OTUD1 promotes hypertensive kidney fibrosis and injury by deubiquitinating CDK9 in renal epithelial cells.

Hypertensive renal disease (HRD) contributes to the progression of kidney dysfunction and ultimately leads to end-stage renal disease. Understanding the mechanisms underlying HRD is critical for the development of therapeutic strategies. Deubiquitinating enzymes (DUBs) have been recently highlighted in renal pathophysiology. In this study, we investigated the role of a DUB, OTU Domain-Containing Protein 1 (OTUD1), in HRD models. HRD was induced in wild-type or Otud1 knockout mice by chronic infusion of angiotensin II (Ang II, 1 µg/kg per min) through a micro-osmotic pump for 4 weeks. We found that OTUD1 expression levels were significantly elevated in the kidney tissues of Ang II-treated mice. Otud1 knockout significantly ameliorated Ang II-induced HRD, whereas OTUD1 overexpression exacerbated Ang II-induced kidney damage and fibrosis. Similar results were observed in TCMK-1 cells but not in SV40 MES-13 cells following Ang II (1 µM) treatment. In Ang II-challenged TCMK-1 cells, we demonstrated that OTUD1 bound to CDK9 and induced CDK9 deubiquitination: OTUD1 catalyzed K63 deubiquitination on CDK9 with its Cys320 playing a critical role, promoting CDK9 phosphorylation and activation to induce inflammatory responses and fibrosis in kidney epithelial cells. Administration of a CDK9 inhibitor NVP-2 significantly ameliorated Ang II-induced HRD in mice. This study demonstrates that OTUD1 mediates HRD by targeting CDK9 in kidney epithelial cells, suggesting OTUD1 is a potential target in treating this disease.

15-Lipoxygenase worsens renal fibrosis, inflammation, and metabolism in a murine model of ureteral obstruction.

15-Lipoxygenase (15-LO) is a nonheme iron-containing dioxygenase that has both pro- and anti-inflammatory roles in many tissues and disease states. 15-LO is thought to influence macrophage phenotype, and silencing 15-LO reduces fibrosis after acute inflammatory triggers. The goal of the present study was to determine whether altering 15-LO expression influences inflammation and fibrogenesis in a murine model of unilateral ureteral obstruction (UUO). C57BL/6J mice, 15-LO knockout (Alox15-/-) mice, and 15-LO transgenic overexpressing (15LOTG) mice were subjected UUO, and kidneys were analyzed at 3, 10, and 14 days postinjury. Histology for fibrosis, inflammation, cytokine quantification, flow cytometry, and metabolomics were performed on injured tissues and controls. PD146176, a specific 15-LO inhibitor, was used to complement experiments involving knockout animals. Compared with wild-type animals undergoing UUO, Alox15-/- mouse kidneys had less proinflammatory, profibrotic message along with less fibrosis and macrophage infiltration. PD146176 inhibited 15-LO and resulted in reduced fibrosis and macrophage infiltration similar to Alox15-/- mice. Flow cytometry revealed that Alox15-/- UUO-injured kidneys had a dynamic change in macrophage phenotype, with an early blunting of CD11bHiLy6CHi "M1" macrophages and an increase in anti-inflammatory CD11bHiLy6CInt "M2c" macrophages and reduced expression of the fractalkine receptor chemokine (C-X3-C motif) receptor 1. Many of these findings were reversed when UUO was performed on 15LOTG mice. Metabolomics analysis revealed that wild-type kidneys developed a glycolytic shift postinjury, while Alox15-/- kidneys exhibited increased oxidative phosphorylation. In conclusion, 15-LO manipulation by genetic or pharmacological means induces dynamic changes in the inflammatory microenvironment in the UUO model and appears to be critical in the progression of UUO-induced fibrosis.NEW & NOTEWORTHY 15-Lipoxygenase (15-LO) has both pro- and anti-inflammatory functions in leukocytes, and its role in kidney injury and repair is unexplored. Our study showed that 15-LO worsens inflammation and fibrosis in a rodent model of chronic kidney disease using genetic and pharmacological manipulation. Silencing 15-LO promotes an increase in M2c-like wound-healing macrophages in the kidney and alters kidney metabolism globally, protecting against anaerobic glycolysis after injury.

