Chronic exposure of human mesangial cells to high glucose environments activates the p38 MAPK pathway.

BACKGROUND: High glucose (HG) environments activate several protein kinase pathways in mesangial cells, including the mitogen-activated protein kinase (MAPK) pathway, ERK. The p38 MAPK pathway is activated by events that occur in the setting of diabetes, such as protein kinase C (PKC) up-regulation and cellular stresses (osmotic stress and redox changes). Substrates of activated p38 MAPK include transcription factors that are involved in the microvascular complications of diabetes. This current study investigated the mechanisms of HG-mediated activation of p38 MAPK in cultured human mesangial cells (HMCs) and the effects of p38 MAPK activation on the transcription factor activator protein-1 (AP-1). METHODS: HMCs were cultured in 5 mmol/L D-glucose [normal glucose (NG)] or 30 mmol/L D-glucose (HG) for seven days. Cells were also treated with HG for brief periods of time (0.5 to 4 hours) to assess the acute effects of HG on p38 MAPK. Using Western blotting of HMC lysates, changes in the tyrosine and threonine phosphorylation of p38 MAPK were measured. The kinase activity of immunoprecipitated p38 MAPK was determined by an in vitro assay that measured the phosphorylation and activation of MAPKAP kinase-2, an intermediary signaling protein downstream of p38 MAPK. To investigate the role of osmotic stress in HG activation of p38 MAPK, cells were acutely treated with mannitol (25 to 250 mOsm/L x 5 to 60 min) or were grown seven days in media supplemented with mannitol at concentrations iso-osmotic to HG media. To investigate the role of PKC in HG-mediated p38 MAPK activation, HMCs were treated with the PKC inhibitors GF 109203X, Ro 32-0432, or rottlerin during the last several hours of HG treatment. HG conditioned cells were also treated with the antioxidants L-N-acetylcysteine (L-NAC) or diphenyliodonium (DPI) prior to harvest. To determine a functional significance of HG-mediated p38 MAPK activation, the DNA binding of the transcription factor complex AP-1 was measured by electrophoretic mobility shift assay. RESULTS: The p38 MAPK pathway was not activated by the acute addition of HG to the HMCs. However, activation of p38 MAPK in HMCs grown seven days in HG was demonstrated by increased tyrosine and threonine phosphorylation of p38 MAPK proteins and increased kinase activity of immunoprecipitated p38 MAPK. As assessed by a kinase assay, p38 MAPK activity in cells grown in HG for seven days exceeded that of NG cells by more than 250%. This difference was not due to differences in the amount of p38 MAPK protein between the treatment groups. Acute osmotic activation of p38 MAPK occurred at extremely high mannitol concentrations (250 mOsm/L) that exceeded the osmotic stress of acute HG. Furthermore, in cells grown for seven days in mannitol at concentrations similar to HG, p38 MAPK activity was similar to control values. Phorbol ester (PMA) treatment stimulated a twofold increase in p38 MAPK activity. The addition of GFX or Ro 32-0432 to HG cells, at concentrations that inhibited PMA activation of p38 MAPK, did not inhibit the glucose-mediated p38 MAPK activation. Rottlerin, a PKC delta inhibitor, also failed to reverse the HG-mediated p38 MAPK activation. Treatment of HG cells with L-NAC or DPI inhibited the HG-mediated p38 MAPK phosphorylation. As we have previously shown, DNA binding of the transcription factor complex AP-1 was increased in HG cells. This binding was reversed by treatment of the HG cells with the p38 MAPK inhibitor SB 203580. CONCLUSIONS: Chronic exposure of HMC to HG concentrations activates the p38 MAPK pathway. This activation appears to be independent of changes in the amount of total p38 MAPK produced by the cells, independent of chronic osmotic stress and independent of PKC activation. The reversal of p38 MAPK by L-NAC and DPI suggests the glucose-mediated p38 MAPK activation may occur via reactive oxygen species. The activity of AP-1, a transcription factor complex that regulates several genes involved in diabetic nephropathy, is reversed when the p38 MAPK pathway is inhibited. These findings suggest the p38 MAPK pathway may be an important pathway involved in diabetic complications.

Transcutaneous oxygen tension in patients with calciphylaxis.

