Pax protein depletion in proximal tubules triggers conserved mechanisms of resistance to acute ischemic kidney injury preventing transition to chronic kidney disease.

Acute kidney injury (AKI) is a common condition that lacks effective treatments. In part, this shortcoming is due to an incomplete understanding of the genetic mechanisms that control pathogenesis and recovery. Identifying the molecular and genetic regulators unique to nephron segments that dictate vulnerability to injury and regenerative potential could lead to new therapeutic targets to treat ischemic kidney injury. Pax2 and Pax8 are homologous transcription factors with overlapping functions that are critical for kidney development and are re-activated in AKI. Here, we examined the role of Pax2 and Pax8 in recovery from ischemic AKI and found them upregulated after severe AKI and correlated with chronic injury. Surprisingly, proximal-tubule-selective deletion of Pax2 and Pax8 resulted in a less severe chronic injury phenotype. This effect was mediated by protection against the acute insult, similar to pre-conditioning. Prior to injury, Pax2 and Pax8 mutant mice develop a unique subpopulation of proximal tubule cells in the S3 segment that displayed features usually seen only in acute or chronic injury. The expression signature of these cells was strongly enriched with genes associated with other mechanisms of protection against ischemic AKI including caloric restriction, hypoxic pre-conditioning, and female sex. Thus, our results identified a novel role for Pax2 and Pax8 in mature proximal tubules that regulates critical genes and pathways involved in both the injury response and protection from ischemic AKI.

Pax Protein Depletion in Proximal Tubules Triggers Conserved Mechanisms of Resistance to Acute Ischemic Kidney Injury and Prevents Transition to Chronic Kidney Disease.

Acute kidney injury (AKI) is a common condition that lacks effective treatments. In part this shortcoming is due to an incomplete understanding of the genetic mechanisms that control pathogenesis and recovery. Pax2 and Pax8 are homologous transcription factors with overlapping functions that are critical for kidney development and are re-activated in AKI. In this report, we examined the role of Pax2 and Pax8 in recovery from ischemic AKI. We found that Pax2 and Pax8 are upregulated after severe AKI and correlate with chronic injury. Surprisingly, we then discovered that proximal-tubule-selective deletion of Pax2 and Pax8 resulted in a less severe chronic injury phenotype. This effect was mediated by protection against the acute insult, similar to preconditioning. Prior to injury, Pax2 and Pax8 mutant mice develop a unique subpopulation of S3 proximal tubule cells that display features usually seen only in acute or chronic injury. The expression signature of these cells was strongly enriched with genes associated with other mechanisms of protection against ischemic AKI including caloric restriction, hypoxic preconditioning, and female sex. Taken together, our results identify a novel role for Pax2 and Pax8 in mature proximal tubules that regulates critical genes and pathways involved in both injury response and protection from ischemic AKI. TRANSLATIONAL STATEMENT: Identifying the molecular and genetic regulators unique to the nephron that dictate vulnerability to injury and regenerative potential could lead to new therapeutic targets to treat ischemic kidney injury. Pax2 and Pax8 are two homologous nephron-specific transcription factors that are critical for kidney development and physiology. Here we report that proximal-tubule-selective depletion of Pax2 and Pax8 protects against both acute and chronic injury and induces an expression profile in the S3 proximal tubule with common features shared among diverse conditions that protect against ischemia. These findings highlight a new role for Pax proteins as potential therapeutic targets to treat AKI.

Proximal tubule LPA1 and LPA2 receptors use divergent signaling pathways to additively increase profibrotic cytokine secretion.

