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T cells mediate organ injury and repair. A proportion of unconventional kidney T cells called double-negative (DN) T cells (TCR+ CD4− CD8−), with anti-inflammatory properties, were previously demonstrated to protect from early injury in moderate experimental acute kidney injury (AKI). However, their role in repair after AKI has not been studied. We hypothesized that DN T cells mediate repair after severe AKI. C57B6 mice underwent severe (40 min) unilateral ischemia-reperfusion injury (IRI). Kidney DN T cells were studied by flow cytometry and compared with gold-standard anti-inflammatory CD4+ regulatory T cells (Tregs). In vitro effects of DN T cells and Tregs on renal tubular epithelial cell (RTEC) repair after injury were quantified with live-cell analysis. DN T cells, Tregs, CD4, or vehicle were adoptively transferred after severe AKI. Glomerular filtration rate (GFR) was measured using fluorescein isothiocyanate (FITC)-sinistrin. Fibrosis was assessed with Masson's trichrome staining. Profibrotic genes were measured with qRT-PCR. Percentages and the numbers of DN T cells substantially decreased during repair phase after severe AKI, as well as their activation and proliferation. Both DN T cells and Tregs accelerated RTEC cell repair in vitro. Post-AKI transfer of DN T cells reduced kidney fibrosis and improved GFR, as did Treg transfer. DN T cell transfer lowered transforming growth factor (TGF)β1 and α-smooth muscle actin (αSMA) expression. DN T cells reduced effector-memory CD4+ T cells and IL-17 expression. DN T cells undergo quantitative and phenotypical changes after severe AKI, accelerate RTEC repair in vitro as well as improve GFR and renal fibrosis in vivo. DN T cells have potential as immunotherapy to accelerate repair after AKI. NEW & NOTEWORTHY: Double-negative (DN) T cells (CD4− CD8−) are unconventional kidney T cells with regulatory abilities. Their role in repair from acute kidney injury (AKI) is unknown. Kidney DN T cell population decreased during repair after ischemic AKI, in contrast to regulatory T cells (Tregs) which increased. DN T cell administration accelerated tubular repair in vitro, while after severe in vivo ischemic injury reduced kidney fibrosis and increased glomerular filtration rate (GFR). DN T cell infusion is a potential therapeutic agent to improve outcome from severe AKI. [ABSTRACT FROM AUTHOR]

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 (Havcrl) 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-l, 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-I 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. ischemia-reperfusion injury; gene expression profiling; chronic kidney disease doi: 10.1152/ajprenal.00619.2009.

Early exposure to germs modifies kidney damage and inflammation after experimental ischemia-reperfusion injury. Am J Physiol Renal Physiol 297: F1457-F1465, 2009. First published August 12, 2009; doi:10.1152/ajprenal.90769.2008.--Kidney ischemia-reperfusion injury (IRI) is, in part, mediated by immune and inflammatory factors. Since microbial stimuli are known to alter immune and inflammatory responses, we hypothesized that differences in perinatal microbial status would modify renal injury following IRI. We performed bilateral renal IRI on 6-wk-old germ-free and control mice and studied the effects on kidney lymphocyte trafficking, cytokines, function, and structure. Compared with control mice, normal kidneys of germ-free mice exhibited more NKT cells and lower IL-4 levels. Postischemia, more CD8 T cells trafficked into postischemic kidneys of germ-free mice compared with control mice. Renal structural injury and functional decline following IRI were more severe in germ-free mice compared with control mice. When germ-free mice were conventionalized with the addition of bacteria to their diet, the extent of renal injury after IRI became equivalent to age-matched control mice, with similar numbers and phenotypes of T cells and NKT cells, as well as cytokine expression in both normal kidneys and postischemic kidneys of conventionalized germ-free mice and age-matched control mice. Thus microbial stimuli influence the phenotype of renal lymphocytes and the expression of cytokines of normal kidneys and also modulate the outcome of IRI. germ-free status; immune modulation doi: 10.1152/ajprenal.90769.2008.

Distant organ effects of acute kidney injury (AKI) are a leading cause of morbidity and mortality. While little is known about the underlying mechanisms, limited data suggest a role for inflammation and apoptosis. Utilizing a lung candidate gene discovery approach in a mouse model of ischemic AKI-induced lung dysfunction, we identified prominent lung activation of 66 apoptosis-related genes at 6 and/or 36 h following ischemia, of which 6 genes represent the tumor necrosis factor receptor (TNFR) superfamily, and another 23 genes are associated with the TNFR pathway. Given that pulmonary apoptosis is an important pathogenic mechanism of acute lung injury (ALl), we hypothesized that AKI leads to pulmonary proapoptotic pathways that facilitate lung injury and inflammation. Functional correlation with 1) terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and 2) active caspase-3 (aC3) activity, immunoblotting, and immunohistochemistry (IHC) identified kidney IRI-induced pulmonary apoptosis at 24 h, and colocalization studies with CD34 identified predominantly endothelial apoptosis. Mice were treated with the caspase inhibitor Z-VAD-FMK (0.25 mg ip) or vehicle 1 h before and 8 h after sham or kidney IRI, and bronchoalveolar lavage fluid protein was measured at 36 h as a surrogate for lung leak. Caspase inhibition reduced lung microvascular changes after kidney IRI. The pulmonary apoptosis seen in wild-type control mice during AKI was absent in [TNFR.sup.-/-] mice. Using an initial genomic approach to discovery followed by a mechanistic approach to disease targeting, we demonstrate that pulmonary endothelial apoptosis is a direct mediator of the distant organ dysfunction during experimental AKI. lung injury; AKI; inflammation; TNF receptor

