Preferences regarding genetic research results: comparing veterans and nonveterans responses.

OBJECTIVE: Communicating genetic research results to participants presents ethical challenges. Our objectives were to examine participants' preferences in receiving future genetic research results and to compare preferences reported by veteran and nonveterans participants. METHODS: Secondary analysis was performed on data collected in 2000-2004 from 1,575 consent forms signed by Mexican-American participants enrolled in 2 genetic family studies (GFS) in San Antonio: The Family Investigation of Nephropathy and Diabetes (FIND) and the Extended FIND (EFIND). The consent forms for these studies contained multiple-choice questions to examine participants' preferences about receiving their (1) clinical lab results and (2) future genetic research results. The FIND and EFIND databases had information on subjects' demographic characteristics and some selected clinical variables. We identified veterans using the Veterans Health Administration's (VHA's) centralized data repository. We compared veterans' and nonveterans' preferences using Student's t test for continuous variables and χ² test for discrete variables. A logistic regression analyzed subjects' preference for receiving their research results, controlling for other socio-demographic and clinical variables. RESULTS: The sample included 275 (18%) veterans and 1,247 (82%) nonveterans. Our results indicated a strong desire among the majority of participants 1,445 (95%) in getting their clinical lab research results. Likewise, 93% expressed interest in being informed about their future genetic results. There was no significant difference in veterans' and nonveterans' preference to disclosure of the research results (χ² test; p > 0.05). Regression analysis showed no significant relationship (p = 0.449) between the outcome (receiving research results) and veterans' responses after controlling for demographics and educational levels. CONCLUSION: Participants believed they would prefer receiving their genetic research results. Veterans are similar to nonveterans in their preferences. Offering genetic research results to participants should be based on well defined and structured plans to enhance interpretation of genetic data.

Risk of type 2 diabetes among individuals with high and low glomerular filtration rates.

AIMS/HYPOTHESIS: Metabolic abnormalities frequently develop prior to the diagnosis of type 2 diabetes and chronic kidney disease. However, it is not known whether GFR predicts the onset of type 2 diabetes. METHODS: Incident diabetes was ascertained in the Insulin Resistance Atherosclerosis Study (IRAS) (n = 864; age 40-69 years; median follow-up 5.2 years [4.5-6.6 years]; 141 incident cases of diabetes). GFR was estimated by the Modification of Diet in Renal Disease equation. We assessed the relationship between GFR and incident diabetes by logistic regression analysis. Results were adjusted for age, sex, ethnicity, clinic location, BMI, systolic blood pressure, antihypertensive treatment, family history of diabetes, insulin sensitivity and secretion, albumin to creatinine ratio, and levels of triacylglycerols, HDL-cholesterol, plasminogen activator inhibitor-1, and fasting and 2 h glucose. RESULTS: The relationship between GFR and incident diabetes was not linear. This relationship was statistically significant (p = 0.039) using a restricted cubic polynomial spline for GFR as a regression modelling strategy. Participants were stratified by GFR quintiles. Mean values for GFR from the first to the fifth quintile were 60.8, 71.6, 79.8, 88.2 and 109.0 ml min(-1) 1.73 m(-2). Relative to the fourth quintile, the odds ratios of incident diabetes for the first, second, third and fifth quintiles were 2.32 (95% CI 1.06-5.05), 1.76 (95% CI 0.80-3.88), 1.26 (95% CI 0.56-2.84) and 2.59 (95% CI 1.18-5.65), respectively. CONCLUSIONS/INTERPRETATION: Individuals in the upper and lower ranges of GFR are at increased risk of future diabetes. GFR and type 2 diabetes may share common pathogenic mechanisms.

PDGFR-A is a therapeutic target in alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma is an aggressive skeletal muscle cancer of childhood. Our initial studies of rhabdomyosarcoma gene expression for patients enrolled in a national clinical trial suggested that platelet-derived growth factor receptor A (PDGFR-A) may be a mediator of disease progression and metastasis. Using our conditional mouse tumor models that authentically recapitulate the primary mutations and metastatic progression of alveolar rhabdomyosarcomas in humans, we found by immunoblotting and immunokinase assays that PDGFR-A and its downstream effectors, mitogen-activated protein kinase and Akt, were highly activated in both primary and metastatic tumors. Inhibition of PDGFR-A by RNA interference, small molecule inhibitor or neutralizing antibody had a dramatic effect on tumor cell growth both in vitro and in vivo, although resistance evolved in one-third of tumors. These results establish proof-of-principal for PDGFR-A as a therapeutic target in alveolar rhabdomyosarcoma.