Calciphylaxis is a severe complication of chronic renal failure, confined almost exclusively to patients on dialysis therapy. Histological characteristics of calciphylaxis include small-vessel calcifications of skin, subcutaneous tissue, and visceral organs. These vascular changes promote tissue ischemia that often results in tissue necrosis. In this study, we investigated the extent of skin ischemia in patients with calciphylaxis by means of transcutaneous oxygen tension (TCPO(2)) measurement, a noninvasive test that accurately assesses skin oxygenation. TCPO(2) levels were measured in 21 patients with calciphylaxis and 21 age- and sex-matched patients without evidence of calciphylaxis (controls). TCPO(2) levels were measured bilaterally at the chest, anterior abdomen, and upper thigh while patients breathed room air and after a 30-minute exposure to 100% fraction of inspired oxygen (FIO(2)). Compared with controls, patients with calciphylaxis showed significantly lower TCPO(2) levels at each body region. In both controls and patients with calciphylaxis, lower TCPO(2) levels correlated with increased weight and use of hemodialysis. No correlation with serum parathyroid hormone (PTH), serum calcium, or serum phosphorus values was present, although 39% of the patients with calciphylaxis had markedly elevated PTH values (sixfold greater than normal; >300 pg/dL). Low TCPO(2) levels in patients with calciphylaxis were documented in body regions with and without skin lesions. In patients with calciphylaxis, extremely low TCPO(2) values (

Renoprotection: one or many therapies?

BACKGROUND: Renal disease that progresses to end-stage renal disease (ESRD) imposes a great burden on the affected individual and on society, which mainly bears the cost of ESRD (currently more than $10 billion to treat about 333,000 patients annually in the U.S.). Thus, there is a great need to identify therapies that arrest the progression mechanisms common to all forms of renal disease. Progress is being made. Perhaps the most visible advance is the randomized controlled trials (RCT) demonstrating the renoprotective effects of angiotensin-converting enzyme (ACE) inhibitors. There are also numerous other promising renoprotective therapies. Unfortunately, testing each therapy in RCT is not feasible. Thus the nephrologist has two choices: restrict renoprotective therapy to those shown to be effective in RCT, or expand the use of renoprotective therapies to include those that, although unproven, are plausibly effective and prudent to use. The goal of this work is to provide the documentation needed for the nephrologist to choose between these strategies. METHODS: This work first describes the mechanisms believed to be involved in the progression of renal disease. Based largely on this information, 18 separate interventions that slow the progression are described. Each intervention is assigned a level of recommendation (Level 1 is the highest and Level 3 the lowest) according to the strength of evidence supporting its renoprotective efficacy. RESULTS: The number of interventions at each level of recommendation are: Level 1, N = 4; Level 2, N = 4; Level 3, N = 10. Our own experience with the multiple-risk-factor intervention is that most patients can achieve the majority of the Level 1 and 2 interventions, and many of the Level 3 interventions. We recommend the expanded renoprotection strategy. CONCLUSION: This work advances the hypothesis that, until better information becomes available, a broad-based, multiple-risk-factor intervention intended to slow the progression of renal disease can be justified in those with progressive nephropathies. This work is intended primarily for clinical nephrologists and thus each recommended intervention is described in substantial practical detail.

A cyclopentenone prostaglandin activates mesangial MAP kinase independently of PPARgamma.

The mitogen-activated protein (MAP) kinases mediate the response of renal glomerular mesangial cells to a variety of physiologic and pathologic stimuli. This investigation examines the effect of the cyclopentenone prostaglandin 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) on MAP kinases in human mesangial cells. We show that 15d-PGJ2 dose-dependently increases the extracellular signal-regulated kinase (ERK) activity of human mesangial cells, but has no effect on Jun-NH2-terminal kinase or p38 MAP kinase. Despite the fact that 15d-PGJ2 is a peroxisome proliferator-activated receptor (PPAR) ligand, and PPARgamma is shown to be expressed by mesangial cells, the thiazolidinedione PPARgamma agonist ciglitazone does not activate ERK. Additionally, a synthetic PPARgamma antagonist does not attenuate the activation of ERK by 15d-PGJ2. 15d-PGJ2-mediated ERK activation is however blocked by the MEK inhibitor PD 098059, appears to require phosphatidylinositol-3 kinase, but is independent of protein kinase C activation. These results demonstrate a novel effect of 15d-PGJ2 to induce ERK in human mesangial cells independently of PPARgamma.

