Therapies to limit the life-threatening vascular leak observed in patients with acute lung injury (ALI) are currently lacking. We explored the effect of simvastatin, a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor that mediates endothelial cell barrier protection in vitro, in a murine inflammatory model of ALI. C57BL/6J mice were treated with simvastatin (5 or 20 mg/kg body wt via intraperitoneal injection) 24 h before and again concomitantly with intratracheally administered LPS (2 microg/g body wt). Inflammatory indexes [bronchoalveolar lavage (BAL) myeloperoxidase activity and total neutrophil counts assessed at 24 h with histological confirmation] were markedly increased after LPS alone but significantly reduced in mice that also received simvastatin (20 mg/kg; approximately 35-60% reduction). Simvastatin also decreased BAL albumin (approximately 50% reduction) and Evans blue albumin dye extravasation into lung tissue (100%) consistent with barrier protection. Finally, the sustained nature of simvastatin-mediated lung protection was assessed by analysis of simvastatin-induced gene expression (Affymetrix platform). LPS-mediated lung gene expression was significantly modulated by simvastatin within a number of gene ontologies (e.g., inflammation and immune response, NF-kappaB regulation) and with respect to individual genes implicated in the development or severity of ALI (e.g., IL-6, Toll-like receptor 4). Together, these findings confirm significant protection by simvastatin on LPS-induced lung vascular leak and inflammation and implicate a potential role for statins in the management of ALI.

Studies in humans and animal models have demonstrated that acute kidney injury (AKI) has a significant effect on the function of extrarenal organs. The combination of AKI and lung dysfunction is associated with 80% mortality; the lung, because of its extensive capillary network, is a prime target for AKI-induced effects. The study presented here tested the hypothesis that AKI leads to a vigorous inflammatory response and produces distinct genomic signatures in the kidney and lung. In a murine model of ischemic AKI, prominent global transcriptomic changes and histologic injury in both kidney and lung tissues were identified. These changes were evident at both early (6 h) and late (36 h) timepoints after 60-min bilateral kidney ischemia and were more prominent than similar timepoints after sham surgery or 30 min of ischemia. The inflammatory transcriptome (109 genes) of both organs changed with marked similarity, including the innate immunity genes Cd14, Socs3, Saa3, Lcn2, and Il1r2. Functional genomic analysis of these genes suggested that IL-10 and IL-6 signaling was involved in the distant effects of local inflammation, and this was supported by increased serum levels of IL-10 and IL-6 after ischemia-reperfusion. In summary, this is the first comprehensive analysis of concomitant inflammation-associated transcriptional changes in the kidney and a remote organ during AKI. Functional genomic analysis identified potential mediators that connect local and systemic inflammation, suggesting that this type of analysis may be a useful discovery tool for novel biomarkers and therapeutic drug development. Clinical studies have revealed a strong association between AKI and dysfunction of extrarenal organs, and more recently animal research has shown a significant causal effect of AKI on distant organ dysfunction. [1][2][3][4][5][6][7] Since the availability of dialysis, AKIassociated distant organ dysfunction constitutes the major cause of death in these patients, with the mortality rate still in the 50% range. Despite this frustrating outcome, little is known about the potential pathophysiological interactions between the kidney and extrarenal organs in critically ill patients. Numerous recent studies have demonstrated that outcomes of AKI are heavily dependent upon the severity of comorbid conditions. 8 -10 Isolated AKI has a much better prognosis than AKI associated with multiple organ failure, 11,12 and the presence of renal insufficiency continues to be a sensitive marker for poor outcome in the hospitalized patient. 13 Thus, there is an urgent need to study the systemic effects of AKI, and modern discovery tools have the potential to unveil novel diagnostic and therapeutic targets.Inflammation is a major component of the initiation and exacerbation of kidney injury during AKI, 14,15 and local inflammation of kidney tissues could be a source of the development of inflamma-