High d(+)glucose concentration inhibits RANKL-induced osteoclastogenesis.

Diabetes is a chronic disease associated with hyperglycemia and altered bone metabolism that may lead to complications including osteopenia, increased risk of fracture and osteoporosis. Hyperglycemia has been implicated in the pathogenesis of diabetic bone disease; however, the biologic effect of glucose on osteoclastogenesis is unclear. In the present study, we examined the effect of high d(+)glucose (d-Glc) and l(-)glucose (l-Glc; osmotic control) on RANKL-induced osteoclastogenesis using RAW264.7 cells and Bone Marrow Macrophages (BMM) as models. Cells were exposed to sustained high glucose levels to mimic diabetic conditions. Osteoclast formation was analyzed using tartrate resistant acid phosphatase (TRACP) assay, expression of calcitonin receptor (CTR) and cathepsin K mRNAs, and cultures were examined for reactive oxygen species (ROS) using dichlorodihydrofluorescein diacetate (DCF-DA) fluorescence, caspase-3 and Nuclear Factor kappaB (NF-kappaB) activity. Cellular function was assessed using a migration assay. Results show, for the first time, that high d-Glc inhibits osteoclast formation, ROS production, caspase-3 activity and migration in response to RANKL through a metabolic pathway. Our findings also suggest that high d-Glc may alter RANKL-induced osteoclast formation by inhibiting redox-sensitive NF-kappaB activity through an anti-oxidative mechanism. This study increases our understanding of the role of glucose in diabetes-associated bone disease. Our data suggest that high glucose levels may alter bone turnover by decreasing osteoclast differentiation and function in diabetes and provide new insight into the biologic effects of glucose on osteoclastogenesis.

Ablation of the gene encoding p66Shc protects mice against AGE-induced glomerulopathy by preventing oxidant-dependent tissue injury and further AGE accumulation.

AIMS/HYPOTHESIS: AGEs have been implicated in renal disease associated with ageing, diabetes and other age-related disorders. Reactive oxygen species (ROS) promote formation of AGEs, which cause AGE-receptor-mediated ROS generation with activation of signalling pathways leading to tissue injury and further AGE accumulation. ROS generation is regulated by the Src homology 2 domain-containing transforming protein C1 (Shc1) isoform p66(Shc), whose deletion has been shown to protect from tissue injury induced by ageing, diabetes, hyperlipidaemia and ischaemia-reperfusion by preventing oxidative stress. This study was aimed at assessing the role of p66(Shc) in the modulation of oxidative stress and oxidant-dependent renal injury induced by AGEs. METHODS: For 10 weeks, male p66 (shc) knockout (KO) and wild-type (WT) mice were injected with 60 microg/day albumin modified or unmodified by N epsilon-(carboxymethyl) lysine (CML). Mice were then killed for the assessment of renal function and structure, as well as systemic and renal tissue oxidative stress. RESULTS: Upon CML injection, KO mice, in contrast to WT mice, showed no or only mild forms of proteinuria, glomerular hypertrophy, mesangial expansion, glomerular sclerosis, renal/glomerular cell apoptosis and extracellular matrix upregulation. Moreover, KO mice had lower circulating and tissue AGEs than WT mice and unchanged plasma isoprostane 8-epi-prostaglandin-F(2alpha) levels, renal/glomerular CML, 4-hydroxy-2-nonenal, AGE receptor and NAD(P)H oxidase 4 (NOX4) content (and expression of the corresponding genes), and nuclear factor kappaB activation (NFkappaB). Mesangial cells from KO mice exposed to CML showed no or slight increase in ROS levels and NFkappaB activation, again at variance with WT cells. CONCLUSIONS/INTERPRETATION: These data indicate that p66(Shc) participates in the pathogenesis of AGE-dependent glomerulopathy by mediating AGE-induced tissue injury and further AGE formation through ROS-dependent mechanisms involving NFkappaB activation and upregulation of Nox4 expression and NOX4 production.