Ulinastatin Attenuates LPS-Induced Inflammation and Inhibits Endoplasmic Reticulum Stress-Induced Apoptosis in Renal Tubular Epithelial Cells via Regulation of the TLR4/NF-κB and Nrf2/HO-1 Pathways.

Acute kidney injury (AKI) is one of the most common diseases in patients treated in intensive care units. This study was intended to explore the underlying mechanism by which ulinastatin (UTI) influenced the inflammation and apoptosis of renal tubular epithelial cells, HK-2.The effects of UTI on the cell viability of HK-2 cells were first measured by MTT and lactate dehydrogenase (LDH) detection kit. The apoptosis and inflammation of HK-2 cells were then determined by TUNEL, western blot, ELISA, and RT-qPCR. Then, the proteins in the Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2)/Heme oxygenase 1 (HO-1) signaling pathways were measured by western blot for confirming the relationship between UTI and these pathways. Finally, Nrf-2 inhibitor ML385 and TLR4 activator CCL-34 were respectively used on LPS-induced HK-2 cells exposed to UTI for the conduction of gain-of-function and loss-of-function assays.UTI treatment boosted the cell viability of HK-2 cells damaged by LPS. Furthermore, UTI exposure cut down the apoptosis rate and inhibited the expression inflammatory factors of HK-2 cells induced by LPS. UTI treatment decreased the expression of proteins in the TLR4/NF-κB pathway, increased the HO-1 expression, and prompted the translocation of Nrf2 from the cytoplasm to the nucleus. The alleviated effects of UTI on inflammation and apoptosis LPS-induced HK-2 cells were abolished by ML385 and TLR4, respectively.UTI attenuates LPS-induced inflammation and inhibits endoplasmic reticulum stress-induced apoptosis in renal tubular epithelial cells by regulating TLR4/NF-κB and Nrf2/HO-1 pathways.

CDK9 inhibition improves diabetic nephropathy by reducing inflammation in the kidneys.

Diabetic nephropathy (DN) is a chronic inflammatory renal disease induced by hyperglycemia. Recent studies have implicated cyclin-dependent kinase 9 (CDK9) in inflammatory responses and renal fibrosis. In this study, we explored a potential role of CDK9 in DN by using cultured mouse mesangial cell line SV40 MES-13 and streptozotocin-induced type 1 mouse model of diabetes. We inhibited CDK9 in mice and in cultured cells by a highly selective CDK9 inhibitor, LDC000067 (LDC), and evaluated inflammatory and fibrogenic outcome by mRNA and protein analyses. Our studies show that treatment of diabetic mice with LDC significantly inhibits the levels of inflammatory cytokines and fibrogenic genes in kidney specimens. These reductions were associated with improved renal function. We also found that LDC treatment suppressed MAPK-AP1 activation. We then confirmed the involvement of CDK9 in cultured SV40 MES-13 cells and showed that deficiency in CDK9 prevents glucose-induced inflammatory and fibrogenic proteins. This protection was also afforded by suppression of MAPK-AP1. Taken together, our results how that hyperglycemia activates CDK9-MAPK-AP1 axis in kidneys to induce inflammation and fibrosis, leading to renal dysfunction. Our findings also suggest that CDK9 may serve as a potential therapeutic target for DN.

Protective role of DJ-1 in endotoxin-induced acute kidney injury.

Acute kidney injury (AKI) is a frequent complication of sepsis and an important cause of morbidity and mortality worldwide. A cornerstone of sepsis-associated AKI is dysregulated inflammation, leading to increased tissue oxidative stress and free radical formation, which leads to multiple forms of cell death. DJ-1 is a peroxiredoxin protein with multiple functions, including its ability to control cellular oxidative stress. Although DJ-1 is expressed prominently by renal tubules, its role in AKI has not been investigated. In the present study, we examined the effect of DJ-1 deficiency in a murine model of endotoxin-induced AKI. Endotoxemia induced greater kidney injury in DJ-1-deficient mice. Furthermore, DJ-1 deficiency increased renal oxidative stress associated with increased renal tubular apoptosis and with expression of death domain-associated protein (DAXX). Similar to the in vivo model, in vitro experiments using a medullary collecting duct cell line (mIMCD3) and cytotoxic serum showed that serum obtained from wild-type mice resulted in increased expression of s100A8/s100A9, DAXX, and apoptosis in DJ-1-deficient mIMCD3 cells. Our findings demonstrate a novel renal protective role for renal tubular DJ-1 during endotoxemia through control of oxidative stress, renal inflammation, and DAXX-dependent apoptosis.