Oxidative stress is involved in acute kidney injury due to ischemia-reperfusion and chemotherapy-induced nephrotoxicity. To investigate their basic mechanisms we studied the role of nuclear factor-erythroid 2-p45-related factor 2 (Nrf2), a redox-sensitive transcription factor that regulates expression of several antioxidant and cytoprotective genes. We compared the responses of Nrf2-knockout mice and their wild-type littermates in established mouse models of ischemia-reperfusion injury and cisplatin-induced nephrotoxicity. Several Nrf2-regulated genes encoding antioxidant enzymes/proteins were significantly upregulated in the kidneys of wild type but not Nrf2-knockout mice following renal ischemia. Renal function, histology, vascular permeability, and survival were each significantly worse in the Nrf2 knockout mice. Further, proinflammatory cytokine and chemokine expression tended to increase after ischemia in the knockout compared to the wild-type mice. Treatment of the knockout mice with the antioxidants N-acetyl-cysteine or glutathione improved renal function. The knockout mice were more susceptible to cisplatin-induced nephrotoxicity, and this was blunted by N-acetyl-cysteine pretreatment. Our study demonstrates that Nrf2-deficiency enhances susceptibility to both ischemic and nephrotoxic acute kidney injury, and identifies this transcription factor as a potential therapeutic target in these injuries.

Hassoun HT, Grigoryev DN, Lie ML, Liu M, Cheadle C, Tuder RM, Rabb H. Ischemic acute kidney injury induces a distant organ functional and genomic response distinguishable from bilateral nephrectomy. Am J Physiol Renal Physiol 293: F30-F40, 2007. First published February 27, 2007 doi:10.1152/ajprenal.00023.2007.-Acute kidney injury (AKI) is associated with significant mortality, which increases further when combined with acute lung injury. Experiments in rodents have shown that kidney ischemia-reperfusion injury (IRI) facilitates lung injury and inflammation. To identify potential ischemia-specific lung molecular pathways involved, we conducted global gene expression profiling of lung 6 or 36 h following 1) bilateral kidney IRI, 2) bilateral nephrectomy (BNx), and 3) sham laparotomy in C57BL/6J mice. Bronchoalveolar lavage fluid analysis revealed increased total protein, and lung histology revealed increased cellular inflammation following IRI, but not BNx, compared with sham controls. Total RNA from whole lung was isolated and hybridized to 430MOEA (22,626 genes) GeneChips (n ϭ 3/group), which were analyzed by robust multichip average and significance analysis of microarrays and linked to gene ontology (GO) terms using MAPPFinder. The microarray power analysis predicted that the false discovery rate (q Ͻ 1%) and Ն50%-fold change compared with sham would represent significant changes in gene expression. Analysis identified 266 and 455 ischemia-specific, AKI-associated lung genes with increased expression and 615 and 204 with decreased expression at 6 and 36 h, respectively, compared with sham controls. Real-time PCR analysis validated select array changes in lung serum amyloid A3 and endothelin-1. GO analysis revealed significant activation (Z Ͼ 1.95) of several proinflammatory and proapoptotic biological processes. Ischemic AKI induces functional and transcriptional changes in the lung distinct from those induced by uremia alone. Further investigation using this lung molecular signature induced by kidney IRI will provide mechanistic insights and new therapies for critically ill patients with AKI.ischemia-reperfusion; lung injury; microarray ISCHEMIC ACUTE KIDNEY INJURY (AKI) occurs in various clinical settings including shock, sepsis, transplantation, and vascular surgery. Despite advances in renal replacement therapy, the mortality of patients with AKI has remained high over the past few decades, and renal insufficiency continues to be a sensitive marker for a poor outcome in critically ill patients (2, 22). Epidemiological studies have identified an association between AKI and dysfunction of extrarenal organs (4). Given the persistent AKI-associated poor clinical outcomes, there is a need to study the systemic and remote organ effects that occur in the context of AKI.Acute lung injury (ALI) is a significant cause of respiratory failure in the intensive care unit and carries a substantial mortality of 30 -40% (8). When combined with AKI, mortality approaches 80% (26). The clinical presentation of AKI-associate...