Pauci-immune ANCA-positive crescentic glomerulonephritis associated with metastatic adenocarcinoma of the lung.

A 66-year-old woman developed rapidly progressive renal failure several days after she was diagnosed with non-small cell carcinoma of the lung. Antineutrophil cytoplasmic antibody test performed as an indirect immunofluorescence assay was positive with a perinuclear pattern of staining (pANCA). The patient did not improve with hemodialysis treatment and died on the second day after admission to the hospital. A complete autopsy was performed and showed metastatic adenocarcinoma of the lung and pauci-immune crescentic glomerulonephritis. A literature search showed only 7 previously reported cases of malignant tumors associated with ANCA-positive pauci-immune crescentic glomerulonephritis. The clinicopathologic findings of the current and all previously reported cases and possible relationship between ANCA-positive glomerulonephritis and malignancy are discussed.

Remission of nephrotic syndrome in type 1 diabetes: long-term follow-up of patients in the Captopril Study.

In 1994, we reported a 3.4 +/- 0.8 year follow-up of the eight patients who experienced remission of nephrotic syndrome during the Collaborative Study Group-sponsored, multicenter trial of captopril therapy in patients with type 1 diabetes with nephropathy (Captopril Study). Of the 409 patients randomized to treatment on the Captopril Study, 108 had nephrotic syndrome (24-hour proteinuria >/= 3.5 g of protein) at baseline. Of these 108 patients, 8 experienced remission of nephrotic syndrome (proteinuria

The mitogen-activated protein kinase p38 is necesssary for interleukin 1beta-induced monocyte chemoattractant protein 1 expression by human mesangial cells.

Mitogen-activated protein (MAP) kinases have been suggested as potential mediators for interleukin 1beta (IL-1beta)-induced gene activation. This study investigated the role of the MAP kinases p38 and ERK2 in IL-1beta-mediated expression of the chemokine MCP-1 by human mesangial cells. Phosphorylation of p38 kinase, which is necessary for activation, increased significantly after IL-1beta treatment. p38 kinase immunoprecipitated from IL-1beta-treated cells phosphorylated target substrates to a greater extent than p38 kinase from controls. SB 203580, a selective p38 kinase inhibitor, was used to examine the role of p38 kinase in MCP-1 expression. SB 203580 decreased IL-1beta-induced MCP-1 mRNA and protein levels, but did not affect MCP-1 mRNA stability. Because NF-kappaB is necessary for MCP-1 gene expression, the effect of p38 kinase inhibition on IL-1beta induction of NF-kappaB was measured. SB 203580 (up to 25 microM) had no effect on IL-1beta-induced NF-kappaB nuclear translocation or DNA binding activity. Our previous work showed that IL-1beta also activates the MAP kinase ERK2 in human mesangial cells. PD 098059, a selective inhibitor of the ERK activating kinase MEK1, had no effect on IL-1beta-induced MCP-1 mRNA or protein levels, or on IL-1beta activation of NF-kappaB. These data indicate that p38 kinase is necessary for the induction of MCP-1 expression by IL-1beta, but is not involved at the level of cytoplasmic activation of NF-kappaB. In contrast, ERK2 does not mediate IL-1beta induced MCP-1 gene expression.

Receptors for the anaphylatoxin C5a (CD88) on human mesangial cells.

In these studies, we determined whether there are receptors for the anaphylatoxin C5a (C5aR, CD88) on human mesangial cells (HMC). To prepare Abs to C5aR, we first synthesized an immunogenic peptide spanning residues 8-32 of the molecule, and this peptide was used to immunize rabbits. Anti-C5aR antiserum, but not preimmune serum, stained fixed and unfixed HMC in culture. By Western blotting anti-C5aR, Abs identified a 49.6-kDa protein in HMC. By reverse-transcription PCR, a cDNA product of 558 bp was amplified corresponding to the expected size of C5aR cDNA. A cDNA of the same size was amplified simultaneously from human PBL. Restriction mapping of the products amplified from HMC and from PBL gave restriction fragments of the same size. Incubation of HMC with increasing doses of C5a caused a progressive increase in the levels of the transcription factors activator protein-1 (AP-1) and cAMP response element binding protein (CREB), but C5a had no effect on the level of nuclear factor-kappaB (NF-kappaB). The effects of C5a on AP-1 were concentration and time dependent and peaked after 60 min. In contrast, the C5a metabolite C5adesArg had no significant effect on AP-1 levels. Preincubation of HMC with rabbit anti-C5aR antiserum inhibited partially the effect of C5a on AP-1. However, anti-C5aR Abs alone had no appreciable effects on AP-1. C5a caused a significant up-regulation of mRNA for the early response genes c-jun and c-fos on HMC. These results provide evidence for the presence of C5aR in adult HMC in culture and indicate that, after binding to C5aR, the anaphylatoxin C5a causes significant up-regulation of certain transcription factors and early response genes.