Dicer deficiency in proximal tubules exacerbates renal injury and tubulointerstitial fibrosis and upregulates Smad2/3.

Renal fibrosis is a common pathological feature in chronic kidney disease (CKD), including diabetic kidney disease (DKD) and obstructive nephropathy. Multiple microRNAs have been implicated in the pathogenesis of both DKD and obstructive nephropathy, although the overall role of microRNAs in tubular injury and renal fibrosis in CKD is unclear. Dicer (a key RNase III enzyme for microRNA biogenesis) was specifically ablated from kidney proximal tubules in mice via the Cre-lox system to deplete micoRNAs. Proximal tubular Dicer knockout (PT- Dicer KO) mice and wild-type (WT) littermates were subjected to streptozotocin (STZ) treatment to induce DKD or unilateral ureteral obstruction (UUO) to induce obstructive nephropathy. Renal hypertrophy, renal tubular apoptosis, kidney inflammation, and tubulointerstitial fibrosis were examined. Compared with WT mice, PT- Dicer KO mice showed more severe tubular injury and renal inflammation following STZ treatment. These mice also developed higher levels of tubolointerstitial fibrosis. Meanwhile, PT- Dicer KO mice had a significantly higher Smad2/3 expression in kidneys than WT mice (at 6 mo of age) in both control and STZ-treated mice. Similarly, UUO induced more severe renal injury, inflammation, and interstitial fibrosis in PT- Dicer KO mice than WT. Although we did not detect obvious Smad2/3 expression in sham-operated mice (2-3 mo old), significantly more Smad2/3 was induced in obstructed PT- Dicer KO kidneys. These results supported a protective role of Dicer-dependent microRNA synthesis in renal injury and fibrosis development in CKD, specifically in DKD and obstructive nephropathy. Depletion of Dicer and microRNAs may upregulate Smad2/3-related signaling pathway to enhance the progression of CKD.

Inhibition of HDAC enhances STAT acetylation, blocks NF-κB, and suppresses the renal inflammation and fibrosis in Npr1 haplotype male mice.

Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) plays a critical role in the regulation of blood pressure and fluid volume homeostasis. Mice lacking functional Npr1 (coding for GC-A/NPRA) exhibit hypertension and congestive heart failure. However, the underlying mechanisms remain largely less clear. The objective of the present study was to determine the physiological efficacy and impact of all-trans-retinoic acid (ATRA) and sodium butyrate (NaBu) in ameliorating the renal fibrosis, inflammation, and hypertension in Npr1 gene-disrupted haplotype (1-copy; +/-) mice (50% expression levels of NPRA). Both ATRA and NaBu, either alone or in combination, decreased the elevated levels of renal proinflammatory and profibrotic cytokines and lowered blood pressure in Npr1+/- mice compared with untreated controls. The treatment with ATRA-NaBu facilitated the dissociation of histone deacetylase (HDAC) 1 and 2 from signal transducer and activator of transcription 1 (STAT1) and enhanced its acetylation in the kidneys of Npr1+/- mice. The acetylated STAT1 formed a complex with nuclear factor-κB (NF-κB) p65, thereby inhibiting its DNA-binding activity and downstream proinflammatory and profibrotic signaling cascades. The present results demonstrate that the treatment of the haplotype Npr1+/- mice with ATRA-NaBu significantly lowered blood pressure and reduced the renal inflammation and fibrosis involving the interactive roles of HDAC, NF-κB (p65), and STAT1. The current findings will help in developing the molecular therapeutic targets and new treatment strategies for hypertension and renal dysfunction in humans.

Inflammation and Edema in the Lung and Kidney of Hemorrhagic Shock Rats Are Alleviated by Biliary Tract External Drainage via the Heme Oxygenase-1 Pathway.

