Epicatechin limits renal injury by mitochondrial protection in cisplatin nephropathy.

Cisplatin nephropathy can be regarded as a mitochondrial disease. Intervention to halt such deleterious injury is under investigation. Recently, the flavanol (-)-epicatechin emerges as a novel compound to protect the cardiovascular system, owing in part to mitochondrial protection. Here, we have hypothesized that epicatechin prevents the progression of cisplatin-induced kidney injury by protecting mitochondria. Epicatechin was administered 8 h after cisplatin injury was induced in the mouse kidney. Cisplatin significantly induced renal dysfunction and tubular injury along with an increase in oxidative stress. Mitochondrial damages were also evident as a decrease in loss of mitochondrial mass with a reduction in the oxidative phosphorylation complexes and low levels of MnSOD. The renal damages and mitochondrial injuries were significantly prevented by epicatechin treatment. Consistent with these observations, an in vitro study using cultured mouse proximal tubular cells demonstrated that cisplatin-induced mitochondrial injury, as revealed by a decrease in mitochondrial succinate dehydrogenase activity, an induction of cytochrome c release, mitochondrial fragmentation, and a reduction in complex IV protein, was prevented by epicatechin. Such a protective effect of epicatechin might be attributed to decreased oxidative stress and reduced ERK activity. Finally, we confirmed that epicatechin did not perturb the anticancer effect of cisplatin in HeLa cells. In conclusion, epicatechin exhibits protective effects due in part to its ability to prevent the progression of mitochondrial injury in mouse cisplatin nephropathy. Epicatechin may be a novel option to treat renal disorders associated with mitochondrial dysfunction.

Nicorandil, a K(atp) channel opener, alleviates chronic renal injury by targeting podocytes and macrophages.

Nicorandil exhibits a protective effect in the vascular system, which is thought to be due to vasodilatation from opening ATP-dependent potassium channels and donation of nitric oxide. Recently, nicorandil was shown to be renoprotective in models of acute kidney injury and glomerulonephritis. However, the specific mechanisms of renoprotection are unclear. We evaluated the effect of nicorandil on the rat remnant kidney model of chronic kidney disease. Blood pressure was unchanged by a 10-wk course of nicorandil, while albuminuria was significantly reduced. Glomerular injury and tubulointerstitial injury were also ameliorated by nicorandil. Oxidative stress, as noted by renal nitrotyrosine level and urine 8-hydroxy-2'-deoxyguanosine, were elevated in this model and was significantly reduced by nicorandil treatment. Treatment was associated with maintenance of the mitochondrial antioxidant, manganese SOD, in podocytes and with suppression of xanthine oxidase expression in infiltrating macrophages. Interestingly, these two cell types express sulfonylurea receptor 2 (SUR2), a binding site of nicorandil in the ATP-dependent K channel. Consistently, we found that stimulating SUR2 with nicorandil prevented angiotensin II-mediated upregulation of xanthine oxidase in the cultured macrophage, while xanthine oxidase expression was rather induced by blocking SUR2 with glibenclamide. In conclusion, nicorandil reduces albuminuria and ameliorates renal injury by blocking oxidative stress in chronic kidney disease.

Nicorandil as a novel therapy for advanced diabetic nephropathy in the eNOS-deficient mouse.

Nicorandil is an orally available drug that can act as a nitric oxide donor, an antioxidant, and an ATP-dependent K channel activator. We hypothesized that it may have a beneficial role in treating diabetic nephropathy. We administered nicorandil to a model of advanced diabetic nephropathy (the streptozotocin-induced diabetes in mice lacking endothelial nitric oxide synthase, eNOSKO); controls included diabetic eNOS KO mice without nicorandil and nondiabetic eNOS KO mice treated with either nicorandil or vehicle. Mice were treated for 8 wk. Histology, blood pressure, and renal function were determined. Additional studies involved examining the effects of nicorandil on cultured human podocytes. Here, we found that nicorandil did not affect blood glucose levels, blood pressure, or systemic endothelial function, but significantly reduced proteinuria and glomerular injury (mesangiolysis and glomerulosclerosis). Nicorandil protected against podocyte loss and podocyte oxidative stress. Studies in cultured podocytes showed that nicorandil likely protects against glucose-mediated oxidant stress via the ATP-dependent K channel as opposed to its NO-stimulating effects. In conclusion, nicorandil may be beneficial in diabetic nephropathy by preserving podocyte function. We recommend clinical trials to determine whether nicorandil may benefit diabetic nephropathy or other conditions associated with podocyte dysfunction.