We hypothesized that the influence of acute kidney injury (AKI) on the sensitivity of the lung to an injurious process varies with the severity of the injurious process. Thus, we thought that AKI would exacerbate lung injury from low degrees of lung trauma but attenuate lung injury from higher degrees of lung trauma. C57BL/6 mice underwent AKI (30-min kidney ischemia) or sham surgery, followed at 24 h by 4 h of spontaneous breathing (SB), mechanical ventilation with low tidal volume (7 ml/kg, LTV), or mechanical ventilation with high tidal volume (30 ml/kg, HTV). Compared with LTV, median bronchoalveolar lavage (BAL) protein leak was significantly lower with SB and greater with HTV in both sham and AKI mice. Compared with LTV, median Evans blue dye-labeled albumin extravasation in lungs (L-EBD) was also significantly lower with SB and greater with HTV. L-EBD showed a significant interaction between ventilatory mode and kidney health, such that AKI attenuated the L-EBD rise seen in HTV vs. LTV sham mice. An interaction between ventilatory mode and kidney health could also be seen in BAL neutrophil number (PMN). Thus, AKI attenuated the BAL PMN rise seen in HTV vs. LTV sham mice. These data support the presence of a complex interaction between mechanical ventilation and AKI in which the sensitivity of the lung to trauma varies with the magnitude of the trauma and may involve a modification of pulmonary neutrophil activity by AKI. acute kidney injury; Evans blue dye

Acute lung injury (ALI) in combination with acute kidney injury carries a mortality approaching 80% in the intensive care unit. Recently, attention has focused on the interaction of the lung and kidney in the setting of ALI and mechanical ventilation (MV). Small animal models of ALI and MV have demonstrated changes in inflammatory mediators, water channels, apoptosis, and function in the kidney early in the course of injury. The purpose of this investigation was to test the hypothesis that ALI and injurious MV cause early, measurable changes in kidney structure and function in a canine HC1 aspiration model of ALI when hemodynamics and arterial blood gas tensions are carefully controlled. Intratracheal HC1 induced profound ALI as demonstrated by increased shunt fraction and airway pressures compared with sham injury. Shaminjured animals had similar mean arterial pressure and arterial P[co.sub.2] and HC[O.sub.3] levels compared with injured animals. Measurements of renal function including renal blood flow, urine flow, serum creatinine, glomerular filtration rate, urine albumin-to-creatinine ratio, and kidney histology scores were not different between groups. With maintenance of hemodynamic parameters and alveolar ventilation, ALI and injurious MV do not alter kidney structure and function early in the course of injury in this acid aspiration canine model. Kidney injury in large animal models may be more similar to humans and may differ from results seen in small animal models. mechanical ventilation; biotrauma; renal function

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 = 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 < 1%) and [greater than or equal to]50%-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 > 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 doi: 10.1152/ajpregu.00023.2007

Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury in both native kidneys and renal allografts. Disruption of the actin cytoskeleton is a key event with multiple repercussions on cell adhesion and function during IRI. However, receptors involved in regulating cytoskeletal repair following injury have not been identified. In an in vivo model of renal IRI, we used multiprobe RNase protection assay to examine the expression of Eph receptor tyrosine kinases, key regulators of actin dynamics in embryonic development. We found that one receptor in particular, EphA2, was strongly upregulated in the kidney following IRI. Ephrins, the cell-bound ligands of Eph receptors, were also strongly expressed. In cultured renal tubular cells, diverse injurious stimuli mimicking IRI also resulted in upregulation of EphA2 protein expression. Upregulation of EphA2 was inhibited by the Src kinase inhibitor PP2. Conversely, overexpression of Src kinases strongly enhanced the expression of endogenous EphA2 as well as the activity of a human EphA2 promoter construct. Activation of the Erk pathway was necessary, but not sufficient for full induction of EphA2 upreglation by Src kinases. Stimulation of renal tubular epithelial cells with the EphA2 ligand ephrin-A1 caused tyrosine phosphorylation of endogenous EphA2, paxillin, and an unidentified ~65-kDa protein and resulted in increased cortical F-actin staining. In summary, under in vitro conditions mimicking IRI, EphA2 expression is strongly upregulated through a Src kinase-dependent pathway. Interactions between upregulated EphA2 and its ephrin ligands may provide critical cell contact-dependent, bidirectional cues for cytoskeletal repair in renal IRI. epithelial injury; tyrosine kinases; MDCK; IMCD-3

