BRG1 increases transcription of proinflammatory genes in renal ischemia.

Acute kidney injury stimulates renal production of inflammatory mediators, including TNF-alpha and monocyte chemoattractant protein 1 (MCP-1). These responses reflect, in part, injury-induced transcription of proinflammatory genes by proximal tubule cells. Because of the compact structure of chromatin, a series of events at specified loci remodel chromatin to provide access for transcription factors and RNA polymerase II (Pol II). Here, we examined the role of Brahma-related gene-1 (BRG1), a chromatin remodeling enzyme, in the transcription of TNF-alpha and MCP-1 in response to renal ischemia. Two hours after renal ischemic injury in mice, renal TNF-alpha and MCP-1 mRNA increased and remained elevated for at least 1 wk. Matrix chromatin immunoprecipitation assays revealed sustained increases in Pol II at these genes, suggesting that the elevated mRNA levels were, at least in part, transcriptionally mediated. The profile of BGR1 binding to the genes encoding TNF-alpha and MCP-1 resembled Pol II recruitment. Knockdown of BRG1 by small interfering RNA blocked an ATP depletion-induced increase in TNF-alpha and MCP-1 transcription in a human proximal tubule cell line; this effect was associated with decreased recruitment of BRG1 and Pol II to these genes. In conclusion, BRG1 promotes increased transcription of TNF-alpha and MCP-1 by the proximal tubule in response to renal ischemia.

High ambient glucose augments angiotensin II-induced proinflammatory gene mRNA expression in human mesangial cells: effects of valsartan and simvastatin.

BACKGROUND: Hyperglycemia may potentiate the adverse renal effects of angiotensin II (AII). In the kidney, the major target of AII action is the glomerular mesangial cell, where its hemodynamic and proinflammatory action contributes to renal injury. AII action is mediated by several types of cell receptors. Among those, the AT1 receptor has been best studied using specific AII receptor blockers (ARBs). These agents have emerged as major new modalities in the prevention and amelioration of renal disease where the ARB renoprotective anti-inflammatory properties could be more important than previously appreciated. Like the ARBs, statins may also modulate inflammatory responses that are renoprotective and complement their cholesterol-lowering effects. AIM: The aim of this project was to (i) identify a repertoire of proinflammatory mesangial cell AII-inducible mRNAs; (ii) determine if the AII-induced proinflammatory mRNA responses depend on ambient glucose, and (iii) test the anti-inflammatory effectiveness of an ARB, valsartan, either alone or in combination with a statin, simvastatin. RESULTS/CONCLUSIONS: Using high-density microarrays and real-time PCR we identified several AII-inducible proinflammatory mesangial genes that exhibited augmented mRNA responses in high-glucose milieu. Valsartan blocked the AII-induced mRNA expression of proinflammatory genes (i.e. MCP-1, LIF and COX-2) maintained in normal and high glucose. These observations add to the mounting evidence that ARBs have anti-inflammatory effects in the kidney, a beneficial effect that may be more important in protecting renal function in diabetic patients. While simvastatin inhibited expression of some mRNAs encoding chemokines/cytokines, it enhanced expression of mRNA encoding COX-2, a key mediator of inflammation. Thus, the non-cholesterol effects of statins on inflammatory responses appear complex.

Renal ischemia-induced cholesterol loading: transcription factor recruitment and chromatin remodeling along the HMG CoA reductase gene.

Acute kidney injury evokes renal tubular cholesterol synthesis. However, the factors during acute kidney injury that regulate HMG CoA reductase (HMGCR) activity, the rate-limiting step in cholesterol synthesis, have not been defined. To investigate these factors, mice were subjected to 30 minutes of either unilateral renal ischemia or sham surgery. After 3 days, bilateral nephrectomy was performed and cortical tissue extracts were prepared. The recruitment of RNA polymerase II (Pol II), transcription factors (SREBP-1, SREBP-2, NF-kappaB, c-Fos, and c-Jun), and heat shock proteins (HSP-70 and heme oxygenase-1) to the HMGCR promoter and transcription region (start/end exons) were assessed by Matrix ChIP assay. HMGCR mRNA, protein, and cholesterol levels were determined. Finally, histone modifications at HMGCR were assessed. Ischemia/reperfusion (I/R) induced marked cholesterol loading, which corresponded with elevated Pol II recruitment to HMGCR and increased expression levels of both HMGCR protein and mRNA. I/R also induced the binding of multiple transcription factors (SREBP-1, SREBP-2, c-Fos, c-Jun, NF-kappaB) and heat shock proteins to the HMGCR promoter and transcription regions. Significant histone modifications (increased H3K4m3, H3K19Ac, and H2A.Z variant) at these loci were also observed but were not identified at either the 5' and 3' HMGCR flanking regions (+/-5000 bps) or at negative control genes (beta-actin and beta-globin). In conclusion, I/R activates the HMGCR gene via multiple stress-activated transcriptional and epigenetic pathways, contributing to renal cholesterol loading.