The lung and kidney are two organs that are easily affected by hemorrhagic shock (HS). We investigated roles of biliary tract external drainage (BTED) in inflammation and edema of the lung and kidney in HS and its relationship with the heme oxygenase-1 (HO-1) pathway. Rat models of HS were induced by drawing blood from the femoral artery until a mean arterial pressure (MAP) of 40 ± 5 mmHg was achieved. A MAP of 40 ± 5 mmHg was maintained for 60 min. Thirty-six Sprague-Dawley rats were randomized to the following groups: sham group; HS group; HS + zinc protoporphyrin IX (ZnPP), a specific HO-1 inhibitor, group; HS + BTED group; HS + BTED + ZnPP group; and HS + BTED + bile infusion (BI) group. HO-1 levels, aquaporin-1 levels, and ratios of dry/wet in the lung and kidney increased markedly after BTED, but tumor necrosis factor-α and myeloperoxidase levels in the lung and kidney decreased significantly after BTED under HS conditions. Under the condition that HO-1 was inhibited by ZnPP, all these effects induced by BTED disappeared in the lung and kidney. These results demonstrated that inflammation and edema of the lung and kidney of HS rats are alleviated by BTED via the HO-1 pathway.

Signaling Rho-kinase mediates inflammation and apoptosis in T cells and renal tubules in cisplatin nephrotoxicity.

Nephrotoxicity is a frequent complication of cisplatin-induced chemotherapy, in which T cells are known to promote acute kidney injury (AKI). Apoptosis and necrosis of tubules and inflammatory events also contribute to the nephrotoxicity. A delineation of the mechanisms that underlie the inappropriate renal and tubular inflammation can thus provide important insights into potential therapies for cisplatin-induced AKI. Rho-kinases are known to act as molecular switches controlling several critical cellular functions, including cell migration, cytokine production, and apoptosis. Here, we show that the Rho-kinase inhibitor fasudil attenuated cisplatin nephrotoxicity, resulting in less histological damage, improved renal function, and the infiltration of fewer leukocytes into the kidney. Renal nuclear factor-κB activation and apoptosis were reduced, and the expressions of proinflammatory renal cytokine and chemokine mRNA were decreased. Urinary and renal kidney injury molecule-1 (Kim-1) expression was also reduced, a finding that is consistent with diminished kidney injury. In the current study, we also showed that fasudil could be protective of the impaired tubules. In vitro, fasudil reduced the apoptosis (annexin-V+PI cells) and cytokine production (tumor necrosis factor+ cells) in T cells and the apoptosis (annexin-V+PI cells) and tubular damage (Kim-1+ cells) in proximal tubular cells by flow cytometric analysis. As Rho-kinase plays an important role in promoting cisplatin nephrotoxicity, inhibiting Rho-kinase may be a therapeutic strategy for preventing cisplatin-induced AKI.

Isolation and sequencing of swine carbonic anhydrase VI, an enzyme expressed in the swine kidney.

BACKGROUND: Carbonic anhydrase VI (CA-VI) is produced by the salivary gland and is secreted into the saliva. Although CA-VI is found in the epithelial cells of distal straight tubule of swine kidneys, the exact function of CA-VI in the kidneys remains unclear. RESULTS: CA-VI was located in the epithelial cells of distal straight tubule of swine kidneys.A full-length cDNA clone of CA-VI was generated from the swine parotid gland by reverse transcription polymerase chain reaction, using degenerate primers designed based on conserved regions of the same locus in human and bovine tissues. The cDNA sequence was 1348 base pairs long and was predicted to encode a 317 amino acid polypeptide with a putative signal peptide of 17 amino acids. The deduced amino acid sequence of mature CA-VI was most similar (77.4%) to that of human CA-VI. CA-VI expression was confirmed in both normal and nephritic kidneys, as well as parotid. As the primers used in this study spanned two exons, the influence of genomic DNA was not detected. The expression of CA-VI was demonstrated in both normal and nephritic kidneys, and mRNA of CA-VI in the normal kidneys which was the normalised to an endogenous ß-actin was 0.098 ± 0.047, while it was significantly lower in the diseased kidneys (0.012 ± 0.007). The level of CA-VI mRNA in normal kidneys was 19-fold lower than that of the parotid gland (1.887). CONCLUSIONS: The localisation of CA-VI indicates that it may play a specialised role in the kidney.

Integrin-linked kinase: a new member of the kinases involved in hypertensive end-organ damage?

Integrin-linked kinase predominantly localizes at focal adhesions to regulate actin cytoskeletal dynamics, including cell migration and matrix remodelling. Although recent studies have suggested both physiological and pathophysiological roles of integrin-linked kinase in the cardiovascular and renal system, its involvement in hypertensive organ dysfunctions, such as those that occur in kidney, has not been investigated. In the present issue of Clinical Science, Alique and co-workers have demonstrated that angiotensin II-induced renal inflammatory responses were attenuated in mice with conditional deficiency of integrin-linked kinase, which were associated with suppression of nuclear factor κB activation and reactive oxygen species generation but not hypertension. The significance, potential mechanisms and future direction are presented and discussed in this Commentary.