Lysophosphatidic acid (LPA) increases platelet-derived growth factor-B (PDGFB) and connective tissue growth factor (CTGF) production and secretion by proximal tubule (PT) cells through LPA2 receptor-Gqα-αvß6-integrin-mediated activation of transforming growth factor-ß1 (TGFB1). LPA2, ß6-integrin, PDGFB, and CTGF increase in kidneys after ischemia-reperfusion injury (IRI), coinciding with fibrosis. The TGFB1 receptor antagonist SD-208 prevents increases of ß6-integrin, TGFB1-SMAD signaling, and PDGFB/CTGF expression after IRI and ameliorates fibrosis (Geng H, Lan R, Singha PK, Gilchrist A, Weinreb PH, Violette SM, Weinberg JM, Saikumar P, Venkatachalam MA. Am J Pathol 181: 1236-1249, 2012; Geng H, Lan R, Wang G, Siddiqi AR, Naski MC, Brooks AI, Barnes JL, Saikumar P, Weinberg JM, Venkatachalam MA. Am J Pathol 174: 1291-1308, 2009). We report now that LPA1 receptor signaling through epidermal growth factor receptor (EGFR)-ERK1/2-activator protein-1 cooperates with LPA2-dependent TGFB1 signaling to additively increase PDGFB/CTGF production and secretion by PT cells. Conversely, inhibition of both pathways results in greater suppression of PDGFB/CTGF production and secretion and promotes greater PT cellular differentiation than inhibiting one pathway alone. Antagonism of the LPA-generating enzyme autotaxin suppressed signaling through both pathways. After IRI, kidneys showed not only more LPA2, nuclear SMAD2/3, and PDGFB/CTGF but also increased LPA1 and autotaxin proteins, together with enhanced EGFR/ERK1/2 activation. Remarkably, the TGFB1 receptor antagonist SD-208 prevented all of these abnormalities excepting increased LPA2. SD-208 inhibits only one arm of LPA signaling: LPA2-Gqα-αvß6-integrin-dependent production of active TGFB1 and its receptor-bound downstream effects. Consequently, far-reaching protection by SD-208 against IRI-induced signaling alterations and tubule-interstitial pathology is not fully explained by our data. TGFB1-dependent feedforward modulation of LPA1 signaling is one possibility. SD-208 effects may also involve mitigation of injury caused by IRI-induced TGFB1 signaling in endothelial cells and monocytes. Our results have translational implications for using TGFB1 receptor antagonists, LPA1 and LPA2 inhibitors concurrently, and autotaxin inhibitors in acute kidney injury to prevent the development of chronic kidney disease.

Proximal Tubular Oxidative Metabolism in Acute Kidney Injury and the Transition to CKD.

The proximal tubule relies on oxidative mitochondrial metabolism to meet its energy needs and has limited capacity for glycolysis, which makes it uniquely susceptible to damage during AKI, especially after ischemia and anoxia. Under these conditions, mitochondrial ATP production is initially decreased by several mechanisms, including fatty acid-induced uncoupling and inhibition of respiration related to changes in the shape and volume of mitochondria. Glycolysis is initially insufficient as a source of ATP to protect the cells and mitochondrial function, but supplementation of tricarboxylic acid cycle intermediates augments anaerobic ATP production, and improves recovery of mitochondrial oxidative metabolism. Incomplete recovery is characterized by defects of respiratory enzymes and lipid metabolism. During the transition to CKD, tubular cells atrophy but maintain high expression of glycolytic enzymes, and there is decreased fatty acid oxidation. These metabolic changes may be amenable to a number of therapeutic interventions.

Mitochondrial quality control in kidney injury and repair.

Mitochondria are essential for the activity, function and viability of eukaryotic cells and mitochondrial dysfunction is involved in the pathogenesis of acute kidney injury (AKI) and chronic kidney disease, as well as in abnormal kidney repair after AKI. Multiple quality control mechanisms, including antioxidant defence, protein quality control, mitochondrial DNA repair, mitochondrial dynamics, mitophagy and mitochondrial biogenesis, have evolved to preserve mitochondrial homeostasis under physiological and pathological conditions. Loss of these mechanisms may induce mitochondrial damage and dysfunction, leading to cell death, tissue injury and, potentially, organ failure. Accumulating evidence suggests a role of disturbances in mitochondrial quality control in the pathogenesis of AKI, incomplete or maladaptive kidney repair and chronic kidney disease. Moreover, specific interventions that target mitochondrial quality control mechanisms to preserve and restore mitochondrial function have emerged as promising therapeutic strategies to prevent and treat kidney injury and accelerate kidney repair. However, clinical translation of these findings is challenging owing to potential adverse effects, unclear mechanisms of action and a lack of knowledge of the specific roles and regulation of mitochondrial quality control mechanisms in kidney resident and circulating cell types during injury and repair of the kidney.

Correction to: Phenytoin inhibits necroptosis.

The name of the one of the authors was misspelt. The author's surname is Rodriguez, not Rodriquez as originally published. This has been corrected in both the PDF and HTML versions of the Article.

Phenytoin inhibits necroptosis.