Evaluation of tight junction protein 1 encoding zona occludens 1 as a candidate gene for albuminuria in a Mexican American population.

Albuminuria, a hallmark of diabetic nephropathy, has been shown to be significantly heritable in multiple studies. Therefore, the identification of genes that affect susceptibility to albuminuria may lead to novel avenues of intervention. Current evidence suggests that the podocyte and slit diaphragm play a key role in controlling the selective sieve of the glomerular filtration barrier, and podocyte-specific genes have been identified that are necessary for maintaining its integrity. We therefore investigated the role of gene variants of tight junction protein (TJP1) which encodes another slit diaphragm-associated protein zona occludens 1 as risk factors for albuminuria in the San Antonio Family Diabetes/Gallbladder Study (SAFDGS), which consists of extended Mexican-American families with a high prevalence of type 2 diabetes. Albuminuria, defined as an albumin (mg/dl) to creatinine (mg/dl) ratio (ACR) of 0.03, which is approximately equivalent to a urinary albumin excretion (UAE) >30 mg/day, was present in a total of 14.9% of participants, and 31% had type 2 diabetes. The TJP1 exons, flanking intronic sequence, and putative proximal promoter regions were investigated in this population. Twentynine polymorphisms, including 7 nonsynonymous SNPs, were identified and genotyped in all subjects of this study for association analysis. Three sets of correlated SNPs, which include 3 exonic SNPs, were nominally associated with ACR (p value range 0.007-0.049); however, the association with the discrete trait albuminuria was not significant (p value range 0.094-0.338). We conclude that these variants in TJP1 do not appear to be major determinants for albuminuria in the SAFDGS; however, they may play a minor role in its severity in this Mexican-American population. Further examination of the TJP1 gene region in this and other cohorts will be useful to determine whether ZO-1 plays a significant role in glomerular permselectivity.

Angiotensin II inhibits insulin-stimulated phosphorylation of eukaryotic initiation factor 4E-binding protein-1 in proximal tubular epithelial cells.

Interaction between angiotensin II, which binds a G-protein-coupled receptor, and insulin, a ligand for receptor tyrosine kinase, was examined in renal proximal tubular epithelial cells. Augmented protein translation by insulin involves activation of eukaryotic initiation factor 4E (eIF4E) which follows the release of the factor from a heterodimeric complex by phosphorylation of its binding protein, 4E-BP1. Angiotensin II (1 nM) or insulin (1 nM) individually stimulated 4E-BP1 phosphorylation. However, pre-incubation with angiotensin II abrogated insulin-induced phosphorylation of 4E-BP1, resulting in persistent binding to eIF4E. Although angiotensin II and insulin individually activated phosphoinositide 3-kinase and extracellular signal-regulated kinase (ERK)-1/-2-type mitogen-activated protein (MAP) kinase, pre-incubation with angiotensin II abolished insulin-induced stimulation of these kinases, suggesting more proximal events in insulin signalling may be intercepted. Pretreatment with angiotensin II markedly inhibited insulin-stimulated tyrosine phosphorylation of insulin-receptor beta-chain and insulin-receptor substrate 1. Losartan prevented angiotensin II inhibition of insulin-induced ERK-1/-2-type MAP kinase activation and 4E-BP1 phosphorylation, suggesting mediation of the effect of angiotensin II by its type 1 receptor. Insulin-stimulated de novo protein synthesis was also abolished by pre-incubation with angiotensin II. These data show that angiotensin II inhibits 4E-BP1 phosphorylation and stimulation of protein synthesis induced by insulin by interfering with proximal events in insulin signalling. Our data provide a mechanistic basis for insulin insensitivity induced by angiotensin II.

Angiotensin II activates Akt/protein kinase B by an arachidonic acid/redox-dependent pathway and independent of phosphoinositide 3-kinase.