Evidence for functional heterogeneity of circulating B-type natriuretic peptide.

OBJECTIVES: These studies describe molecular forms of circulating B-type natriuretic peptide (BNP) as well as their biological activity. BACKGROUND: Increased circulating levels of immunoreactive BNP correlate with the severity of heart failure and are considered a sensitive biomarker. However, little is known about the molecular forms of circulating BNP and their biological activity. METHODS: Western blot analysis was used to characterize immunoreactive BNP species in heart failure plasma. Recombinant proBNP was assessed for reactivity in commercially available BNP assays and cell activity by cyclic guanosine monophosphate production in vascular cells. RESULTS: Heart failure plasma contained both low- (LMW-BNP) and high-molecular-weight (HMW-BNP) forms. The LMW-BNP migrated similarly to a 32-amino acid BNP standard, whereas HMW-BNP, when deglycosylated, was similar to deglycosylated recombinant proBNP. Recombinant proBNP and BNP were equally recognized by the Triage BNP assay (Biosite, San Diego, California). Furthermore, recombinant proBNP and BNP were both recognized by the Advia Centaur BNP test (Bayer Diagnostics, Tarrytown, New York), but only recombinant proBNP was recognized by the Elecsys NTproBNP assay (Roche Diagnostics, Indianapolis, Indiana). Recombinant proBNP exerted significantly less biological activity than BNP on human endothelial and vascular smooth muscle cells. Comparison of effective concentration (50%) values indicates that proBNP is 6- to 8-fold less potent than BNP in these human cells. CONCLUSIONS: This study demonstrates that proBNP, constituting a substantial portion of immunoreactive BNP in heart failure plasma, possesses significantly lower biological activity than the processed 32-amino acid hormone. These results implicate a discordance in heart failure between the high circulating levels of immunoreactive BNP and hormone activity, suggesting that some patients may be in a state of natriuretic peptide deficiency.

Antitumor activity of TGF-beta inhibitor is dependent on the microenvironment.

Pancreatic cancer is one of the deadliest forms of cancer and effective treatment remains a clinical challenge. Transforming growth factor-beta (TGF-beta) has important roles in primary tumor progression and in promoting metastasis, and has become an attractive target for therapy. Previously, we reported that treatment of pancreatic cancer cells in vitro with SD-208, a small molecule inhibitor of the TGF-beta receptor I kinase (TGF-betaRI), inhibited expression of genes associated with tumor progression and inhibited invasiveness in a cell-based assay. In a demonstration of efficacy of TGF-beta signaling inhibition in an in vivo model of pancreatic cancer, we showed significantly reduced primary tumor weight and decreased incidence of metastasis in the Panc-1 orthotopic xenograft model of established pancreatic cancer. In this report, we extend these in vivo findings to examine the mechanistic consequences of TGF-betaRI inhibition on Panc-1 primary tumors and their microenvironment in situ. In a longitudinal study of TGF-betaRI inhibition in the Panc-1 orthotopic model, we show that SD-208 treatment significantly reduced tumor growth measured as bioluminescence intensity throughout the study. Histological evaluation revealed that SD-208 treatment reduced proliferation and induced apoptosis in the primary tumors, and reduced fibrosis in the tumor microenvironment. An immune contribution (greater B-cell infiltration in SD-208-treated tumors) was also suggested by the histological analyses. SD-208 not only blocked direct TGF-beta signaling in Panc-1 primary tumors (reduced phospho SMAD2/3), but also down-regulated the expression of TGF-beta-regulated genes (PAI-1 and COL7A1). Taken together, our results indicate that a TGF-betaRI kinase inhibitor has a potential therapeutic benefit for pancreatic cancer patients.

Vascular endothelial growth factor administration does not improve microvascular disease in the salt-dependent phase of post-angiotensin II hypertension.

