhanced in situ phosphorylation of the 80 kd myristoylated alanine-rich C kinase substrate protein, a specific endogenous substrate of PKC in MC. The same concentrations of LDL subsequently (18 to 72 hours) enhanced fibronectin synthesis, as reflected by increased incorporation of labeled methionine into fibronectin. GF 109203X, a selective inhibitor of PKC, blocked increases in both PKC activity and fibronectin synthesis induced by LDL in MCs. Furthermore, prior downregulation of PKC to less than 1% of basal activity by exposure of MCs to 0.5 mumol/L phorbol myristate acetate (PMA) also prevented LDL stimulation of fibronectin synthesis. The activation of PKC by LDL seen after 15 minutes of exposure was transient and was not observed after 4 or 48 hours of exposure of MCs to LDL. However, exposure to LDL for 48 hours, but not for 15 minutes or 4 hours, increased both maximal PKC responses to phorbol dibutyrate (PDBu) and tritiated PDBu binding to MCs by 30%. These findings suggest that chronic exposure to LDL increases the total PKC content in MCs and thereby might modulate responses to other PKC agonists. Neither the cyclooxygenase inhibitor piroxicam nor the thromboxane/prostaglandin endoperoxide receptor blocker Sq-29548 altered LDL stimulation of fibronectin synthesis in MCs, suggesting that this action of LDL was not mediated by changes in MC eicosanoid generation. By contrast, antibody to TGF-beta blocked LDL stimulation of fibronectin synthesis in MCs. TGF-beta bioactivity, determined with the mink lung epithelial cell assay, was two to three times higher in the medium of MCs cultured with LDL for 24 to 48 hours as compared with corresponding control values. Total TGF-beta bioactivity examined after heat activation of latent TGF-beta was also two times higher in the medium of MCs exposed to LDL as compared with that of controls. Prior down-regulation of PKC by exposure of MCs to PMA blocked the increases in TGF-beta bioactivity induced by LDL.(ABSTRACT TRUNCATED AT 400 WORDS)

Thromboxane stimulation of mesangial cell fibronectin synthesis is signalled by protein kinase C and modulated by cGMP.

Thromboxane (TX) has been implicated in the pathogenesis of glomerulosclerosis in several models of glomerular injury. In the present study, we examined the role of the protein kinase C (PKC) signalling system in expression of the action of the TXA2/PGH2 analogue U-46619 to stimulate fibronectin (Fn) synthesis in cultured rat mesangial cells (MC), and the influence of cGMP on this MC response. U-46619 activated PKC and enhanced Fn synthesis in MC in a time and concentration dependent fashion. Both responses to U-46619 were blocked by GF 109203X, a selective inhibitor of PKC activity, as well as by calphostin C and staurosporine, PKC inhibitors structurally distinct from GFX. Down-regulation of PKC by prior sustained exposure of MC to 0.5 microM phorbol myristate acetate similarly blocked increases in Fn synthesis induced by U-46619. The TXA2/PGH2 receptor antagonist Sq-29548 also prevented activation of PKC and stimulation of Fn synthesis by U-46619, consistent with transduction of these responses via specific high affinity TXA2/PGH2 receptors on MC. Addition of exogenous 8-Br-cGMP or stimulation of endogenous cGMP generation with atrial natriuretic peptide (ANP) suppressed both U-46619 activation of PKC and stimulation of Fn synthesis. cGMP did not alter TXA2/PGH2 receptor number of affinity in MC, but significantly suppressed phorbol ester activation of PKC. Thus, cGMP inhibition of U-46619 actions is expressed at steps distal to TX receptor binding and may involve effects at and proximal to activation of PKC. Interactions between the PKC and cGMP cellular signalling systems may be important determinants of MC matrix protein production in response to TX.

Loss of protein kinase C delta isozyme immunoreactivity in human adenocarcinomas.

Protein kinase C (PKC) has been implicated in the control of colonic epithelial proliferative activity and in the process of malignant transformation. In the present study, we assessed by histone IIIS phosphorylation in vitro, total PKC activity, and the subcellular distribution of this activity in human adenocarcinomas and surrounding uninvolved mucosa from six patients. In these same tissues, we also examined the isozyme profile of PKC by immunoblotting. Total PKC activity and the subcellular distribution of PKC activity was not significantly different in mucosa compared to corresponding values in the tumors. Extracts of both human mucosa and tumors reacted with antibody to PKC isozymes alpha, beta, delta and zeta but did not react with antibody to the gamma and epsilon isozymes. The antibodies employed were directed against rabbit brain PKC (alpha, beta, gamma) or peptide sequences deduced from rat cDNA (gamma, delta, epsilon, and zeta). Accordingly, the apparent absence of the epsilon isozyme in human mucosa and adenocarcinoma may be due to failure to conserve the relevant sequence rather than to loss of the isozyme per se. No statistically significant differences were noted in subcellular distribution of any of the isozymes in the tumors compared to mucosa. However, the subcellular distribution of the delta isozyme was highly variable in the tumors. Total PKC beta immunoreactivity and that of the soluble, but not particulate, fraction were both significantly lower in homogenates of adenocarcinomas compared to corresponding values in surrounding mucosa, when expressed as a function of protein. However, these differences in PKC beta were abolished when results were expressed as a function of tissue DNA content.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired nitric oxide-dependent cyclic guanosine monophosphate generation in glomeruli from diabetic rats. Evidence for protein kinase C-mediated suppression of the cholinergic response.