Receptor-interacting protein kinases 1 and 3 (RIPK1/3) have best been described for their role in mediating a regulated form of necrosis, referred to as necroptosis. During this process, RIPK3 phosphorylates mixed lineage kinase domain-like (MLKL) to cause plasma membrane rupture. RIPK3-deficient mice have recently been demonstrated to be protected in a series of disease models, but direct evidence for activation of necroptosis in vivo is still limited. Here, we sought to further examine the activation of necroptosis in kidney ischemia-reperfusion injury (IRI) and from TNFα-induced severe inflammatory response syndrome (SIRS), two models of RIPK3-dependent injury. In both models, MLKL-ko mice were significantly protected from injury to a degree that was slightly, but statistically significantly exceeding that of RIPK3-deficient mice. We also demonstrated, for the first time, accumulation of pMLKL in the necrotic tubules of human patients with acute kidney injury. However, our data also uncovered unexpected elevation of blood flow in MLKL-ko animals, which may be relevant to IRI and should be considered in the future. To further understand the mode of regulation of cell death by MLKL, we screened a panel of clinical plasma membrane channel blockers and we found phenytoin to inhibit necroptosis. However, we further found that phenytoin attenuated RIPK1 kinase activity in vitro, likely due to the hydantoin scaffold also present in necrostatin-1, and blocked upstream necrosome formation steps in the cells undergoing necroptosis. We further report that this clinically used anti-convulsant drug displayed protection from kidney IRI and TNFα-induces SIRS in vivo. Overall, our data reveal the relevance of RIPK3-pMLKL regulation for acute kidney injury and identifies an FDA-approved drug that may be useful for immediate clinical evaluation of inhibition of pro-death RIPK1/RIPK3 activities in human diseases.

Citrate shows protective effects on cardiovascular and renal function in ischemia-induced acute kidney injury.

BACKGROUND: Ischemia and reperfusion (I/R) is one of the major causes of acute kidney injury (AKI). Citrate reduces hypoxia-induced mitochondrial energetic deficits in isolated proximal tubules. Moreover, citrate anticoagulation is now frequently used in renal replacement therapy. In the present study a rat model of I/R-induced AKI was utilized to examine renal protection by citrate in vivo. METHODS: AKI was induced by bilateral renal clamping (40 min) followed by reperfusion (3 h). Citrate was infused at three different concentrations (0.3 mmol/kg/h; 0.6 mmol/kg/h and 1.0 mmol/kg/h) continuously for 60 min before and 45 min after ischemia. Plasma calcium concentrations were kept stable by infusion of calcium gluconate. The effect of citrate was evaluated by biomonitoring, blood and plasma parameters, histopathology and tissue ATP content. RESULTS: In comparison to the normoxic control group bilateral renal ischemia led to an increase of creatinine and lactate dehydrogenase activity and a decrease in tissue ATP content and was accompanied by a drop in mean arterial blood pressure. Infusion of 1.0 mmol/kg/h citrate led to lower creatinine and reduced LDH activity compared to the I/R control group and a tendency for higher tissue ATP content. Pre-ischemic infusion of 1.0 mmol/kg/h citrate stabilized blood pressure during ischemia. CONCLUSIONS: Citrate has a protective effect during I/R-induced AKI, possibly by limiting the mitochondrial deficit as well as by beneficial cardiovascular effects. This strengthens the rationale of using citrate in continuous renal replacement therapy and encourages consideration of citrate infusion as a therapeutic treatment for AKI in humans.

Tissue-specific metabolic reprogramming drives nutrient flux in diabetic complications.

Diabetes is associated with altered cellular metabolism, but how altered metabolism contributes to the development of diabetic complications is unknown. We used the BKS db/db diabetic mouse model to investigate changes in carbohydrate and lipid metabolism in kidney cortex, peripheral nerve, and retina. A systems approach using transcriptomics, metabolomics, and metabolic flux analysis identified tissue-specific differences, with increased glucose and fatty acid metabolism in the kidney, a moderate increase in the retina, and a decrease in the nerve. In the kidney, increased metabolism was associated with enhanced protein acetylation and mitochondrial dysfunction. To confirm these findings in human disease, we analyzed diabetic kidney transcriptomic data and urinary metabolites from a cohort of Southwestern American Indians. The urinary findings were replicated in 2 independent patient cohorts, the Finnish Diabetic Nephropathy and the Family Investigation of Nephropathy and Diabetes studies. Increased concentrations of TCA cycle metabolites in urine, but not in plasma, predicted progression of diabetic kidney disease, and there was an enrichment of pathways involved in glycolysis and fatty acid and amino acid metabolism. Our findings highlight tissue-specific changes in metabolism in complication-prone tissues in diabetes and suggest that urinary TCA cycle intermediates are potential prognostic biomarkers of diabetic kidney disease progression.