Renal microvascular injury and tubulointerstitial inflammation may provide a potential mechanism for the development of salt-sensitive hypertension. Therefore, we hypothesized that vascular endothelial growth factor (VEGF) administration would prevent the development of salt-sensitive hypertension induced by ANG II. Infusion of ANG II in rats for 2 wk led to an elevation in blood pressure and an increase in blood urea nitrogen. Prominent tubular injury, focal areas of peritubular capillary loss accompanied by a decrease in urinary nitrites, thickening of the afferent arteriole, and an elevation in systemic and renal VEGF protein levels also occurred. In separate studies, animals were infused with ANG II and then placed on a low-salt diet for 1 wk. At this point, the animals were paired on the basis of weight and blood pressure and treated with either VEGF(121) or vehicle subcutaneously for 8 wk while being fed a high-salt diet. During the treatment period, a spontaneous improvement in many parameters, including both renal function and healing of the peritubular capillaries, occurred to the same degree in both vehicle- and VEGF(121)-treated rats. VEGF(121) significantly reduced blood pressure and accelerated the recovery of tubular injury. In contrast, vehicle-treated rats demonstrated a persistent increase in afferent arteriolar media-to-lumen ratio, which was further enhanced in rats treated with VEGF(121). Therefore, VEGF therapy has only limited benefits on the healing of renal lesions in the salt-dependent phase of post-ANG II-mediated hypertension.

Normalizing the bone marrow microenvironment with p38 inhibitor reduces multiple myeloma cell proliferation and adhesion and suppresses osteoclast formation.

The multiple myeloma (MM) bone marrow (BM) microenvironment plays a critical role in supporting tumor growth and survival as well as in promoting formation of osteolytic lesions. Recent results suggest that the p38 mitogen-activated protein kinase (MAPK) is an important factor in maintaining this activated environment. In this report, we demonstrate that the p38alpha MAPK inhibitor, SCIO-469, suppresses secretion of the tumor-supportive factors IL-6 and VEGF from BM stromal cells (BMSCs) as well as cocultures of BMSCs with MM cells, resulting in reduction in MM cell proliferation. Additionally, we show that SCIO-469 prevents TNFalpha-induced adhesion of MM cells to BMSCs through an ICAM-1- and VCAM-1-independent mechanism. Microarray analysis revealed a novel set of TNFalpha-induced chemokines in BMSCs that is strongly inhibited by SCIO-469. Furthermore, reintroduction of chemokines CXCL10 and CCL8 to BMSCs overcomes the inhibitory effect of SCIO-469 on TNFalpha-induced MM adhesion. Lastly, we show that SCIO-469 inhibits secretion and expression of the osteoclast-activating factors IL-11, RANKL, and MIP-1alpha as well as prevents human osteoclast formation in vitro. Collectively, these results suggest that SCIO-469 treatment can suppress factors in the bone marrow microenvironment to inhibit MM cell proliferation and adhesion and also to alleviate osteolytic activation in MM.

Preventive and therapeutic potential of p38 alpha-selective mitogen-activated protein kinase inhibitor in nonobese diabetic mice with type 1 diabetes.

Mitogen-activated protein kinases (MAPKs) and heat shock proteins (HSPs) are ubiquitous proteins that function within T cells in both normal and stress-related pathophysiological states, including type 1 diabetes. The nonobese diabetic (NOD) mouse spontaneously develops T cell-mediated autoimmune pancreatic beta cell destruction that is similar to type 1 diabetes in humans. Because p38 MAPKs have been shown to modulate T cell function, we studied the effects of a p38alpha MAPK-selective inhibitor, indole-5-carboxamide (SD-169), on the development and progression of type 1 diabetes in the NOD mouse. In preventive treatment studies, SD-169 significantly reduced p38 and HSP60 expression in T cells of the pancreatic beta islets. Following treatment, the incidence of diabetes as determined by blood glucose levels was significantly lower, and immuno-histochemistry of pancreatic beta islet tissue demonstrated significant reduction in CD5+ T cell infiltration in the SD-169 treatment group as compared with untreated NOD mice. In therapeutic studies using mildly and moderately hyperglycemic NOD mice, SD-169 treatment lowered blood glucose and improved glucose homeostasis. Furthermore, following cessation of SD-169 treatment, NOD mice showed significant arrest of diabetes. In conclusion, we report that this p38alpha-selective inhibitor prevents the development and progression of diabetes in NOD mice by inhibiting T cell infiltration and activation, thereby preserving beta cell mass via inhibition of the p38 MAPK signaling pathway. These results have bearing on current prophylactic and therapeutic protocols using p38alpha-selective inhibitors in the prediabetic period for children at high risk of type 1 diabetes, in the honeymoon period, and for adults with latent autoimmune diabetes.