Severe ischemia-reperfusion injury (IRI) predisposes to king-term impairment in kidney function both in patients and experimentally through unknown mechanisms. Given emerging evidence implicating lymphocytes in the pathogenesis of early injury to kidney, liver, and lung after IRI, we hypothesized that kidney IRI would potentially release or expose normally sequestered antigens that would lead to proliferation of antigen-recognizing lymphocytes. This, in turn, would directly participate in progressive kidney injury. To test this hypothesis, we purified splenic lymphocytes from C57BL/6 mice with severe renal IRI or sham operation 6 wk postischemia and transferred these cells to normal mice. Donor mice with IRI had significant fibrosis and cellular inflammation. The recipient mice were followed for 6 or 12 wk. Donor lymphocytes were found to traffic into recipient kidney. Twelve weeks after transfer, kidneys from mice which received IRI-primed lymphocytes exhibited significantly increased urinary albumin excretion compared with lymphocytes from sham mice. Splenic [CD3.sup.+], [CD4.sup.+], [CD3.sup.+] [CD25.sup.+], and [CD4.sup.+] [CD44.sup.+] counts were significantly increased in mice after lymphocyte transfer from IRI mice vs. mice with lymphocytes from sham mice. These data demonstrate that lymphocytes from IRI mice can traffic to recipient kidney and directly mediate albuminuria. These data identify a novel mechanism by which initial kidney injury predisposes to long-term dysfunction and identify lymphocytes as potential therapeutic targets for progressive renal diseases. chronic kidney disease; inflammation; autoimmunity

In kidneys subjected to ischemic reperfusion injury (IRI) stathmin, a tubulin-binding protein involved in the regulation of mitosis, is expressed in dedifferentiated and proliferating renal tubule cells during the recovery phase. To ascertain the role of stathmin in the recovery from ischemic kidney injury, stathmin-deficient (OP18-/-) and wild-type (WT) animals were subjected to experimental IRI. At 3, 7, and 14 days after reperfusion serum samples and kidneys were collected for the examination of parameters of renal function, morphology, and recovery. Our studies indicate that on day 14 after reperfusion OP18-/- mice have significant renal failure, whereas the creatinine levels of WT animals have returned to baseline. Compared with WT animals OP18-/- mice had more extensive tubular fibrosis. The examination of proliferating cell nuclear antigen expression indicated that OP18-/- animals have increased proliferative or DNA repair activity for a more prolonged duration. The OP18-/- animals also had an increased number of tubules with apoptotic cells. These results suggest that in stathmin-deficient mice subjected to IRI, the aberrant regulation of cell cycle progression, not observed under normal conditions, impairs or at least delays the process of tubular repair and recovery after acute renal injury. acute renal failure; cell proliferation; renal function; fibrosis

Renal ischemia-reperfusion injury (IRI) is the leading cause of acute kidney injury [AKI; acute renal failure (ARF)] in native kidneys and delayed graft function in deceased donor kidney transplants. Serum creatinine rises late after renal IRI, which results in delayed diagnosis. There is an important need to identify novel biomarkers for early diagnosis and prognosis in renal IRI. Given the inflammatory pathophysiology of renal IRI, we used a protein array to measure 18 cytokines and chemokines in a mouse model of renal IRI at 3, 24, and 72 h postischemia. A rise in renal keratinocyte-derived chemokine (KC) was the earliest and most consistent compared with other molecules, with 3-h postischemia values being 9- and 13-fold greater than sham and normal animals, respectively. Histological changes were evident within 1 h of IRI but serum creatinine only increased 24 h after IRI. With the use of an ELISA, KC levels in serum and urine were highest 3 h postischernia, well before a significant rise in serum creatinine. The human analog of KC, Gro-[alpha], was markedly elevated in urine from humans who received deceased donor kidney transplants that required dialysis, compared with deceased donor kidney recipients with good graft function and live donor recipients with minimal ischemia. Measurement of KC and its human analog, Gro-[alpha], could serve as a useful new biomarker for ischemic ARF. growth-related oncogene-[alpha]; transplantation; acute renal failure; allograft

Ischemic renal injury can be classified into the initiation and extension phase followed by the recovery phase. The recovery phase is characterized by increased dedifferentiated and mitotic cells in the damaged tubules. Suppression subtractive hybridization was performed by using RNA from normal and ischemic kidneys to identify the genes involved in the physiological response to ischemia-reperfusion injury (IRI). The expression of stathmin mRNA increased by fourfold at 24 h of reperfusion. The stathmin mRNA did not increase in sodium-depleted animals or in animals with active, persistent injury secondary to cis-platinum. Immunofluorescent labeling demonstrated that the expression of stathmin increased dramatically at 48 h of reperfusion. Labeling with antibodies to stathmin and proliferating cell nuclear antigen (PCNA) indicates that the expression of stathmin was induced before the upregulation of PCNA and that all PCNA-positive cells expressed stathmin. Double immunofluorescent labeling demonstrated the colocalization of stathmin with vimentin, a marker of dedifferentiated cells. Stathmin expression was also significantly enhanced in acute tubular necrosis in humans. On the basis of its induction profile in IRI, the data indicating its enhanced expression in proliferating cells and regenerating organs, we propose that stathmin is a marker of dedifferentiated, mitotically active epithelial cells that may contribute to tubular regeneration and could prove useful in distinguishing the injury phase from recovery phase in IRI. acute renal failure; acute tubular necrosis; ischemia-reperfusion injury; cis-platinum nephrotoxicity; sodium depletion; tubule regeneration