Obstructive sleep apnea (OSA), a condition tightly linked to obesity, leads to chronic intermittent hypoxia (CIH) during sleep. There is emerging evidence that OSA is independently associated with insulin resistance and fatty liver disease, suggesting that OSA may affect hepatic lipid metabolism. To test this hypothesis, leptin-deficient obese (ob/ob) mice were exposed to CIH during the light phase (9 AM-9 PM) for 12 wk. Liver lipid content and gene expression profile in the liver (Affymetrix 430 GeneChip with real-time PCR validation) were determined on completion of the exposure. CIH caused a 30% increase in triglyceride and phospholipid liver content (P < 0.05), whereas liver cholesterol content was unchanged. Gene expression analysis showed that CIH upregulated multiple genes controlling 1) cholesterol and fatty acid biosynthesis [malic enzyme and acetyl coenzyme A (CoA) synthetase], 2) predominantly fatty acid biosynthesis (acetyl-CoA carboxylase and stearoyl-CoA desaturases 1 and 2), and 3) triglyceride and phospholipid biosynthesis (mitochondrial glycerol-3-phosphate acyltransferase). A majority of overexpressed genes were transcriptionally regulated by sterol regulatory element-binding protein (SREBP) 1, a master regulator of lipogenesis. A 2.8-fold increase in SREBP-1 gene expression in CIH was confirmed by real-time PCR (P = 0.001). Expression of major genes of cholesterol biosynthesis, SREBP-2 and 3-hydroxy-3-methylglutaryl-CoA reductase, was unchanged. In conclusion, we have shown that CIH may exacerbate preexisting fatty liver of obesity via upregulation of the pathways of lipid biosynthesis in the liver.

The statins, a class of HMG-CoA reductase inhibitors, directly affect multiple vascular processes via inhibition of geranylgeranylation, a covalent modification essential for Rho GTPase interaction with cell membrane-bound activators. We explored simvastatin effects on endothelial cell actomyosin contraction, gap formation, and barrier dysfunction produced by the edemagenic agent, thrombin. Human pulmonary artery endothelial cells exposed to prolonged simvastatin treatment (5 microM, 16 h) demonstrated significant reductions in thrombin-induced (1 U/ml) barrier dysfunction ( approximately 70% inhibition) with accelerated barrier recovery, as measured by transendothelial resistance. Furthermore, simvastatin attenuated basal and thrombin-stimulated (1 U/ml, 5 min) myosin light chain diphosphorylation and stress fiber formation while dramatically increasing peripheral immunostaining of actin and cortactin, an actin-binding protein, in conjunction with increased Rac GTPase activity. As both simvastatin-induced Rac activation and barrier protection were delayed (maximal after 16 h), we assessed the role of gene expression and protein translation in the simvastatin response. Simultaneous treatment with cycloheximide (10 microg/ml, 16 h) abolished simvastatin-mediated barrier protection. Robust alterations were noted in the expression of cytoskeletal proteins (caldesmon, integrin beta4), thrombin regulatory elements (PAR-1, thrombomodulin), and signaling genes (guanine nucleotide exchange factors) in response to simvastatin by microarray analysis. These novel observations have broad clinical implications in numerous vascular pathobiologies characterized by alterations in vascular integrity including inflammation, angiogenesis, and acute lung injury.

. TNF-␣ increases tyrosine phosphorylation of vascular endothelial cadherin and opens the paracellular pathway through fyn activation in human lung endothelia.

Acute kidney injury (AKI) is being increasingly shown to be a risk factor for chronic kidney disease (CKD), but little is known about the possible mechanistic links. We hypothesized that analysis of the genomic signature in the repair stage after AKI would reveal pathways that could link AKI and CKD. Unilateral renal pedicle clamping for 45 min was performed in male C57BL/6J mice. Mice were euthanized at 3, 10, and 28 days after ischemia-reperfusion injury (IRI). Total RNA was isolated from kidney and analyzed using an Illumina mouse array. Among 24,600 tested genes, 242, 146, and 46 genes were upregulated at days 3, 10, and 28 after IRI, and 85, 35, and 0 genes were downregulated, respectively. Gene ontology analysis showed that gene expression changes were primarily related to immune and inflammatory pathways both early and late after AKI. The most highly upregulated genes late after AKI were hepatitis A virus cellular receptor 1 (Havcr1) and lipocalin 2 (Lcn2), which code for kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), respectively. This was unexpected since they are both primarily potential biomarkers of the early stage of AKI. Furthermore, increases observed in gene expression in amiloride binding protein 1, vascular cell adhesion molecule-1, and endothelin 1 could explain the salt-sensitive hypertension that can follow AKI. These data suggested that 1) persistent inflammation and immune responses late after AKI could contribute to the pathogenesis of CKD, 2) late upregulation of KIM-1 and NGAL could be a useful marker for sustained renal injury after AKI, and 3) hypertension-related gene changes could underlie mechanisms for persistent renal and vascular injury after AKI.