Affymetrix oligonucleotide analysis of gene expression in the injured heart.

Microarrays have helped researchers gain much insight into gene expression profiles in the context of many diseases including those in the injured heart. Our genomic investigations have been focused on elucidation of host gene responses to enterovirus infection. We have gained valuable technical expertise in using Affymetrix oligonucleotide arrays, also known as GeneChips, and cDNA spotted arrays to probe differential gene expression in both cultured cells and in heart tissue. Here, we provide a technique-focused supplement to the Affymetrix GeneChip Expression Analysis Manual for sample preparation, processing, and array hybridization. We provide expanded explanations to highlight important points within the existing protocol and offer variations to standard procedures when appropriate. For investigators using myocardial tissues for microarray experiments, we further address the necessity of and methods for in situ flushing of the vasculature, tissue homogenization, and considerations for limits of expression detection in rare cells. It is our intention to provide useful technical information, based on our experience, to assist those researchers using Affymetrix GeneChips in their own genomic research.

Role of ERK1/2 and p38 mitogen-activated protein kinases in the regulation of thrombospondin-1 by TGF-beta1 in rat proximal tubular cells and mouse fibroblasts.

Thrombospondin-1 (TSP-1) inhibits angiogenesis and activates latent TGF-beta1, both of which are strongly associated with progression of renal disease. Recently, it was reported that Smad2 but not Smad3 regulates TSP-1 expression in response to TGF-beta1 in rat tubular epithelial cells as well as in mouse fibroblasts. This study investigated the role of ERK1/2 and p38 mitogen-activated protein kinases (MAPK). TGF-beta1 activated both ERK1/2 and p38 in the rat proximal tubular cell line NRK52E. Blocking ERK1/2 and p38 inhibited TGF-beta1-induced TSP-1 mRNA and protein expression. Next, the cross-talk between Smad2 and ERK1/2 or p38 was examined. Whereas blocking of ERK1/2 or p38 failed to inhibit TGF-beta1-induced Smad2 activation, inhibition of Smad2 by Smad7 overexpression inhibited the phosphorylation of ERK1/2 but not p38 in response to TGF-beta1. Similar results were observed using mouse fibroblasts from Smad2 knockout embryos, in that TGF-beta1 was able to activate p38 but not ERK1/2 in this cell line. In conclusion, TSP-1 expression is regulated by both ERK1/2 and p38 MAPK in rat proximal tubular cells and mouse fibroblasts in response to TGF-beta1. The ERK1/2 activation is dependent on Smad2 activation, whereas the p38 activation occurs independent of Smad2. Because TSP-1 is a major antiangiogenic molecule and an activator of TGF-beta1, this provides an important insight to the mechanism by which TGF-beta1 may mediate interstitial fibrosis and progressive renal disease.

TGF-beta induces proangiogenic and antiangiogenic factors via parallel but distinct Smad pathways.

BACKGROUND: Angiogenesis has a key role in numerous disease processes. One of the most important angiogenic factors is vascular endothelial growth factor (VEGF-A), whereas thrombospondin-1 (TSP-1) is a major antiangiogenic factor. Recent studies have shown that VEGF-A as well as TSP-1 is regulated by transforming growth factor-beta1 (TGF-beta1), but the mechanism remains unclear. METHODS: We examined the role of TGF-beta1 and its signaling pathways in mediating expression of these two molecules. Rat proximal tubular cells (NRK52E) were stimulated with TGF-beta1 to induce VEGF-A and TSP-1 synthesis. To clarify roles of receptor-activated Smads (R-Smads), we blocked Smad signaling using overexpression of the inhibitory Smad, Smad7, and by using fibroblasts from wild-type or knockout mice. To confirm the antiantigenic role of Smads, soluble Flt-1 regulation in response to TGF-beta1 was also examined. In addition, the effect of conditioned media from NRK52E and Smad knockout cells was examined on endothelial cell proliferation. RESULTS: Induction of VEGF-A and TSP-1 by TGF-beta1 in NRK52E cells was associated with activation of pathway-restricted R-Smads (Smad2 and 3) and blocking these Smads by overexpression of Smad7 blocked their induction. By using of Smad knockout cells, Smad3 was shown to have a key role in the stimulation of VEGF-A expression whereas Smad2 was critical for TSP-1 expression. Consistent with the hypothesis that Smad2 has an antiangiogenic function, we also demonstrated that Smad2, but not Smad3, mediated the expression of VEGF-A antagonist, soluble VEGF-A receptor sFlt-1, in response to TGF-beta1. Conditioned media from NRK52E, which was stimulated by TGF-beta1 for 24 hours, did not induce endothelial cell proliferation. However, conditioned media from Smad2 knockout induced endothelial cell proliferation, whereas endothelial cell proliferation was inhibited by Smad3 knockout-derived conditioned media. CONCLUSION: R-Smads have distinct roles in mediating the expression of pro- and antiangiogenic growth factors in response to TGF-beta1.