Integrin-linked kinase plays a key role in the regulation of angiotensin II-induced renal inflammation.

ILK (integrin-linked kinase) is an intracellular serine/threonine kinase involved in cell-matrix interactions. ILK dysregulation has been described in chronic renal disease and modulates podocyte function and fibrosis, whereas data about its role in inflammation are scarce. AngII (angiotensin II) is a pro-inflammatory cytokine that promotes renal inflammation. AngII blockers are renoprotective and down-regulate ILK in experimental kidney disease, but the involvement of ILK in the actions of AngII in the kidney has not been addressed. Therefore we have investigated whether ILK signalling modulates the kidney response to systemic AngII infusion in wild-type and ILK-conditional knockout mice. In wild-type mice, AngII induced an inflammatory response, characterized by infiltration of monocytes/macrophages and lymphocytes, and up-regulation of pro-inflammatory factors (chemokines, adhesion molecules and cytokines). AngII activated several intracellular signalling mechanisms, such as the NF-κB (nuclear factor κB) transcription factor, Akt and production of ROS (reactive oxygen species). All these responses were prevented in AngII-infused ILK-deficient mice. In vitro studies characterized further the mechanisms regulating the inflammatory response modulated by ILK. In cultured tubular epithelial cells ILK blockade, by siRNA, inhibited AngII-induced NF-κB subunit p65 phosphorylation and its nuclear translocation. Moreover, ILK gene silencing prevented NF-κB-related pro-inflammatory gene up-regulation. The results of the present study demonstrate that ILK plays a key role in the regulation of renal inflammation by modulating the canonical NF-κB pathway, and suggest a potential therapeutic target for inflammatory renal diseases.

Protective role of the endothelial isoform of nitric oxide synthase in ANG II-induced inflammatory responses in the kidney.

In the present study, we examine the hypothesis that the nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays a protective role in the development of ANG II-induced hypertension and renal injury by minimizing oxidative stress and the inflammation induced by TNF-α. Systolic blood pressure (SBP) and renal injury responses to chronic infusions of ANG II (via implanted minipumps) were evaluated for 2 wk in wild-type (WT) and in eNOS knockout mice (KO) cotreated with or without a superoxide (O2(-)) scavenger, tempol (400 mg/l in the drinking water), or a TNF-α receptor blocker, etanercept (5 mg/kg/day ip). In study 1, when ANG II was given at a dose of 25 ng/min, it increased mean SBP in WT mice (Δ36 ± 3 mmHg; n = 7), and this effect was attenuated in mice pretreated with tempol (Δ24 ± 3 mmHg; n = 6). In KO mice (n = 9), this dose of ANG II resulted in severe renal injury associated with high mortality. To avoid this high mortality in KO, study 2 was conducted with a lower dose of ANG II (10 ng/min) that increased SBP slightly in WT (Δ17 ± 7 mmHg; n = 6) but exaggeratedly in KO (Δ48 ± 12 mmHg, n = 6) associated with severe renal injury. Cotreatment with either tempol (n = 6) or etanercept (n = 6) ameliorated the hypertensive, as well as the renal injury responses in KO compared with WT. These data demonstrate a protective role for eNOS activity in preventing renal inflammatory injury and hypertension induced by chronic increases in ANG II.

Loss of GSTM1, a NRF2 target, is associated with accelerated progression of hypertensive kidney disease in the African American Study of Kidney Disease (AASK).