Angiotensin II (Ang II) exerts contractile and trophic effects in glomerular mesangial cells (MCs). One potential downstream target of Ang II is the protein kinase Akt/protein kinase B (PKB). We investigated the effect of Ang II on Akt/PKB activity in MCs. Ang II causes rapid activation of Akt/PKB (5-10 min) but delayed activation of phosphoinositide 3-kinase (PI3-K) (30 min). Activation of Akt/PKB by Ang II was not abrogated by the PI3-K inhibitors or by the introduction of a dominant negative PI3-K, indicating that in MCs, PI3-K is not an upstream mediator of Akt/PKB activation by Ang II. Incubation of MCs with phospholipase A2 inhibitors also blocked Akt/PKB activation by Ang II. AA mimicked the effect of Ang II. Inhibitors of cyclooxygenase-, lipoxyogenase-, and cytochrome P450-dependent metabolism did not influence AA-induced Akt/PKB activation. However, the antioxidants N-acetylcysteine and diphenylene iodonium inhibited both AA- and Ang II-induced Akt/PKB activation. Dominant negative mutant of Akt/PKB or antioxidants, but not the dominant negative form of PI3-K, inhibited Ang II-induced protein synthesis and cell hypertrophy. These data provide the first evidence that Ang II induces protein synthesis and hypertrophy in MCs through AA/redox-dependent pathway and Akt/PKB activation independent of PI3-K.

Insulin-like growth factor-I induces renal cell hypertrophy via a calcineurin-dependent mechanism.

Insulin-like growth factor-I (IGF-I) may play an important role in the development of renal hypertrophy. In this study we determined the effect of IGF-I on cultured mesangial cells (MCs) and examined activation of key signaling pathways. IGF-I induced hypertrophy as determined by an increase in cell size and an increase in protein to DNA ratio and increased accumulation of extracellular matrix (ECM) proteins. IGF-I also activated both Erk1/Erk2 MAPK and phosphatidylinositol 3-kinase (PI3K) in MCs. Inhibition of either MAPK or PI3K, however, had no effect on IGF-I-induced hypertrophy or ECM production. Next, we examined the effect of IGF-I on activation of the calcium-dependent phosphatase calcineurin. IGF-I treatment stimulated calcineurin activity and increased the protein levels of calcineurin and the calcineurin binding protein, calmodulin. Cyclosporin A, an inhibitor of calcineurin, blocked both IGF-I-mediated hypertrophy and up-regulation of ECM. In addition, calcineurin resulted in sustained Akt activation, indicating possible cross-talk with other signaling pathways. Finally, IGF-I treatment resulted in the calcineurindependent nuclear localization of NFATc1. Therefore, IGF-I induces hypertrophy and increases ECM accumulation in MCs. IGF-I-mediated hypertrophy is associated with activation of Erk1/Erk2 MAPK and PI3K but does not require either of these pathways. Instead, IGF-I mediates hypertrophy via a calcineurin-dependent pathway.

Ceramide blocks PDGF-induced DNA synthesis in mesangial cells via inhibition of Akt kinase in the absence of apoptosis.

The mechanism of action of ceramide in glomerular mesangial cells has not been studied. We investigated the effect of C2 ceramide on the mitogenic signal transduction pathways induced by PDGF in mesangial cells. Increasing concentrations of C2 ceramide inhibited PDGF-induced DNA synthesis in a dose-dependent manner with maximum inhibition at 15 microM. This inhibition of DNA synthesis was associated with attenuation of PDGF-induced early response gene c-fos transcription. PDGF receptor beta immunecomplex kinase assay showed no inhibitory effect of C2 ceramide on PDGF receptor tyrosine kinase activity. We have recently shown that the mitogenic effect of PDGF is mediated by the enzyme phosphatidylinositol (PI) 3 kinase in mesangial cells. C2 ceramide had no effect on PDGF-induced PDGFR-associated PI 3 kinase activity. These data indicate that inhibitory effect of C2 on PDGF-induced DNA synthesis is likely due to post-receptor and post-PI 3 kinase events. To address the mechanism of C2-mediated inhibition of DNA synthesis, we investigated the downstream target of PI 3 kinase, Akt. PDGF time-dependently increased Akt kinase activity in a PI 3 kinase-dependent manner. Incubation of mesangial cells with C2 ceramide inhibited PDGF-induced Akt activity. Akt kinase inhibits apoptosis of cells via phosphorylation of multiple proapoptotic proteins. However, inhibition of Akt activity by C2 ceramide did not induce apoptosis in mesangial cells. These data provide the first evidence that in mesangial cells, ceramide cross-talks with PI 3 kinase-dependent Akt kinase to inhibit PDGF-induced DNA synthesis without inducing apoptosis.