Rat fetal kidney mRNA was analyzed by RT-PCR to identify protein kinases. This screening demonstrated expression of a protein kinase consistent with SK2, a group II germinal center kinase and homolog of human Ste20-like kinase (SLK). SK2 mRNA, protein expression, and kinase activity were increased in rat fetal kidney homogenates (embryonic days 17-21) compared with adult controls. In adult kidneys subjected to cross-clamping of the renal artery, followed by reperfusion, SK2 mRNA, protein expression, and kinase activity were increased compared with untreated contralateral controls. By immunohistochemistry, SK2 expression was evident mainly in the cytoplasm of tubular epithelial cells in fetal and adult kidneys. There was also some expression in developing and mature podocytes, but staining of the interstitium was negative. In cultured renal tubular epithelial cells, SK2 kinase activity was increased after incubation with serum, or alter exposure to chemical anoxia plus reexposure to glucose. Stable overexpression of SLK reduced cell proliferation and increased apoptosis and exacerbated apoptosis and necrosis after chemical anoxia plus reexposure to glucose. Thus SK2 is a renal epithelial protein kinase whose expression and activity are increased during development and recovery from acute renal failure, where tubular epithelial regeneration may recapitulate developmental processes. The actions of SK2 appear to be antiproliferative and may facilitate cell injury. protein kinases; renal development; renal failure; signal transduction

Recent data support a modulatory role for CD4 T cells in experimental renal ischemia-reperfusion injury (IRI). CD4 T cells can functionally differentiate to either a Th1 (IFN-[gamma] producing) or the counterbalancing Th2 (IL-4) phenotype. The enzymes signal transducers and activators of transcription (STAT) 4 and STAT6 regulate Th1 or Th2 differentiation and cytokine production, respectively. We therefore hypothesized that mice that were STAT4 deficient would be protected from renal IRI and that STAT6-deficient mice would have a more severe course. Intracellular cytokine staining of splenocytes from STAT4-/- or STAT6-/- exhibited distinct IFN-[gamma] and IL-4 cytokine expression profiles. STAT6-/- had markedly worse renal function and tubular injury postischemia compared with wild type. STAT4-/had only mildly improved function. Renal phagocyte infiltration and ICAM-1 upregulation were similar in STAT4-/-, STAT6-/-, and wild type. To evaluate if the mechanism of the marked worsening in the STAT6-/- mice could be due to IL-4 deficiency, IL-4-deficient mice were studied and had similar postischemic phenotype to STAT6-/- mice. These data demonstrate that the STAT6 pathway has a major protective role in renal IRI. IL-4 deficiency is a likely mechanism underlying the STAT6 effect. A 'yin-yang' role for inflammation is emerging in renal IRI, similar to recent observations in atherosclerosis. Th1/Th2 cells; inflammation; interleukin-4

Acute renal failure (ARF) commonly occurs after whole body ischemia. Most experimental models of ARF have relied on the isolated renal artery clamping model; however, there is a pressing need to develop and understand the pathogenesis of new models with more 'clinical relevance.' We evaluated a new murine model of ARF after whole body ischemia reperfusion injury (WBIRI). WBIRI was induced by an infusion of potassium chloride and a cardiac arrest period of 10 min. Resuscitation was achieved by cardiac compressions, ventilation, epinephrine, and fluids. WBIRI leads to a significant increase in serum creatinine (SCr) and renal tubular injury by 24 h. Renal myeloperoxidase (MPO) levels increased at 24 h after WBIRI. Increased expression of the proinfiammatory genes, ICAM-1 and IL-6, was also observed in the kidney following WBIRI. On the basis of recent data that T cells are important mediators of isolated renal IRI, WBIRI was evaluated in T cell-deficient nu/nu mice. T cell-deficient mice had a significantly reduced rise in SCr and decreased tubular injury compared with wild-type mice. T cell-deficient mice had a decrease in ICAM-1 expression after WBIRI, but no decrease in renal MPO. This study describes a new, clinically relevant, model of ARF after WBIRI in mice and identifies the T cell as an important mediator of renal injury following WBIRI. Reduced ICAM-1 expression may provide a mechanism for this involvement. lymphocytes; cardiac arrest; kidney dysfunction