Oxidative stress is acknowledged to play a role in kidney disease progression. Genetic variants that affect the capacity to handle oxidative stress may therefore influence the outcome of kidney disease. We examined whether genetic variants of the GSTM1 gene, a member of a superfamily of glutathione S-transferases, influence the course of kidney disease progression in participants of the African American Study of Kidney Disease (AASK) trial. Groups with and without the common GSTM1 null allele, GSTM1(0), differed significantly in the time to a glomerular filtration rate (GFR) event or dialysis (P = 0.04) and in the time to GFR event, dialysis, or death (P = 0.02). The hazard ratios (HR) for the time to a GFR event or dialysis in those with two or one null allele relative to those possessing none were 1.88 [95% confidence interval (CI), 1.07 to 3.30, P = 0.03] and 1.68 (95% CI, 1.00 to 2.84, P < 0.05), respectively. For the time to GFR event, dialysis, or death, the HR for two null alleles was 2.06 (95% CI, 1.20 to 3.55, P = 0.01) and for one null allele 1.70 (95% CI, 1.02 to 2.81, P = 0.04). We demonstrated that GSTM1 directly regulates intracellular levels of 4-hydroxynonenal (4-HNE) in vascular smooth muscle cells. Furthermore, we showed that renal 4-HNE levels and GSTM1 are both increased after reduction of renal mass (RRM) in the mouse. We conclude that GSTM1 is normally upregulated in chronic kidney disease (CKD) in a protective response to increased oxidative stress. A genetic variant that results in loss of GSTM1 activity may be deleterious in CKD.

Poly(ADP-ribose) polymerase 1 activation is required for cisplatin nephrotoxicity.

Apoptosis, necrosis, and inflammation are hallmarks of cisplatin nephrotoxicity; however, the role and mechanisms of necrosis and inflammation remains undefined. As poly(ADP-ribose) polymerase 1 (PARP1) inhibition or its gene deletion is renoprotective in several renal disease models, we tested whether its activation may be involved in cisplatin nephrotoxicity. Parp1 deficiency was found to reduce cisplatin-induced kidney dysfunction, oxidative stress, and tubular necrosis, but not apoptosis. Moreover, neutrophil infiltration, activation of nuclear factor-κB, c-Jun N-terminal kinases, p38 mitogen-activated protein kinase, and upregulation of proinflammatory genes were all abrogated by Parp1 deficiency. Using proximal tubule epithelial cells isolated from Parp1-deficient and wild-type mice and pharmacological inhibitors, we found evidence for a PARP1/Toll-like receptor 4/p38/tumor necrosis factor-α axis following cisplatin injury. Furthermore, pharmacological inhibition of PARP1 protected against cisplatin-induced kidney structural/functional damage and inflammation. Thus, our findings suggest that PARP1 activation is a primary signal and its inhibition/loss protects against cisplatin-induced nephrotoxicity. Targeting PARP1 may offer a potential therapeutic strategy for cisplatin nephrotoxicity.

Focal adhesion kinase signaling mediates acute renal injury induced by ischemia/reperfusion.

Renal ischemia/reperfusion (I/R) injury is associated with cell matrix and focal adhesion remodeling. Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that localizes at focal adhesions and regulates their turnover. Here, we investigated the role of FAK in renal I/R injury, using a novel conditional proximal tubule-specific fak-deletion mouse model. Tamoxifen treatment of FAK(loxP/loxP)//γGT-Cre-ER(T2) mice caused renal-specific fak recombination (FAK(ΔloxP/ΔloxP)) and reduction of FAK expression in proximal tubules. In FAK(ΔloxP/ΔloxP) mice compared with FAK(loxP/loxP) controls, unilateral renal ischemia followed by reperfusion resulted in less tubular damage with reduced tubular cell proliferation and lower expression of kidney injury molecule-1, which was independent from the postischemic inflammatory response. Oxidative stress is involved in the pathophysiology of I/R injury. Primary cultured mouse renal cells were used to study the role of FAK deficiency for oxidative stress in vitro. The conditional fak deletion did not affect cell survival after hydrogen peroxide-induced cellular stress, whereas it impaired the recovery of focal adhesions that were disrupted by hydrogen peroxide. This was associated with reduced c-Jun N-terminal kinase-dependent phosphorylation of paxillin at serine 178 in FAK-deficient cells, which is required for focal adhesion turnover. Our findings support a role for FAK as a novel factor in the initiation of c-Jun N-terminal kinase-mediated cellular stress response during renal I/R injury and suggest FAK as a target in renal injury protection.

Deletion of soluble epoxide hydrolase gene improves renal endothelial function and reduces renal inflammation and injury in streptozotocin-induced type 1 diabetes.