Activation of renal signaling pathways in db/db mice with type 2 diabetes.

BACKGROUND: Altered regulation of signaling pathways may contribute to the pathogenesis of renal disease. We examined renal cortical signaling pathways in type 2 diabetes. METHODS: The status of renal cortical signaling pathways was examined in control and db/db mice with type 2 diabetes in the early phase of diabetic nephropathy associated with renal matrix expansion and albuminuria. RESULTS: Tyrosine phosphorylation of renal cortical proteins was increased in diabetic mice. Renal cortical activities of phosphatidylinositol 3-kinase (PI 3-kinase) in antiphosphotyrosine immunoprecipitates, Akt (PKB), and ERK1/2-type mitogen-activated protein (MAP) kinase activities were significantly augmented sixfold (P < 0.01), twofold (P < 0.0003), and sevenfold (P < 0.001), respectively, in diabetic mice compared with controls. A part of the increased renal cortical PI 3-kinase activity was due to insulin receptor activation, as PI 3-kinase activity associated with beta chain of the insulin receptor was increased nearly fourfold (P < 0.0235). Additionally, the kinase activity of the immunoprecipitated insulin receptor beta chain was augmented in the diabetic renal cortex, and tyrosine phosphorylation of the insulin receptor was increased. In the liver, activities of PI 3-kinase in the antiphosphotyrosine immunoprecipitates and Akt also were increased threefold (P < 0.05) and twofold (P < 0.0002), respectively. However, there was no change in the hepatic insulin receptor-associated PI 3-kinase activity. Additionally, the hepatic ERK1/2-type MAP kinase activity was inhibited by nearly 50% (P < 0.01). CONCLUSIONS: These studies demonstrate that a variety of receptor signaling pathways are activated in the renal cortex of mice with type 2 diabetes, and suggest a role for augmented insulin receptor activity in nephropathy of type 2 diabetes.

Agonist-specific regulation of monocyte chemoattractant protein-1 expression by cyclooxygenase metabolites in hepatic stellate cells.

Activated hepatic stellate cells (HSC) regulate the liver "wound-healing" response through expression of chemokines, including monocyte chemoattractant protein-1 (MCP-1), which participate in the formation of the inflammatory infiltrate during liver injury. Cyclooxygenase (COX) catalyzes the conversion of arachidonic acid into prostaglandins, which may contribute to the inflammatory response. In this study, we investigated the effects of COX inhibitors on the expression of MCP-1 in cultured HSC. Pretreatment of HSC with nonspecific COX inhibitors such as indomethacin or ibuprofen markedly reduced the expression of MCP-1 caused by exposure to tumor necrosis factor alpha (TNF-alpha) or interleukin-1alpha (IL-1alpha). NS-398, a specific COX-2 inhibitor, also resulted in a dose-dependent inhibition of MCP-1 gene and protein expression. These effects were dependent on reduced MCP-1 transcription, as established using a reporter plasmid. In contrast, the up-regulation of MCP-1 expression caused by interferon gamma (IFN-gamma) was not sensitive to COX inhibitors. Quiescent HSC did not show detectable expression of COX-2, which became evident after activation in culture, and while TNF-alpha and IL-1alpha markedly increased the expression of COX-2, IFN-gamma did not have any effects. Pretreatment of HSC with the stable cyclic adenosine monophosphate (cAMP) analog, 8-bromo cAMP, reverted the effects of the COX-2 inhibitor, but not of a nuclear factor-kappaB (NF-kappaB) inhibitor, demonstrating that prostaglandins modulate MCP-1 expression via production of cAMP. On the other hand, the action of NF-kappaB inhibitors was negligible in IFN-gamma-stimulated cells. These findings indicate that cross-talk between cytokines and a prostaglandin-cAMP pathway differentially regulates the proinflammatory potential of HSC, contributing to the modulation of liver tissue inflammation.

Insulin regulation of protein translation repressor 4E-BP1, an eIF4E-binding protein, in renal epithelial cells.