Ischemia-reperfusion injury (IRI) is the major cause of acute renal failure in native and allograft kidneys. Identifying the molecules and pathways involved in the pathophysiology of renal IRI will yield valuable new diagnostic and therapeutic information. To identify differentially regulated genes in renal IRI, RNA from rat kidneys subjected to an established renal IRI protocol (bilateral occlusion of renal pedicles for 30 min followed by reperfusion) and time-matched kidneys from sham-operated animals was subjected to suppression subtractive hybridization. The level of spermidine/spermine [N.sup.1]-acetyltransferase (SSAT) mRNA, an essential enzyme for the catabolism of polyamines, increased in renal IRI. SSAT expression was found throughout normal kidney tubules, as detected by nephron segment RT-PCR. Northern blots demonstrated that the mRNA levels of SSAT are increased by greater than threefold in the renal cortex and by fivefold in the renal medulla at 12 h and returned to baseline at 48 h after ischemia. The increase in SSAT mRNA was paralleled by an increase in SSAT protein levels as determined by Western blot analysis. The concentration of putrescine in the kidney increased by ~4- and ~7.5-fold at 12 and 24 h of reperfusion, respectively, consistent with increased functional activity of SSAT. To assess the specificity of SSAT for tubular injury, a model of acute renal failure from [Na.sup.+] depletion (without tubular injury) was studied; SSAT mRNA levels remained unchanged in rats subjected to [Na.sup.+] depletion. To distinguish SSAT increases from the effects of tubular injury vs. uremic toxins, SSAT was increased in cis-platinum-treated animals before the onset of renal failure. The expression of SSAT mRNA and protein increased by ~3.5- and > 10-fold, respectively, in renal tubule epithelial cells subjected to ATP depletion and metabolic poisoning (an in vitro model of kidney IRI). Our results suggest that SSAT is likely a new marker of tubular cell injury that distinguishes acute prerenal from intrarenal failure. acute renal failure; polyamines; putrescine; spermidine/ spermine [N.sup.1]-acetyltransferase

L-selectin is unlikely to play a significant pathological role in renal ischemic-reperfusion injury (IRI) in heart and skeletal muscles. A study using L-selectin-deficient and control mice with ischemia reveals that pathological assessments of these two groups vary very little from each other. Both groups show similar elevations of serum creatinine and blood urea nitrogen, and tubular necrosis postischemia. They are also comparable with regard to the postischemic increase in peritubular neutrophils.

Pretreatment of uninephrectomized Sprague-Dawley rats with monoclonal antibodies (MAbs) against the adhesion molecules CD11a and CD11b or control MAbs, and evaluation of serum creatinine (SCr), kidney histopathological damage scores (PDS) and complete blood count before and 24 h after ischemia of 60 min help determine the involvement of CD11a and CD11b in ischemic-reperfusion organ injury in the kidney. Both anti-CD11a- and the anti-CD11b-treated groups exhibit lower values of mean SCr and PDS 24 hours post-ischemia compared to the control-MAP-treated animals, indicating that ischemic acute renal failure in rats is mediated by the CD11/CD18 leukocyte adhesion pathway.

Nuclear factor erythroid 2-related factor 2 (Nrf2) and hypoxia-inducible factor-1α (HIF1α) transcription factors protect against ischemic acute kidney injury (AKI) by upregulating metabolic and cytoprotective gene expression. In this study, we tested the hypothesis that Nrf2 is required for HIF1α-mediated hypoxic responses using Nrf2-sufficient (wild-type) and Nrf2-deficient (Nrf2–/–) primary murine renal/kidney tubular epithelial cells (RTECs) and human immortalized tubular epithelial cells (HK2 cells) with HIF1 inhibition and activation. The HIF1 pathway inhibitor digoxin blocked hypoxia-stimulated HIF1α activation and heme oxygenase (HMOX1) expression in HK2 cells. Hypoxia-mimicking cobalt (II) chloride-stimulated HMOX1 expression was significantly lower in Nrf2–/– RTECs than in wild-type counterparts. Similarly, hypoxia-stimulated HIF1α-dependent metabolic gene expression was markedly impaired in Nrf2–/– RTECs. Nrf2 deficiency impaired hypoxia-induced HIF1α stabilization independent of increased prolyl 4-hydroxylase gene expression. We found decreased HIF1α mRNA levels in Nrf2–/– RTECs under both normoxia and hypoxia-reoxygenation conditions. In silico analysis and chromatin immunoprecipitation assays demonstrated Nrf2 binding to the HIF1α promoter in normoxia, but its binding decreased in hypoxia-exposed HK2 cells. However, Nrf2 binding at the HIF1α promoter was enriched following reoxygenation, demonstrating that Nrf2 maintains constitutive HIF1α expression. Consistent with this result, we found decreased levels of Nrf2 in hypoxia and that were restored following reoxygenation. Inhibition of mitochondrial complex I prevented hypoxia-induced Nrf2 downregulation and also increased basal Nrf2 levels. These results demonstrate a crucial role for Nrf2 in optimal HIF1α activation in hypoxia and that mitochondrial signaling downregulates Nrf2 levels in hypoxia, whereas reoxygenation restores it. Nrf2 and HIF1α interact to provide optimal metabolic and cytoprotective responses in ischemic AKI. [ABSTRACT FROM AUTHOR]