Differential regulation of VEGF by TGF-beta and hypoxia in rat proximal tubular cells.

VEGF expression by proximal tubular epithelial cells may play a critical role in maintaining peritubular capillary endothelium in renal disease. Two major processes involved in renal injury include hypoxia (from vasoconstriction or vascular injury) and transforming growth factor (TGF)-beta-dependent fibrosis, both of which are known to stimulate VEGF. Because the TGF-beta/Smad pathway is activated in hypoxia, we tested the hypothesis that the induction of VEGF in hypoxia could be partially dependent on TGF-beta. Rat proximal tubular (NRK52E) cells treated with TGF-beta under normoxic conditions secreted VEGF at 24 h, and this was significantly reduced by blocking Smad activation by overexpressing the inhibitory Smad7 or by blocking p38 and ERK1/2 MAP kinase activation or protein kinase C activation with specific inhibitors. With acute hypoxia, rat proximal tubular cells also express VEGF mRNA and protein as well as TGF-beta. However, the induction of VEGF occurs before synthesis of TGF-beta and is not blocked by either a TGF-beta antagonist, by Smad7 overexpression, or by blockage of ERK1/2, whereas induction is blocked by PKC inhibition or partially blocked by a p38 inhibitor. Finally, the addition of TGF-beta with hypoxia results in significantly more VEGF expression than either stimulation alone. Thus TGF-beta and hypoxia act via additive/synergistic but distinct pathways to stimulate VEGF in proximal tubular cells, a finding that may be important in understanding how VEGF is stimulated in renal disease.

Increased oxidative stress following acute and chronic high altitude exposure.

The generation of reactive oxygen species is typically associated with hyperoxia and ischemia reperfusion. Recent evidence has suggested that increased oxidative stress may occur with hypoxia. We hypothesized that oxidative stress would be increased in subjects exposed to high altitude hypoxia. We studied 28 control subjects living in Lima, Peru (sea level), at baseline and following 48 h exposure to high altitude (4300 m). To assess the effects of chronic altitude exposure, we studied 25 adult males resident in Cerro de Pasco, Peru (altitude 4300 m). We also studied 27 subjects living in Cerro de Pasco who develop excessive erythrocytosis (hematocrit > 65%) and chronic mountain sickness. Acute high altitude exposure led to increased urinary F(2)-isoprostane, 8-iso PGF(2 alpha) (1.31 +/- 0.8 microg/g creatinine versus 2.15 +/- 1.1, p = 0.001) and plasma total glutathione (1.29 +/- 0.10 micromol versus 1.37 +/- 0.09, p = 0.002), with a trend to increased plasma thiobarbituric acid reactive substance (TBARS) (59.7 +/- 36 pmol/mg protein versus 63.8 +/- 27, p = NS). High altitude residents had significantly elevated levels of urinary 8-iso PGF(2 alpha) (1.3 +/- 0.8 microg/g creatinine versus 4.1 +/- 3.4, p = 0.007), plasma TBARS (59.7 +/- 36 pmol/mg protein versus 85 +/- 28, p = 0.008), and plasma total glutathione (1.29 +/- 0.10 micromol versus 1.55 +/- 0.19, p < 0.0001) compared to sea level. High altitude residents with excessive erythrocytosis had higher levels of oxidative stress compared to high altitude residents with normal hematological adaptation. In conclusion, oxidative stress is increased following both acute exposure to high altitude without exercise and with chronic residence at high altitude.

The role of p38alpha mitogen-activated protein kinase activation in renal fibrosis.