BACKGROUND: Augmented protein translation by insulin involves activation of eukaryotic initiation factor 4E (eIF4E) that follows release of eIF4E from a heterodimeric complex by phosphorylation of its inhibitory binding protein, 4E-BP1. We examined insulin regulation of 4E-BP1 phosphorylation in murine proximal tubular epithelial cells. METHODS AND RESULTS: Insulin (1 nmol/L) increased de novo protein synthesis by 58 +/- 11% (P < 0.001). Insulin also augmented 4E-BP1 phosphorylation and phosphatidylinositol 3-kinase (PI 3-kinase) activity in antiphosphotyrosine immunoprecipitates. This could be prevented by PI 3-kinase inhibitors, Wortmannin, and LY294002. Insulin also activated Akt that lies downstream of PI 3-kinase. Rapamycin abrogated 4E-BP1 phosphorylation in response to insulin, suggesting involvement of mammalian target of rapamycin (mTOR), a kinase downstream of Akt. Insulin-stimulated phosphorylation of 4E-BP1 was also inhibited by PD098059, implying involvement of Erk-1/-2 mitogen-activated protein (MAP) kinase. An increase in Erk-1/-2 type MAP kinase activity by insulin was directly confirmed in an immunokinase assay and was found to be PI 3-kinase dependent. CONCLUSIONS: In proximal tubular epithelial cells, insulin augments 4E-BP1 phosphorylation, which is PI 3-kinase and mTOR dependent. The requirement for Erk-1/-2 MAP kinase activation for 4E-BP1 phosphorylation by insulin suggests a cross-talk between PI 3-kinase and Erk-1/-2-type MAP kinase pathways.

Activation of PLC and PI 3 kinase by PDGF receptor alpha is not sufficient for mitogenesis and migration in mesangial cells.

BACKGROUND: Platelet-derived growth factor (PDGF) isoforms act through two distinct cell surface alpha and beta receptors. Glomerular mesangial cells express both receptors. PDGF BB and AB are potent mitogens for glomerular mesangial cells, and PDGF BB stimulates cell migration in a phosphatidylinositol 3 (PI 3) kinase-dependent manner. In this study, we investigated the effect of PDGF AA on cell migration, PI 3 kinase and phospholipase C (PLC) activation, and the role of these two enzymes in mediating biological responses in these cells in response to all three isoforms. METHODS: 3H-thymidine incorporation and modified Boyden chamber assay were used to determine DNA synthesis and directed migration, respectively, in response to all three PDGF isoforms. Differential activation of alpha and beta receptors was studied by immunecomplex tyrosine kinase assay of corresponding receptor immunoprecipitates. PLC gamma 1 activity was determined by measuring total inositol phosphates in response to different PDGF isoforms. PI 3 kinase activity was determined in antiphosphotyrosine or PDGF receptor immunoprecipitates. RESULTS: Both PDGF BB and AB resulted in stimulation of DNA synthesis and directed migration of mesangial cells. AA was neither chemotactic nor mitogenic. However, all three isoforms increased tyrosine phosphorylation of a 180 kD protein in antiphosphotyrosine immunoprecipitates, suggesting activation of respective receptors. Direct immunecomplex tyrosine kinase assay of alpha and beta receptors demonstrated significant activation of both of these receptors when cells are treated with PDGF BB or AB. PDGF AA increased tyrosine kinase activity of the alpha receptor but not the beta receptor. All three isoforms significantly stimulated the production of inositol phosphates with order of potency being BB > AB > AA. PDGF AA also dose dependently stimulated PI 3 kinase activity measured in antiphosphotyrosine immunoprecipitates of treated cells. A comparison of PI 3 kinase activity in antiphosphotyrosine immunoprecipitates from mesangial cells stimulated with three different PDGF isoforms showed significant activation of this enzyme with a decreasing order of activity: BB > AB > AA. CONCLUSION: Taken together, these data demonstrate that all three isoforms of PDGF significantly stimulate PLC gamma 1 and PI 3 kinase, two enzymes necessary for both DNA synthesis and directed migration. However, activation of alpha receptor by PDGF AA with a subsequent increase in PLC and PI 3 kinase activities is not sufficient to induce these biological responses in mesangial cells. These data indicate that the extent of activation of signal transduction pathways may be a major determinant of the biological activity of different PDGF isoforms in mesangial cells.