Monitoring of blood cyclosporine concentration in steroid-resistant nephrotic syndrome.

OBJECTIVE: Cyclosporine has been used for patients with nephrotic syndrome. Because of substantial inter- and intra-patient variability and a narrow therapeutic window, drug monitoring of cyclosporine is mandatory. To confirm the therapeutic effects of a cyclosporine microemulsion (CSAME), the absorption profile of the agent after preprandial administration was determined in steroid-resistant patients with refractory nephrotic syndrome. METHODS: Fourteen patients were enrolled into the study (mean age, 31.2+/-12; 6 men, 8 women). The patients received 1.5 mg/kg of cyclosporine 30 minutes before breakfast for 6 months. Blood cyclosporine concentration was measured 5 times serially: before administration (C0) and at 1-hour intervals until 4 hours after administration of cyclosporine (C1-C4). In addition, area under the concentration-time curve from 0-4 hours (AUC0-4) was calculated. RESULTS: After 6 months, CSAME showed marked improvement in proteinuria levels (8.3+/-4.8 g/day vs 0.8+/-0.4 g/day, p<0.001). No changes in serum creatinine and urea nitrogen levels were observed. In 83% of the patients, the CSAME peak concentration appeared within 1 hour after administration (C1). A strong positive correlation was noted between AUC0-4 and C1 (R2=0.90312) and C2 (R2=0.78431). The mean steroid (prednisolone) dose was 40 mg/day when CSAME treatment was started, but a lowering of the dose to 17.5 mg/day (p<0.001) was achieved at 6 months after CSAME therapy. CONCLUSION: Preprandial administration of CSAME is effective in steroid-resistant patients with refractory nephrotic syndrome. C1 or C2, but not C0, was a good clinical marker for CSAME exposure.

Growth and development alter susceptibility to acute renal injury.

Many of the studies of acute renal injury have been conducted in young mice usually during their rapid growth phase; yet, the impact of age or growth stage on the degree of injury is unknown. To address this issue, we studied three forms of injury (endotoxemic-, glycerol-, and maleate-induced) in mice ranging in age from adolescence (3 weeks) to maturity (16 weeks). The severity of injury within each model significantly correlated with weight and age. We also noticed a progressive age-dependent reduction in renal cholesterol content, a potential injury modifier. As the animals grew and aged they also exhibited stepwise decrements in the mRNAs of HMG CoA reductase and the low density lipoprotein receptor, two key cholesterol homeostatic genes. This was paralleled by decreased amounts of RNA polymerase II and the transcription factor SREBP1/2 at the reductase and lipoprotein receptor gene loci as measured by chromatin immunoprecipitation. Our study shows that the early phase of mouse growth can profoundly alter renal susceptibility to diverse forms of experimental acute renal injury.

Endotoxin mediates recruitment of RNA polymerase II to target genes in acute renal failure.

Acute renal failure (ARF) sensitizes the kidney to endotoxin (LPS)-driven production of cytokines and chemokines. This study assessed whether this LPS hyperresponsiveness exists at the genomic level. Three heterogeneous mouse models of ARF were studied: Maleate nephrotoxicity, unilateral ureteral obstruction, and LPS preconditioning. In all cases, LPS was injected approximately 18 h after injury was induced, and over the next 0 to 90 min, RNA polymerase II recruitment to the genome at three LPS-responsive genes (TNF-alpha, monocyte chemoattractant-1 [MCP-1], and heme oxygenase-1 [HO-1]) was assessed by chromatin immunoprecipitation. LPS hyperresponsiveness was noted in each model, measured by exaggerated increases in TNF-alpha and MCP-1 mRNA (approximately two to 10 times higher than LPS-injected controls). Corresponding increases in the recruitment of RNA polymerase II to the TNF-alpha and MCP-1 genes were observed, and increased trimethylation of histone 3 lysine 4 (H3K4m3) at these sites may have played a role in this recruitment. Conversely, recruitment of RNA polymerase II to the HO-1 gene was suppressed ("tolerance"), and no increase in H3K4m3 was observed at HO-1 exons. The ARF-induced changes in mRNA did not correlate with mRNA stability, suggesting the mechanistic importance of RNA polymerase II-mediated transcriptional events. In conclusion, LPS hyperresponsiveness after ARF is likely mediated at the genomic level, possibly by H3K4m3.

Maleate nephrotoxicity: mechanisms of injury and correlates with ischemic/hypoxic tubular cell death.