Studies suggest that soluble epoxide hydrolase (sEH) inhibition reduces end-organ damage in cardiovascular diseases. We hypothesize that sEH gene (Ephx2) knockout (KO) improves endothelial function and reduces renal injury in streptozotocin-induced diabetes. After 6 wk of diabetes, afferent arteriolar relaxation to acetylcholine was impaired in diabetic wild-type (WT) mice, as the maximum relaxation was 72% of baseline diameter in the WT but only 31% in the diabetic mice. Ephx2 KO improved afferent arteriolar relaxation to acetylcholine in diabetes as maximum relaxation was 58%. Urinary monocyte chemoattractant protein-1 (MCP-1) excretion significantly increased in diabetic WT mice compared with control (868 ± 195 vs. 31.5 ± 7 pg/day), and this increase was attenuated in diabetic Ephx2 KO mice (420 ± 98 pg/day). The renal phospho-IKK-to-IKK ratio and nuclear factor-κB were significantly decreased, and hemeoxygenase-1 (HO-1) expression increased in diabetic Ephx2 KO compared with diabetic WT mice. Renal NADPH oxidase and urinary thiobarbituric acid reactive substances excretion were reduced in diabetic Ephx2 KO compared with diabetic WT mice. Albuminuria was also elevated in diabetic WT mice compared with control (170 ± 43 vs. 37 ± 13 µg/day), and Ephx2 KO reduced this elevation (50 ± 15 µg/day). Inhibition of sEH using trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (tAUCB) also reduced renal inflammation and injury in diabetic WT mice. Furthermore, inhibition of HO with stannous mesoporphyrin negated the reno-protective effects of tAUCB or Ephx2 KO during diabetes. These data demonstrate that Ephx2 KO improves endothelial function and reduces renal injury during diabetes. Additionally, our data also suggest that activation of HO-1 contributes to improved renal injury in diabetic Ephx2 KO mice.

Glutamine contributes to ameliorate inflammation after renal ischemia/reperfusion injury in rats.

The aim of this study was to investigate the effects of glutamine in an in vivo rat model of renal ischemia/reperfusion (I/R) injury. Male Wistar rats underwent bilateral renal pedicle clamping for 45 min followed by reperfusion for 6 h. Glutamine (1.5 mg/kg) was administered intraperitoneally (i.p.) 15 min prior to reperfusion. Plasma concentrations of urea, creatinine, γ-glutamyl transferase (γ-GT), and aspartate aminotransferase (AST) were measured for the assessment of renal function and reperfusion injury. Markers of oxidative stress, expression of the pro-inflammatory mediators inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), AT-1 expression, and changes in the oxidative stress-sensitive nuclear factor kappa B (NF-κB) signaling pathway were measured to investigate whether glutamine can reduce the renal dysfunction. Kidney myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured for assessment of polymorphonuclear (PMN) cell infiltration and lipid peroxidation, respectively. Renal sections were used for histologic grading of renal injury and for immunohistochemical localization of nitrotyrosine and poly(ADP-ribose) synthetase (PARS). In vivo, glutamine significantly reduced the increase in urea, creatinine, γ-GT, AST, produced by renal ischemia/reperfusion (I/R), suggesting an improvement in both renal function and injury. Glutamine significantly reduced iNOS and NF-κB, kidney MPO activity and MDA levels, indicating a reduction in PMN infiltration and lipid peroxidation, respectively. Glutamine reduced the histological evidence of renal damage associated with I/R and caused a substantial reduction in the staining for nitrotyrosine and PARS, suggesting reduced nitrosative and oxidative stress. Moreover, glutamine attenuated the reduction of COX-2 expression and prevented the increased AT-1 expression after I/R. Our results suggest that glutamine reduces the renal dysfunction and injury associated with I/R of the kidney.

A protective role of Mer receptor tyrosine kinase in nephrotoxic serum-induced nephritis.

The Mer receptor tyrosine kinase is strongly expressed in the glomerulus. We wondered if this molecule might modify immune-mediated glomerular disease through its functions as a receptor for apoptotic cells and immunoregulatory molecule. Mer-knockout (KO) mice showed decreased survival rate and greatly increased proteinuria and serum urea levels compared to wild type (WT) mice by day 3 after injection of NTS. Their glomeruli were hyperplastic and later became necrotic. In the glomerulus of WT mice, a significant increase of Mer expression was observed. Apoptotic bodies were evident in NTS-treated Mer-KO kidneys, but not in normal controls. NTS-treated Mer-KO mice had massive neutrophil infiltration and inflammatory cytokine expression. Mer thus has a critical role in attenuating renal inflammation, both as a receptor for apoptotic cells and as a molecule that downregulates inflammation.