Platelet-derived growth factor receptor beta regulates migration and DNA synthesis in metanephric mesenchymal cells.

Platelet-derived growth factor (PDGF) B-chain and PDGF receptor beta (PDGFR beta) are essential for glomerulogenesis. Mice deficient in PDGF B-chain or PDGFR beta exhibit an abnormal glomerular phenotype characterized by total lack of mesangial cells. In this study, we localized PDGFR beta in the developing rat kidney and explored the biological effects of PDGF in metanephric mesenchymal cells in an attempt to determine the mechanism by which PDGF regulates mesangial cell development. Immunohistochemical and in situ hybridization studies of rat embryonic kidneys reveal that PDGFR beta localizes to undifferentiated metanephric mesenchyme and is later expressed in the cleft of the comma-shaped and S-shaped bodies and in more mature glomeruli in a mesangial distribution. We also isolated and characterized cells from rat metanephric mesenchyme. Metanephric mesenchymal cells express vimentin and alpha-smooth muscle actin but not cytokeratin. These cells also express functional PDGFR beta, as demonstrated by autophosphorylation of the receptor as well as activation of phosphatidylinositol 3 kinase in response to PDGF B-chain homodimer. PDGF B-chain also induces migration and proliferation of metanephric mesenchymal cells. Taken together with the fact that PDGF B-chain is expressed in the glomerular epithelium and mesangial area, as demonstrated in the human embryonic kidney, we suggest that PDGF B-chain acts in a paracrine fashion to stimulate the migration and proliferation of mesangial cell precursors from undifferentiated metanephric mesenchyme to the mesangial area. PDGF B-chain also likely stimulates proliferation of mesangial cell precursors in an autocrine fashion once these cells migrate to the glomerular tuft.

Activation of STAT1 alpha by phosphatase inhibitor vanadate in glomerular mesangial cells: involvement of tyrosine and serine phosphorylation.

Vanadate is an insulinomimetic agent that has potent inhibitory effect on tyrosine phosphatases. We have recently demonstrated that low concentration of vanadate stimulates phosphotyrosine-dependent signal transduction pathways leading to gene expression and DNA synthesis in mesangial cells. To further examine the mechanisms by which vanadate activates mesangial cell, we studied its effect on signal transducer and activators of transcription (STAT). Incubation of lysates from vanadate-stimulated mesangial cells with a specific high affinity sis-inducible DNA element (SIE) resulted in the formation of protein-DNA complex. Supershift analysis using monoclonal antibody against STAT1 alpha showed its exclusive presence in the DNA-protein complex. Incubation of cell lysate with antiphosphotyrosine antibody or with excess phosphotyrosine caused decrease in binding of STAT1 alpha to SIE probe indicating that tyrosine phosphorylation and dimerization of this transcription factor are necessary for its activation. Immunoprecipitation followed by immunecomplex kinase assay showed increased tyrosine kinase activity of Janus kinase 2 (JAK2) in vanadate-treated mesangial cells. The addition of a monoclonal antiphosphoserine antibody to lysates from vanadate-treated mesangial cells results in supershift of protein-DNA complex indicating the presence of serine phosphorylated STAT1 alpha in this complex. Treatment of lystates from vanadated-stimulated mesangial cells with serine phosphatase PP2A causes inhibition of DNA-protein interaction. Collectively, our data indicate that at least one mechanism of activation of mesangial cells during vanadate treatment is increased activation of STAT1 alpha by both tyrosine and serine phosphorylation.

Bone morphogenetic protein-2 inhibits MAPK-dependent Elk-1 transactivation and DNA synthesis induced by EGF in mesangial cells.