Acute kidney injury (AKI) due to cisplatin is a significant problem that limits its use as an effective chemotherapeutic agent. T cell receptor+CD4-CD8- double negative (DN) T cells constitute the major T cell population in the human and mouse kidney, express programmed cell death protein (PD)-1, and protect from ischemic AKI. However, the pathophysiological roles of DN T cells in cisplatin-induced AKI is unknown. In this study, wild-type mice were treated with cisplatin (30 mg/kg) or vehicle, and the effects on kidney DN T cell numbers and function were measured. In vitro experiments evaluated effects of kidney DN T cells on cisplatin-induced apoptosis and PD ligand 1 (PD-L1) in renal epithelial cells. Adoptive transfer experiments assessed the therapeutic potential of DN T cells during cisplatin-induced AKI. Our results show that kidney DN T cell population increased at 24 h and declined by 72 h after cisplatin treatment. Cisplatin treatment increased kidney DN T cell proliferation, apoptosis, CD69, and IL-10 expression, whereas CD62L, CD44, IL-17A, interferon-γ, and TNF-α were downregulated. Cisplatin treatment decreased both PD-1 and natural killer 1.1 subsets of kidney DN T cells with a pronounced effect on the PD-1 subset. In vitro kidney DN T cell coculture decreased cisplatin-induced apoptosis in kidney proximal tubular epithelial cells, increased Bcl-2, and decreased cleaved caspase 3 expression. Cisplatin-induced expression of PD ligand 1 was reduced in proximal tubular epithelial cells cocultured with DN T cells. Adoptive transfer of DN T cells attenuated kidney dysfunction and structural damage from cisplatin-induced AKI. These results demonstrate that kidney DN T cells respond rapidly and play a protective role during cisplatin-induced AKI. [ABSTRACT FROM AUTHOR]

Pathophysiological role of T lymphocytes in renal ischemia-reperfusion injury in mice. Am J Physiol Renal Physiol 279: F525-F531, 2000.--Mononuclear cell infiltrates are found in human renal ischemia-reperfusion injury (IRI), and peritubular T lymphocytes have been identified in experimental IRI. However, the role of T cells in the pathogenesis of renal IRI is unknown. We hypothesized that T cells are one of the important mediators of renal IRI. To test this hypothesis, we used an established mouse model of renal IRI, and evaluated mice with genetically engineered deficiency of both CD4+ and CD8+ T cells. At 48 h postischemia, CD4/CD8-knockout (KO) mice had marked improvement in renal function compared with control C57BL/6 mice (serum creatinine: 0.7 [+ or -] 0.4 vs. 2.5 [+ or -] 0.3 mg/dl, respectively; P [is less than] 0.05). Neutrophil infiltration into postischemic kidney was reduced in CD4/CD8 KO mice, compared with control mice, at both 24 h [polymorphonuclear neutrophils (PMNs)/10 high power fields: 714 [+ or -] 354 vs. 3,514 [+ or -] 660, respectively; P [is less than] 0.05] and 48 h (88 [+ or -] 32 vs. 1,979 [+ or -] 209, respectively; P [is less than] 0.05). Tubular necrosis score in CD4/CD8 KO mice, compared with control mice, was significantly less at 48 h (0.4 [+ or -] 0.1 vs. 2.4 [+ or -] 0.2, respectively; P [is less than] 0.05). Because adhesion between T cells and renal tubular epithelial cells (RTECs) may underlie the pathophysiological role of T cells in renal IRI, we also measured T cell adhesion to primary murine RTECs in vitro. Exposure of RTECs to 2 h of hypoxia followed by 1 h of reoxygenation increased T cell adhesion more than twofold. Phorbol ester treatment, which activates integrins, increased T cell adhesion threefold. These data suggest that T lymphocytes can mediate experimental renal IRI. Moreover, adhesion of infiltrating T cells to renal tubular cells may provide a potential mechanism underlying postischemic tubular dysfunction. kidney inflammation; immune response; mouse model

Ischemia-reperfusion (IR)-induced kidney injury is a major clinical problem, but its underlying mechanisms remain unclear. The transcription factor known as nuclear factor, erythroid 2-like 2 (NFE2L2 or Nrf2) is crucial for protection against oxidative stress generated by prooxidant insults. We have previously shown that Nrf2 deficiency enhances susceptibility to IR-induced kidney injury in mice and that its upregulation is protective. Here, we examined Nrf2 target antioxidant gene expression and the mechanisms of its activation in both human and murine kidney epithelia following acute (2 h) and chronic (12 h) hypoxia and reoxygenation conditions. We found that acute hypoxia modestly stimulates and chronic hypoxia strongly stimulates Nrf2 putative target HMOX1 expression, but not that of other antioxidant genes. Inhibition of AKT1/2 or ERK1/2 signaling blocked this induction; AKT1/2 but not ERK1/2 inhibition affected Nrf2 levels in basal and acute hypoxia-reoxygenation states. Unexpectedly, chromatin immunoprecipitation assays revealed reduced levels of Nrf2 binding at the distal AB1 and SX2 enhancers and proximal promoter of HMOX1 in acute hypoxia, accompanied by diminished levels of nuclear Nrf2. In contrast, Nrf2 binding at the AB1 and SX2 enhancers significantly but differentially increased during chronic hypoxia and reoxygenation, with reaccumulation of nuclear Nrf2 levels. Small interfering-RNA-mediated Nrf2 depletion attenuated acute and chronic hypoxia-inducible HMOX1 expression, and primary Nrf2-null kidney epithelia showed reduced levels of HMOX1 induction in response to both acute and chronic hypoxia. Collectively, our data demonstrate that Nrf2 upregulates HMOX1 expression in kidney epithelia through a distinct mechanism during acute and chronic hypoxia reoxygenation, and that both AKT1/2 and ERK1/2 signaling are required for this process. [ABSTRACT FROM AUTHOR]