Nitric oxide (NO)-dependent cyclic guanosine monophosphate (cGMP) generation was examined in glomeruli isolated from 1-2-wk and 2-mo streptozotocin diabetic (D) and control (C) rats. After 1-2 wk of diabetes, ex vivo basal cGMP generation and cGMP responses to carbamylcholine (CCh) were significantly suppressed in glomeruli from D compared with those from C, whereas cGMP responses to the calcium ionophore A23187 and nitroprusside (NP) did not differ in glomeruli from D vs. those from C. After 2 mo, glomeruli from D did not respond to CCh, and responses to A23187 and NP were suppressed compared with those from C. Differences in basal, CCh, and A23187-responsive cGMP between D and C were abolished by the NO synthetase inhibitor NG-monomethyl-L-arginine. Soluble glomerular guanylate cyclase prepared from either D or C responded indistinguishably to NP, suggesting a role for NO quenching in the suppression of cGMP in intact glomeruli from D. Compared with those from C, glomeruli isolated from D demonstrated increased generation of thromboxane A2 (TXA2) and activation of protein kinase C (PKC). Both the TXA2/endoperoxide receptor antagonist Bay U3405 and inhibitors of PKC activity restored a cGMP response to CCh in glomeruli from D. Conversely, in glomeruli from C, the TXA2/endoperoxide analogue U46619 activated PKC and suppressed the cGMP response to CCh. Both of those actions were blocked by inhibitors of PKC. The results indicate a progressive impairment of NO-dependent cGMP generation in glomeruli from D which may be mediated in part by TXA2 and activation of PKC. This impairment may participate in glomerular injury in diabetes.

Activation of protein kinase C in glomerular cells in diabetes. Mechanisms and potential links to the pathogenesis of diabetic glomerulopathy.

Protein kinase C (PKC) is activated in rat renal glomerulus within a week of induction of experimental diabetes. Studies in isolated glomeruli and in cultured endothelial and mesangial cells have demonstrated that high ambient concentrations of glucose activate PKC and thus implicate hyperglycemia per se as a mediator of PKC activation in glomerular cells in diabetes. High glucose concentrations activate PKC by increasing cellular levels of diacylglycerol (DAG), the major endogenous modulator of this signalling system. In contrast to physiological extracellular stimuli of PKC that increase cellular DAG levels by receptor-mediated enhancement of membrane inositol phospholipid hydrolysis, in glomerular cells high concentrations of glucose increase DAG by de novo synthesis from glycolytic intermediates. Activation of PKC by glucose or other agonists increases the permeability of endothelial cells to albumin and stimulates matrix protein synthesis in mesangial cells; it thereby may be involved in the pathogenesis of both the functional and structural alterations of the glomerulus in diabetes. Recent studies in isolated glomeruli from diabetic rats have also implicated activation of PKC in suppression of nitric oxide (NO)-mediated increases in glomerular cGMP generation in response to cholinergic stimuli. In mesangial cells, cGMP suppresses PKC-mediated increases in matrix protein synthesis. Thus, impaired NO-mediated cGMP generation in glomeruli of diabetic individuals may amplify matrix protein synthesis in response to hyperglycemia and other stimuli of PKC. These and other observations suggest that activation of the PKC system by hyperglycemia may represent an important pathway by which glucotoxicity is transduced in susceptible cells in diabetes.

Eicosanoids in the pathogenesis of the functional and structural alterations of the kidney in diabetes.

Diabetes mellitus alters the cellular production of eicosanoids in a number of tissues, including the kidney, and these agents have in turn been implicated in the pathogenesis of diabetic nephropathy. As delineated in the streptozotocin diabetic rat (SDR) model, a preferential enhancement of glomerular synthesis of the vasodilatory prostaglandins (PGs) PGE2 and PGI2 with concurrent smaller increases in thromboxane (TX)A2 occurs within 1 week after induction of diabetes. This early alteration in glomerular synthesis of eicosanoids in the SDR has been linked to glucose-induced activation of the glomerular protein kinase C signalling system that enhances phospholipase A2 activity and, therefore, release of membrane-bound arachidonic acid for oxygenation. The preferential increase in glomerular production of vasodilatory PGs may contribute to the glomerular hyperfiltration that is characteristic of early diabetes. After more prolonged (months) diabetes in the SDR, glomerular generation and urinary excretion of thromboxane (TX) are preferentially enhanced. Studies with selective inhibitors of TX synthesis in the SDR have implicated this eicosanoid in the pathogenesis of both albuminuria and glomerular structural changes (basement membrane thickening and mesangial matrix expansion). Direct stimulation of matrix protein production has been demonstrated in cultured mesangial cells in response to both TX and high ambient concentrations of glucose. The actions of TX and glucose on mesangial cell matrix production appear to be interactive, with each signalled through distinct pathways of protein kinase C activation.