Bone morphogenetic protein-2 (BMP-2) is a member of the TGFbeta superfamily of growth and differentiation factors. We investigated the effect of BMP-2 on epidermal growth factor (EGF)-induced mitogenic signaling in kidney glomerular mesangial cells. BMP-2 dose-dependently inhibits EGF-induced DNA synthesis. Maximum effect was obtained at a concentration of 100 ng/ml. BMP-2 had no inhibitory effect on the EGF receptor (EGFR)-associated tyrosine kinase activity indicating that inhibition of DNA synthesis is due to regulation of post-receptor signaling event(s). EGF stimulates MAPK activity in mesangial cells in a time-dependent manner. Inhibition of MAPK by the MEK inhibitor PD098059 blocks EGF-induced DNA synthesis indicating the requirement of this enzyme activity in EGF-mediated mitogenic signaling. Furthermore, we show that exposure of mesangial cells to BMP-2 blocks EGF-induced MAPK activity which leads to phosphorylattion of Elk-1 transcription factor. Using a GAL-4 DNA binding-domain-Elk-1 transactivation domain fusion protein-based reporter assay, we demonstrate that BMP-2 inhibits EGF-induced Elk-1-mediated transcription. These data provide the first evidence that BMP-2 signaling in mesangial cells initiates a negative regulatory cross-talk with MAPK-based transcription to inhibit EGF-induced DNA synthesis.

Bone morphogenetic protein 2 inhibits platelet-derived growth factor-induced c-fos gene transcription and DNA synthesis in mesangial cells. Involvement of mitogen-activated protein kinase.

Bone morphogenetic proteins (BMPs) play an important role in nephrogenesis. The biologic effect and mechanism of action of these proteins in the adult kidney has not yet been studied. We investigated the effect of BMP2, a member of these growth and differentiation factors, on mitogenic signal transduction pathways induced by platelet-derived growth factor (PDGF) in glomerular mesangial cells. PDGF is a growth and survival factor for these cells in vitro and in vivo. Incubation of mesangial cells with increasing concentrations of BMP2 inhibited PDGF-induced DNA synthesis in a dose-dependent manner with maximum inhibition at 250 ng/ml. Immune complex tyrosine kinase assay of PDGF receptor beta immunoprecipitates from lysates of mesangial cells treated with PDGF showed no inhibitory effect of BMP2 on PDGF receptor tyrosine phosphorylation. This indicates that the inhibition of DNA synthesis is likely due to postreceptor events. However, BMP2 significantly inhibited PDGF-stimulated mitogen-activated protein kinase (MAPK) activity that phosphorylates the Elk-1 transcription factor, a component of the ternary complex factor. Using a fusion protein-based reporter assay, we also show that BMP2 blocks PDGF-induced Elk-1-mediated transcription. Furthermore, we demonstrate that BMP2 inhibits PDGF-induced transcription of c-fos gene, a natural target of Elk-1 that normally forms a ternary complex that activates the serum response element of the c-fos gene. These data provide the first evidence that in mesangial cells, BMP2 signaling cross-talks with MAPK-based transcriptional events to inhibit PDGF-induced DNA synthesis. One target for this inhibition is the early response gene c-fos.

Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney.

BMP-7, a member of the bone morphogenic protein subfamily (BMPs) of the transforming growth factor-beta superfamily of secreted growth factors, is abundantly expressed in the fetal kidney. The precise role of this protein in renal physiology or pathology is unknown. A cDNA that encodes rat BMP-7 was cloned and used as a probe to localize BMP-7 mRNA expression by in situ hybridization in the adult rat kidney. The highest expression of BMP-7 mRNA could be seen in tubules of the outer medulla. In glomeruli, a few cells, mainly located at the periphery of the glomerular tuft, showed specific and strong signals. Also, high BMP-7 mRNA expression could be localized to the adventitia of renal arteries, as well as to the epithelial cell layer of the renal pelvis and the ureter. Preliminary evidence suggests that BMP-7 enhances recovery when infused into rats with ischemia-induced acute renal failure. We examined BMP-7 mRNA expression in kidneys with acute renal failure induced by unilateral renal artery clamping. BMP-7 mRNA abundance as analyzed by solution hybridization was reduced in ischemic kidneys after 6 and 16 h of reperfusion compared with the contralateral kidney. In situ hybridization in ischemic kidneys showed a marked decrease of BMP-7 mRNA in the outer medulla and in glomeruli. Utilizing rat metanephric mesenchymal cells in culture, we also demonstrate that BMP-7 induces epithelial cell differentiation. Taken together, these data suggest that BMP-7 is important in both stimulating and maintaining a healthy differentiated epithelial cell phenotype.