The role of glycine in regulated cell death.

The cytoprotective effects of glycine against cell death have been recognized for over 28 years. They are expressed in multiple cell types and injury settings that lead to necrosis, but are still not widely appreciated or considered in the conceptualization of cell death pathways. In this paper, we review the available data on the expression of this phenomenon, its relationship to major pathophysiologic pathways that lead to cell death and immunomodulatory effects, the hypothesis that it involves suppression by glycine of the development of a hydrophilic death channel of molecular dimensions in the plasma membrane, and evidence for its impact on disease processes in vivo.

Mitochondrial Pathology and Glycolytic Shift during Proximal Tubule Atrophy after Ischemic AKI.

During recovery by regeneration after AKI, proximal tubule cells can fail to redifferentiate, undergo premature growth arrest, and become atrophic. The atrophic tubules display pathologically persistent signaling increases that trigger production of profibrotic peptides, proliferation of interstitial fibroblasts, and fibrosis. We studied proximal tubules after ischemia-reperfusion injury (IRI) to characterize possible mitochondrial pathologies and alterations of critical enzymes that govern energy metabolism. In rat kidneys, tubules undergoing atrophy late after IRI but not normally recovering tubules showed greatly reduced mitochondrial number, with rounded profiles, and large autophagolysosomes. Studies after IRI of kidneys in mice, done in parallel, showed large scale loss of the oxidant-sensitive mitochondrial protein Mpv17L. Renal expression of hypoxia markers also increased after IRI. During early and late reperfusion after IRI, kidneys exhibited increased lactate and pyruvate content and hexokinase activity, which are indicators of glycolysis. Furthermore, normally regenerating tubules as well as tubules undergoing atrophy exhibited increased glycolytic enzyme expression and inhibitory phosphorylation of pyruvate dehydrogenase. TGF-ß antagonism prevented these effects. Our data show that the metabolic switch occurred early during regeneration after injury and was reversed during normal tubule recovery but persisted and became progressively more severe in tubule cells that failed to redifferentiate. In conclusion, irreversibility of the metabolic switch, taking place in the context of hypoxia, high TGF-ß signaling and depletion of mitochondria characterizes the development of atrophy in proximal tubule cells and may contribute to the renal pathology after AKI.

Long-term response to renal ischaemia in the human kidney after partial nephrectomy: results from a prospective clinical trial.

OBJECTIVE: To assess the 1-year renal functional changes in patients undergoing partial nephrectomy with intra-operative renal biopsies. PATIENTS AND METHODS: A total of 40 patients with a single renal mass deemed fit for a partial nephrectomy were recruited prospectively between January 2009 and October 2010. We performed renal biopsies of normal renal parenchyma and collected serum markers before, during and after surgically induced renal clamp ischaemia during the partial nephrectomy. We then followed patients clinically with interval serum creatinine and physical examination. RESULTS: Peri-operative data from 40 patients showed a transient increase in creatinine levels which did not correlate with ischaemia time. Renal ultrastructural changes were generally mild and included mitochondrial swelling, which resolved at the post-perfusion biopsy. A total of 37 patients had 1-year follow-up data. Creatinine at 1 year increased by 0.121 mg/dL, which represents a 12.99% decrease in renal function from baseline (preoperative creatinine 0.823 mg/dL, estimated glomerular filtration rate = 93.9 mL/min/1.73 m(2) ). The only factors predicting creatinine change on multivariate analysis were patient age, race and ischaemia type, with cold ischaemia being associated with higher creatinine level. Importantly, the duration of ischaemia did not show any significant correlation with renal function change, either as a continuous variable (P = 0.452) or as a categorical variable (P = 0.792). CONCLUSIONS: Our data suggest that limited ischaemia is generally well tolerated in the setting of partial nephrectomy and does not directly correspond to long-term renal functional decline. For surgeons performing partial nephrectomy, the kidney can be safely clamped to ensure optimum oncological outcomes.

Fibrosis without fibroblast TGF-ß receptors?