The posttranscriptional regulation of gene expression occurs through cis RNA regulatory elements by the action of trans factors, which are represented by noncoding RNAs (especially microRNAs) and turnover- and translation-regulatory (TTR) RNAbinding proteins (RBPs). These multifactorial proteins are a group of heterogeneous RBPs primarily implicated in controlling the decay and translation rates of target mRNAs. TTR-RBPs usually shuttle between cellular compartments (the nucleus and cytoplasm) in response to various stimuli and undergo posttranslational modifications such as phosphorylation or methylation to ensure their proper subcellular localization and function. TTR-RBPs are emerging as key regulators of a wide variety of genes influencing kidney physiology and pathology. This review summarizes the current knowledge of TTR-RBPs that influence renal metabolism. We will discuss the role of TTR-RBPs as regulators of kidney ischemia, fibrosis and matrix remodeling, angiogenesis, membrane transport, immunity, vascular tone, hypertension, and acid-base balance as well as anemia, bone mineral disease, and vascular calcification. [ABSTRACT FROM AUTHOR]

Although T cells have been shown to play a direct role in kidney ischemia-reperfusion injury (IRI), little is known about the underlying mechanisms. We hypothesized that studying the transcriptional responses in kidney-infiltrating T cells would help elucidate novel therapeutic targets for kidney IRI. Unilateral renal pedicle clamping for 45 min was performed in male C57BL/6 mice, and CD3+ T cells were isolated from the kidney and purified. Transcriptional activities of T cell were measured by array-based PCR compared between ischemic kidneys and contralateral nonischemic kidneys. Among total of 89 genes analyzed, 24, 22, 24, and 37 genes were significantly changed at 6 h, day 3, day 10, and day 28 after IRI. Genes associated with cytokines, chemokines, and costimulatory molecules were upregulated. Pathway analysis identified CC motif chemokine receptor 5 (CCR5) as a candidate pathophysiological pathway. CCR5 upregulation was validated at the protein level, and CCR5 blockade improved renal function after kidney IRI. Using discovery techniques to identify transcriptional responses in purified kidney-infiltrating cells enabled the elucidation of novel mechanisms and therapeutic targets for IRI. [ABSTRACT FROM AUTHOR]

In this study, we compared the traditional murine model with renal pedicle clamp with models that clamped the renal artery or vein alone as well as to a whole body ischemia-reperfusion injury (WBIRI) model. Male C57BL/6J mice underwent either clamping of the renal artery, vein, or both (whole pedicle) for 30 or 45 min followed by reperfusion, or 10 min of cardiac arrest followed by resuscitation up to 24 h. After 30 min of ischemia, the mice with renal vein clamping showed the mostly increased serum creatinine and the most severe renal tubule injury. After 45 min of ischemia, all mice with renal vasculature clamping had a comparable increase in serum creatinine but the renal tubule injury was most severe in renal artery-clamped mice. Renal arterial blood flow was most decreased in mice with a renal vein clamp compared with a renal artery or pedicle clamp. A 30-or 45-min renal ischemia time led to a significant increase in the protein level of interleukin-6, keratinocyte-derived chemokine (KC), and granular colony-stimulating factor in the ischemic kidney, but the KC was the highest in the renal pedicle-clamped kidney and the lowest in the renal vein-clamped kidney. Of note, 10 min of WBIRI led to kidney dysfunction and structural injury, although less than longer time clamping of isolated renal vasculature. Our data demonstrate important differences in ischemic AKI models. Understanding these differences is important in designing future experimental studies in mice as well as clinical trials in humans. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Kidney ischemia-reperfusion injury (IRI) is, in part, mediated by immune and inflammatory factors. Since microbial stimuli are known to alter immune and inflammatory responses, we hypothesized that differences in perinatal microbial status would modify renal injury following IRI. We performed bilateral renal lRI on 6-wk-old germ-free and control mice and studied the effects on kidney lymphocyte trafficking, cytokines, function, and structure. Compared with control mice, normal kidneys of germ-free mice exhibited more NKT cells and lower IL-4 levels. Postischemia, more CD8 T cells trafficked into postischemic kidneys of germ-free mice compared with control mice. Renal structural injury and functional decline following IRI were more severe in germ-free mice compared with control mice. When germ-free mice were conventionalized with the addition of bacteria to their diet, the extent of renal injury after IRI became equivalent to age-matched control mice, with similar numbers and phenotypes of T cells and NKT cells, as well as cytokine expression in both normal kidneys and postischemic kidneys of conventionalized germ-free mice and age-matched control mice. Thus microbial stimuli influence the phenotype of renal lymphocytes and the expression of cytokines of normal kidneys and also modulate the outcome of IRI. [ABSTRACT FROM AUTHOR]