The p38 mitogen-activated protein kinase (MAPK) pathway transduces external stress stimuli and is important in extracellular matrix synthesis in cell types in vitro; however, its role in renal fibrosis is not known. Explored was the role the p38 MAPK pathway in rat unilateral ureteric obstruction (UUO), a model of renal fibrosis induced by a noninflammatory surgical insult. In a time-course study, a marked increase in phosphorylation (activation) of p38 in both interstitial myofibroblasts and tubules was shown. Rats were then treated daily with a specific inhibitor of p38alpha, NPC 31169, from the time of UUO surgery until being killed 7 d later. Compared with vehicle, NPC 31169-treated rats had a significant reduction in renal fibrosis assessed by interstitial volume, collagen IV deposition, and mRNA levels. This was primarily due to a reduction in the accumulation of interstitial myofibroblasts, as shown by a reduction in the area of immunostaining for alpha-smooth muscle actin and heat shock protein 47. The increase in renal TGF-beta1 mRNA and protein levels in UUO was unaltered with NPC 31169 treatment; however, connective tissue growth factor mRNA was reduced. These results demonstrate that p38alpha MAPK plays an important role in renal fibrosis, acting downstream of TGF-beta1. Blockade of p38 MAPK reduces extracellular matrix production and may be considered a potential therapeutic option in the treatment of renal fibrosis.

B-type natriuretic peptide exerts broad functional opposition to transforming growth factor-beta in primary human cardiac fibroblasts: fibrosis, myofibroblast conversion, proliferation, and inflammation.

The natriuretic peptides, including human B-type natriuretic peptide (BNP), have been implicated in the regulation of cardiac remodeling. Because transforming growth factor-beta (TGF-beta) is associated with profibrotic processes in heart failure, we tested whether BNP could inhibit TGF-beta-induced effects on primary human cardiac fibroblasts. BNP inhibited TGF-beta-induced cell proliferation as well as the production of collagen 1 and fibronectin proteins as measured by Western blot analysis. cDNA microarray analysis was performed on RNA from cardiac fibroblasts incubated in the presence or absence of TGF-beta and BNP for 24 and 48 hours. TGF-beta, but not BNP, treatment resulted in a significant change in the RNA profile. BNP treatment resulted in a remarkable reduction in TGF-beta effects; 88% and 85% of all TGF-beta-regulated mRNAs were affected at 24 and 48 hours, respectively. BNP opposed TGF-beta-regulated genes related to fibrosis (collagen 1, fibronectin, CTGF, PAI-1, and TIMP3), myofibroblast conversion (alpha-smooth muscle actin 2 and nonmuscle myosin heavy chain), proliferation (PDGFA, IGF1, FGF18, and IGFBP10), and inflammation (COX2, IL6, TNFalpha-induced protein 6, and TNF superfamily, member 4). Lastly, BNP stimulated the extracellular signal-related kinase pathway via cyclic guanosine monophosphate-dependent protein kinase signaling, and two mitogen-activated protein kinase kinase inhibitors, U0126 and PD98059, reversed BNP inhibition of TGF-beta-induced collagen-1 expression. These findings demonstrate that BNP has a direct effect on cardiac fibroblasts to inhibit fibrotic responses via extracellular signal-related kinase signaling, suggesting that BNP functions as an antifibrotic factor in the heart to prevent cardiac remodeling in pathological conditions.

Inhibition of p38 mitogen-activated protein kinase augments progression of remnant kidney model by activating the ERK pathway.

p38, a mitogen-activated protein kinase, is a major intracellular signaling molecule involved in inflammation. To test the hypothesis that p38 mediates renal disease progression, we administered a novel p38 alpha inhibitor, NPC31169, to rats with remnant kidneys (RKs). RK rats showed increased p38 activation at 9 weeks (by p38 kinase assay), which was blocked by the inhibitor. In contrast to our expectation, treatment with the NPC31169 resulted in worse renal function, more proteinuria, and more severe glomerulosclerosis and tubulointerstitial injury. p38 inhibition resulted in marked cell proliferation in RK rats, with more proliferating tubular cells, myofibroblasts, and macrophages. In contrast, p38 suppression resulted in less tubular cell apoptosis. Interestingly, Western blot demonstrated increased ERK1/2 phosphorylation in p38-treated rats. No histological changes were observed in p38 inhibited sham-operated rats. Our findings indicate that, whereas blocking p38 usually shows benefit in inflammatory disease, in this model p38 inhibition resulted in accelerated renal progression. We conclude that blocking p38-dependent inflammation may have resulted in enhanced proliferation and increased ERK1/2 activation, and thereby explains the worse renal lesions observed.