Maleate injection causes dose-dependent injury in proximal tubular cells. This study sought to better define underlying pathogenic mechanisms and to test whether maleate toxicity recapitulates critical components of the hypoxic/ischemic renal injury cascade. CD-1 mice were injected with maleate or used as a source for proximal tubule segments (PTS) for in vitro studies. Maleate induced dose-dependent PTS injury [lactate deydrogenase (LDH) release, ATP reductions, nonesterified fatty acid (NEFA) accumulation]. These changes were partially dependent on maleate metabolism (protection conferred by metabolic inhibitors: succinate, acetoacetate). Maleate toxicity reproduced critical characteristics of the hypoxia/ATP depletion-induced injury cascade: 1) glutathione (GSH) conferred protection, but due to its glycine, not cysteine (antioxidant), content; 2) ATP reductions reflected decreased production, not Na-K-ATPase-driven increased consumption; 3) cell death was completely blocked by extracellular acidosis (pH 6.6); 4) intracellular Ca(2+) chelation (BAPTA) mitigated cell death; 5) maleate and hypoxia each caused plasma membrane cholesterol shedding and in both instances, this was completely glycine suppressible; 6) maleate + hypoxia caused neither additive NEFA accumulation nor LDH release, implying shared pathogenic pathways; and 7) maleate, like ischemia, induced renal cortical cholesterol loading; increased HMG CoA reductase (HMGCR) activity (statin inhibitable), increased HMGCR mRNA levels, and increased RNA polymerase II recruitment to the HMGCR locus (chromatin immunoprecipitation, ChIP, assay) were involved. These results further define critical determinants of maleate nephrotoxicity and suggest that it can serve as a useful adjunct for studies of ischemia/ATP depletion-induced, proximal tubule-specific, cell death.

Fatal diffuse pulmonary arterial thrombosis as a complication of nephrotic syndrome.

A 21-year-old man was admitted to our hospital because of leg edema. Because laboratory findings revealed massive proteinuria and hypoproteinemia, he was diagnosed as having nephritic syndrome caused by minimal change disease. He was given a continuous heparin infusion and intravenous steroid therapy, at a prednisolone dose of 1 mg/kg per day, and his condition gradually improved. Five months after discharge, the patient's proteinuria relapsed. He was readmitted to our hospital and we restarted anticoagulant treatment with intravenous heparin and 60 mg prednisolone. On the third hospital day, he complained of chest pain with sudden onset and dyspnea. He quickly developed shock and died. The findings of an autopsy confirmed the presence of diffuse fibrin thrombi in bilateral pulmonary arteries, and we diagnosed the cause of death as diffuse pulmonary artery thrombosis. A coagulation test for activated partial thromboplastin time (aPTT) had already shown that aPTT was prolonged before the initiation of treatment. There may have been a deficit of antithrombin III (ATIII) - a cofactor of heparin - because of the proteinuria; thus, the continuous heparin treatment might not have been effective for the prevention of thrombosis. Alternatives to heparin treatment that do not suppress AT III, such as nafamostat mesilate or argatroban, which do not require the presence of AT III for their anticoagulant action, should be considered in cases similar to the that in the patient reported here. In patients with nephrotic syndrome who exhibit altered coagulation test results, the choice of anticoagulation therapy for treatment of the hypercoagulabilty status associated with nephrotic syndrome should be carefully considered.

Effects of an angiotensin II receptor antagonist and angiotensin-converting enzyme inhibitors on burst forming units-erythroid in chronic hemodialysis patients.

BACKGROUND: Angiotensin-converting enzyme (ACE) inhibitors have been reported to reduce the response to erythropoietin (EPO) administration in chronic hemodialysis patients, but the mechanism for this effect has not yet been clarified. To clarify the mechanism of ACE inhibitors- and angiotensin II type 1 (AT1) receptor antagonist-induced anemia in hemodialysis patients, we examined the effect of ACE inhibitors and AT1 receptor antagonist on burst-forming units-erythroid (BFU-E) in the peripheral blood of hemodialysis patients and healthy controls in vitro. METHODS: Peripheral blood mononuclear cells (PBMNCs) were isolated by gradient centrifugation from 10 patients on regular hemodialysis and 7 healthy control volunteers. A colony assay of hematopoietic progenitors was performed using the methylcellulose culture system. PBMNCs of 1 or 2 x 10(5) were plated in a medium containing EPO with various concentrations of ACE inhibitors or AT1 receptor antagonist and incubated for 14 days. Colonies of BFU-E were counted under an inverted microscope. RESULTS: The PBMNCs from the chronic hemodialysis patients formed fewer BFU-Es than those from healthy volunteers. AT1 receptor antagonist in both healthy volunteers and hemodialysis patients suppressed the number of BFU-Es. The ACE inhibitors produced a smaller effect than the AT1 receptor antagonist. CONCLUSION: AT1 receptor blockade can directly inhibit erythropoiesis in vitro.