A report by Neelisetty et al. suggests that TGFBR2 deletion from matrix-producing interstitial cells results in decreased transforming growth factor-ß (TGF-ß) signaling in the cells but does not decrease renal fibrosis after injury. Considered in the context of TGF-ß signaling in different cell types involved in renal fibrosis and the existence of other ligands that may produce fibrosis, these findings are provocative, but owing to technical issues of recombination efficiency in inducible models of Cre-lox gene deletion, further studies are needed.

Failed Tubule Recovery, AKI-CKD Transition, and Kidney Disease Progression.

The transition of AKI to CKD has major clinical significance. As reviewed here, recent studies show that a subpopulation of dedifferentiated, proliferating tubules recovering from AKI undergo pathologic growth arrest, fail to redifferentiate, and become atrophic. These abnormal tubules exhibit persistent, unregulated, and progressively increasing profibrotic signaling along multiple pathways. Paracrine products derived therefrom perturb normal interactions between peritubular capillary endothelium and pericyte-like fibroblasts, leading to myofibroblast transformation, proliferation, and fibrosis as well as capillary disintegration and rarefaction. Although signals from injured endothelium and inflammatory/immune cells also contribute, tubule injury alone is sufficient to produce the interstitial pathology required for fibrosis. Localized hypoxia produced by microvascular pathology may also prevent tubule recovery. However, fibrosis is not intrinsically progressive, and microvascular pathology develops strictly around damaged tubules; thus, additional deterioration of kidney structure after the transition of AKI to CKD requires new acute injury or other mechanisms of progression. Indeed, experiments using an acute-on-chronic injury model suggest that additional loss of parenchyma caused by failed repair of AKI in kidneys with prior renal mass reduction triggers hemodynamically mediated processes that damage glomeruli to cause progression. Continued investigation of these pathologic mechanisms should reveal options for preventing renal disease progression after AKI.

Synchronized renal tubular cell death involves ferroptosis.

Receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis is thought to be the pathophysiologically predominant pathway that leads to regulated necrosis of parenchymal cells in ischemia-reperfusion injury (IRI), and loss of either Fas-associated protein with death domain (FADD) or caspase-8 is known to sensitize tissues to undergo spontaneous necroptosis. Here, we demonstrate that renal tubules do not undergo sensitization to necroptosis upon genetic ablation of either FADD or caspase-8 and that the RIPK1 inhibitor necrostatin-1 (Nec-1) does not protect freshly isolated tubules from hypoxic injury. In contrast, iron-dependent ferroptosis directly causes synchronized necrosis of renal tubules, as demonstrated by intravital microscopy in models of IRI and oxalate crystal-induced acute kidney injury. To suppress ferroptosis in vivo, we generated a novel third-generation ferrostatin (termed 16-86), which we demonstrate to be more stable, to metabolism and plasma, and more potent, compared with the first-in-class compound ferrostatin-1 (Fer-1). Even in conditions with extraordinarily severe IRI, 16-86 exerts strong protection to an extent which has not previously allowed survival in any murine setting. In addition, 16-86 further potentiates the strong protective effect on IRI mediated by combination therapy with necrostatins and compounds that inhibit mitochondrial permeability transition. Renal tubules thus represent a tissue that is not sensitized to necroptosis by loss of FADD or caspase-8. Finally, ferroptosis mediates postischemic and toxic renal necrosis, which may be therapeutically targeted by ferrostatins and by combination therapy.

Substrate modulation of fatty acid effects on energization and respiration of kidney proximal tubules during hypoxia/reoxygenation.

Kidney proximal tubules subjected to hypoxia/reoxygenation develop a nonesterified fatty acid-induced energetic deficit characterized by persistent partial mitochondrial deenergization that can be prevented and reversed by citric acid cycle substrates. To further assess the role of competition between fatty acids and substrates on inner membrane substrate carriers in the deenergization and the contribution to deenergization of fatty acid effects on respiratory function, digitonin-permeabilized rabbit and mouse tubules were studied using either addition of exogenous oleate after control normoxic incubation or increases of endogenous fatty acids produced by hypoxia/reoxygenation. The results demonstrated major effects of matrix oxaloacetate accumulation on succinate-supported energization and respiration and their modification by fatty acids. Improvements of energization in the presence of fatty acids by glutamate were shown to result predominantly from lowering matrix oxaloacetate rather than from amelioration of transmembrane cycling of fatty acids and uncoupling. Mouse tubules had 2.5 fold higher rates of succinate utilization, which resulted in stronger effects of oxaloacetate accumulation than rabbit tubules. Hypoxia/reoxygenation induced respiratory inhibition that was more severe for complex I-dependent substrates. Fatty acids themselves did not acutely contribute to this respiratory inhibition, but lowering them during 60 min. reoxygenation to allow recovery of ATP during that period alleviated it. These data clarify the basis for the nonesterified fatty acid-induced mitochondrial energetic deficit in kidney proximal tubules that impairs structural and functional recovery and provide insight into interactions that need to be considered in the design of substrate-based interventions to improve mitochondrial function.