Indole-based heterocyclic inhibitors of p38alpha MAP kinase: designing a conformationally restricted analogue.

p38alpha Mitogen Activated Protein Kinase (MAP kinase) is an intracellular soluble serine threonine kinase. p38alpha kinase is activated in response to cellular stresses, growth factors and cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). The central role of p38alpha activation in settings of both chronic and acute inflammation has led efforts to find inhibitors of this enzyme as possible therapies for diseases such as rheumatoid arthritis, where p38alpha activation is thought to play a causal role. Herein, we report structure-activity relationship studies on a series of indole-based heterocyclic inhibitors that led to the design and identification of a new class of p38alpha inhibitors.

Targeted in vivo labeling of receptors for vascular endothelial growth factor: approach to identification of ischemic tissue.

BACKGROUND: A method for identifying tissue experiencing hypoxic stress due to atherosclerotic vascular disease would be clinically useful. Vascular endothelial growth factor-121 (VEGF121) is an angiogenic protein secreted in response to hypoxia that binds to VEGF receptors overexpressed by ischemic microvasculature. We tested the hypothesis that VEGF receptors could serve as markers for ischemic tissue and hence provide a target for imaging such tissue with radiolabeled human VEGF121. METHODS AND RESULTS: A rabbit model of unilateral hindlimb ischemia was created by femoral artery excision (n=14). Control rabbits (n=5) underwent identical surgery without femoral excision. On postoperative day 10, rabbits were intravenously administered 100 microCi of 111In-labeled recombinant human VEGF121, and biodistribution studies and planar imaging were conducted at 3, 24, and 48 hours. On postmortem gamma counting, there was greater accumulation of 111In-labeled VEGF121 in ischemic than in control tissue (P<0.02). Differential uptake of isotope by ischemic muscle was not seen in rabbits injected with 125I-labeled human serum albumin (n=6). Radioactivity imaged in hindlimb regions of interest was significantly higher in ischemic muscle than in sham-operated and contralateral nonoperated hindlimb at 3 hours (P<0.02). Immunohistochemical staining confirmed upregulation of VEGF receptors in ischemic skeletal muscle. CONCLUSIONS: Identification of the ischemic state via targeted radiolabeling of hypoxia-induced angiogenic receptors is possible. This approach could be useful for monitoring the efficacy of revascularization strategies such as therapeutic angiogenesis.

Blockade of p38alpha MAPK ameliorates acute inflammatory renal injury in rat anti-GBM glomerulonephritis.

The p38 mitogen-activated protein kinase (MAPK) pathway is a pro-inflammatory signal transduction pathway. The aim of this study was to examine the role of this pathway in acute renal inflammation. Immunostaining localized components of the p38 MAPK pathway (p38alpha, p-p38, p-ATF-2) in normal glomeruli, to podocytes, and occasional endothelial cells. This study identified an eightfold increase in glomerular activation of p38 MAPK (phosphorylated p38, p-p38) within 3 h of the induction of rat anti-glomerular basement membrane (GBM) glomerulonephritis and localized p-p38 and p-ATF-2 to infiltrating neutrophils, with increased staining of podocytes and endothelial cells. The relevance of these findings to human acute inflammatory renal disease was determined by examination of biopsy specimens. In patients with post-infectious glomerulonephritis, there was an increased number of positive p-p38 glomerular cells, including p-p38 staining of infiltrating neutrophils, compared with normal human kidney. In rats, administration of a specific p38 MAPK inhibitor, NPC 31145, before induction of anti-GBM disease prevented a loss of renal function and substantially reduced proteinuria. The reduction in renal injury was attributed to a 55% reduction in glomerular neutrophil infiltration and a 68% reduction in platelet accumulation. This was associated with an abrogation of glomerular P-selectin immunostaining and inhibition of glomerular P-selectin gene expression. In summary, this study has localized the components of the p38 MAPK pathway to cells in normal and diseased rat and human kidney and identified a number of important mechanisms by which signaling through the p38 MAPK pathway induces inflammatory renal disease. Blockade of the p38 pathway may be a novel therapeutic strategy for the treatment of acute renal inflammation.