Discusses the paracrine and differentiation mechanisms underlying stem cell therapy for the damaged kidney. Association of native kidney acute renal failure (ARF) with high mortality; Functional and structural improvement on recovery from ischemic ARF with the administration of bone marrow-derived mesenchymal stem cells; Alternative mechanism beyond differentiation and paracrine effects.

Acute kidney injury (AKI) due to cisplatin is a significant problem that limits its use as an effective chemotherapeutic agent. T cell receptor + CD4 - CD8 - double negative (DN) T cells constitute the major T cell population in the human and mouse kidney, express programmed cell death protein (PD)-1, and protect from ischemic AKI. However, the pathophysiological roles of DN T cells in cisplatin-induced AKI is unknown. In this study, wild-type mice were treated with cisplatin (30 mg/kg) or vehicle, and the effects on kidney DN T cell numbers and function were measured. In vitro experiments evaluated effects of kidney DN T cells on cisplatin-induced apoptosis and PD ligand 1 (PD-L1) in renal epithelial cells. Adoptive transfer experiments assessed the therapeutic potential of DN T cells during cisplatin-induced AKI. Our results show that kidney DN T cell population increased at 24 h and declined by 72 h after cisplatin treatment. Cisplatin treatment increased kidney DN T cell proliferation, apoptosis, CD69, and IL-10 expression, whereas CD62L, CD44, IL-17A, interferon-γ, and TNF-α were downregulated. Cisplatin treatment decreased both PD-1 and natural killer 1.1 subsets of kidney DN T cells with a pronounced effect on the PD-1 subset. In vitro kidney DN T cell coculture decreased cisplatin-induced apoptosis in kidney proximal tubular epithelial cells, increased Bcl-2, and decreased cleaved caspase 3 expression. Cisplatin-induced expression of PD ligand 1 was reduced in proximal tubular epithelial cells cocultured with DN T cells. Adoptive transfer of DN T cells attenuated kidney dysfunction and structural damage from cisplatin-induced AKI. These results demonstrate that kidney DN T cells respond rapidly and play a protective role during cisplatin-induced AKI.

Although T cells have been shown to play a direct role in kidney ischemia-reperfusion injury (IRI), little is known about the underlying mechanisms. We hypothesized that studying the transcriptional responses in kidney-infiltrating T cells would help elucidate novel therapeutic targets for kidney IRI. Unilateral renal pedicle clamping for 45 min was performed in male C57BL/6 mice, and CD3(+) T cells were isolated from the kidney and purified. Transcriptional activities of T cell were measured by array-based PCR compared between ischemic kidneys and contralateral nonischemic kidneys. Among total of 89 genes analyzed, 24, 22, 24, and 37 genes were significantly changed at 6 h, day 3, day 10, and day 28 after IRI. Genes associated with cytokines, chemokines, and costimulatory molecules were upregulated. Pathway analysis identified CC motif chemokine receptor 5 (CCR5) as a candidate pathophysiological pathway. CCR5 upregulation was validated at the protein level, and CCR5 blockade improved renal function after kidney IRI. Using discovery techniques to identify transcriptional responses in purified kidney-infiltrating cells enabled the elucidation of novel mechanisms and therapeutic targets for IRI.

In this study, we compared the traditional murine model with renal pedicle clamp with models that clamped the renal artery or vein alone as well as to a whole body ischemia-reperfusion injury (WBIRI) model. Male C57BL/6J mice underwent either clamping of the renal artery, vein, or both (whole pedicle) for 30 or 45 min followed by reperfusion, or 10 min of cardiac arrest followed by resuscitation up to 24 h. After 30 min of ischemia, the mice with renal vein clamping showed the mostly increased serum creatinine and the most severe renal tubule injury. After 45 min of ischemia, all mice with renal vasculature clamping had a comparable increase in serum creatinine but the renal tubule injury was most severe in renal artery-clamped mice. Renal arterial blood flow was most decreased in mice with a renal vein clamp compared with a renal artery or pedicle clamp. A 30-or 45-min renal ischemia time led to a significant increase in the protein level of interleukin-6, keratinocyte-derived chemokine (KC), and granular colony-stimulating factor in the ischemic kidney, but the KC was the highest in the renal pedicle-clamped kidney and the lowest in the renal vein-clamped kidney. Of note, 10 min of WBIRI led to kidney dysfunction and structural injury, although less than longer time clamping of isolated renal vasculature. Our data demonstrate important differences in ischemic AKI models. Understanding these differences is important in designing future experimental studies in mice as well as clinical trials in humans.