Ferrostatins inhibit oxidative lipid damage and cell death in diverse disease models.

Ferrostatin-1 (Fer-1) inhibits ferroptosis, a form of regulated, oxidative, nonapoptotic cell death. We found that Fer-1 inhibited cell death in cellular models of Huntington's disease (HD), periventricular leukomalacia (PVL), and kidney dysfunction; Fer-1 inhibited lipid peroxidation, but not mitochondrial reactive oxygen species formation or lysosomal membrane permeability. We developed a mechanistic model to explain the activity of Fer-1, which guided the development of ferrostatins with improved properties. These studies suggest numerous therapeutic uses for ferrostatins, and that lipid peroxidation mediates diverse disease phenotypes.

Two independent pathways of regulated necrosis mediate ischemia-reperfusion injury.

Regulated necrosis (RN) may result from cyclophilin (Cyp)D-mediated mitochondrial permeability transition (MPT) and receptor-interacting protein kinase (RIPK)1-mediated necroptosis, but it is currently unclear whether there is one common pathway in which CypD and RIPK1 act in or whether separate RN pathways exist. Here, we demonstrate that necroptosis in ischemia-reperfusion injury (IRI) in mice occurs as primary organ damage, independent of the immune system, and that mice deficient for RIPK3, the essential downstream partner of RIPK1 in necroptosis, are protected from IRI. Protection of RIPK3-knockout mice was significantly stronger than of CypD-deficient mice. Mechanistically, in vivo analysis of cisplatin-induced acute kidney injury and hyperacute TNF-shock models in mice suggested the distinctness of CypD-mediated MPT from RIPK1/RIPK3-mediated necroptosis. We, therefore, generated CypD-RIPK3 double-deficient mice that are viable and fertile without an overt phenotype and that survived prolonged IRI, which was lethal to each single knockout. Combined application of the RIPK1 inhibitor necrostatin-1 and the MPT inhibitor sanglifehrin A confirmed the results with mutant mice. The data demonstrate the pathophysiological coexistence and corelevance of two separate pathways of RN in IRI and suggest that combination therapy targeting distinct RN pathways can be beneficial in the treatment of ischemic injury.

The RIP1-kinase inhibitor necrostatin-1 prevents osmotic nephrosis and contrast-induced AKI in mice.

The pathophysiology of contrast-induced AKI (CIAKI) is incompletely understood due to the lack of an appropriate in vivo model that demonstrates reduced kidney function before administration of radiocontrast media (RCM). Here, we examine the effects of CIAKI in vitro and introduce a murine ischemia/reperfusion injury (IRI)-based approach that allows induction of CIAKI by a single intravenous application of standard RCM after injury for in vivo studies. Whereas murine renal tubular cells and freshly isolated renal tubules rapidly absorbed RCM, plasma membrane integrity and cell viability remained preserved in vitro and ex vivo, indicating that RCM do not induce apoptosis or regulated necrosis of renal tubular cells. In vivo, the IRI-based CIAKI model exhibited typical features of clinical CIAKI, including RCM-induced osmotic nephrosis and increased serum levels of urea and creatinine that were not altered by inhibition of apoptosis. Direct evaluation of renal morphology by intravital microscopy revealed dilation of renal tubules and peritubular capillaries within 20 minutes of RCM application in uninjured mice and similar, but less dramatic, responses after IRI pretreatment. Necrostatin-1 (Nec-1), a specific inhibitor of the receptor-interacting protein 1 (RIP1) kinase domain, prevented osmotic nephrosis and CIAKI, whereas an inactive Nec-1 derivate (Nec-1i) or the pan-caspase inhibitor zVAD did not. In addition, Nec-1 prevented RCM-induced dilation of peritubular capillaries, suggesting a novel role unrelated to cell death for the RIP1 kinase domain in the regulation of microvascular hemodynamics and pathophysiology of CIAKI.