DNA binding of activator protein-1 is increased in human mesangial cells cultured in high glucose concentrations.

Human mesangial cells (HMC) grown in high glucose environments synthesize excessive amounts of extracellular matrix proteins (ECM). The promoter regions of certain ECM genes contain TPA (phorbol ester)-responsive element (TRE) motifs that bind the transcription factor, activator protein-1 (AP-1), a complex of Jun and other phosphoproteins. AP-1 binding to the TRE promoter is regulated by the quantity, composition and post-translational modifications of proteins in the AP-1 complex. We report an increased binding of AP-1 to TRE oligonucleotides in HMC cultured chronically (5 days) in high glucose environments (30 mM d-glucose). This increased binding is not due to differences in the nuclear quantity of AP-1 proteins or in the composition of the AP-1 complex when compared to AP-1 proteins from cells grown in normal glucose (5 mM d-glucose). A 30 mM l-glucose environment also increased AP-1 binding, but to a degree less than d-glucose. The increased AP-1 binding was partly reversed by treatment of HMC with Calphostin C or Bisindolylmaleimide I suggesting a partial role of the protein kinase C (PKC) pathway in mediating AP-1 binding. AP-1 binding was unaffected by treatment of cells with the MEK inhibitor PD 98059. In addition, increased AP-1 binding persisted for at least 48 hours after media glucose concentrations were normalized. The level of Jun-NH2-terminal kinase (JNK) activity and the phosphorylation of the JNK kinase, SEK1, were unchanged by chronic high glucose concentrations. These studies suggest that in HMC cultured in chronic high glucose, post-translational modifications increase the binding of AP-1 to the TRE motif.

Interleukin-1beta induction of mitogen-activated protein kinases in human mesangial cells. Role of oxidation.

Interleukin-1beta (IL-1beta) significantly influences renal cellular function through the induction of several gene products. The molecular mechanisms involved in gene regulation by IL-1beta are poorly understood; however, the appearance of novel tyrosine phosphoproteins in IL-1beta-treated cells suggests that IL-1beta may function through tyrosine phosphoprotein intermediates. The mitogen-activated protein (MAP) kinases are tyrosine phosphoproteins that could potentially mediate the effects of IL-1beta. Protein tyrosine phosphorylation following IL-1beta treatment may be dependent on redox changes since the IL-1beta receptor is not a protein-tyrosine kinase and oxidation has been shown to induce tyrosine phosphorylation. In this report we demonstrate that conditioning human glomerular mesangial cells with IL-1beta results in the tyrosine phosphorylation and activation of two members of the MAP kinase family, extracellular signal-regulated protein kinase 2 (ERK2) and p54 Jun-NH2-terminal kinase (JNK). This effect of IL-1beta is abrogated by pretreating cells with the antioxidants N-acetyl-L-cysteine or dithiothreitol. Furthermore, the effects of IL-1beta on ERK and JNK activation are reproduced by treating mesangial cells with membrane-permeable oxidants. IL-1beta and oxidants also cause phosphorylation and activation of the upstream ERK regulatory element MAP kinase kinase. Interestingly, IL-1beta, but not exogenous oxidants, causes phosphorylation of the upstream JNK activator, JNK kinase. These data indicate that IL-1beta activates ERK2 through an oxidation-dependent pathway. Exogenous oxidants and IL-1beta activate JNK through different upstream mechanisms; however, antioxidant inhibition of JNK activation indicates that endogenous oxidants may play a role in IL-1beta-induced JNK activation. Thus IL-1beta may affect mesangial cell function by activating MAP kinases, which can then regulate gene transcription. Furthermore, reactive oxygen species released during inflammatory glomerular injury may also affect mesangial function through a MAP kinase signal.