Surgical procedures suppress autophagic flux in the kidney.

Many surgical models are used to study kidney and other diseases in mice, yet the effects of the surgical procedure itself on the kidney and other tissues have not been elucidated. In the present study, we found that both sham surgery and unilateral nephrectomy (UNX), which is used as a model of renal compensatory hypertrophy, in mice resulted in increased mammalian target of rapamycin complex 1/2 (mTORC1/2) in the remaining kidney. mTORC1 is known to regulate lysosomal biogenesis and autophagy. Genes associated with lysosomal biogenesis and function were decreased in sham surgery and UNX kidneys. In both sham surgery and UNX, there was suppressed autophagic flux in the kidney as indicated by the lack of an increase in LC3-II or autophagosomes seen on immunoblot, IF and EM after bafilomycin A1 administration and a concomitant increase in p62, a marker of autophagic cargo. There was a massive increase in pro-inflammatory cytokines, which are known to activate ERK1/2, in the serum after sham surgery and UNX. There was a large increase in ERK1/2 in sham surgery and UNX kidneys, which was blocked by the MEK1/2 inhibitor, trametinib. Trametinib also resulted in a significant decrease in p62. In summary, there was an intense systemic inflammatory response, an ERK-mediated increase in p62 and suppressed autophagic flux in the kidney after sham surgery and UNX. It is important that researchers are aware that changes in systemic pro-inflammatory cytokines, ERK1/2 and autophagy can be caused by sham surgery as well as the kidney injury/disease itself.

Vagus nerve stimulation activates two distinct neuroimmune circuits converging in the spleen to protect mice from kidney injury.

Acute kidney injury is highly prevalent and associated with high morbidity and mortality, and there are no approved drugs for its prevention and treatment. Vagus nerve stimulation (VNS) alleviates inflammatory diseases including kidney disease; however, neural circuits involved in VNS-induced tissue protection remain poorly understood. The vagus nerve, a heterogeneous group of neural fibers, innervates numerous organs. VNS broadly stimulates these fibers without specificity. We used optogenetics to selectively stimulate vagus efferent or afferent fibers. Anterograde efferent fiber stimulation or anterograde (centripetal) sensory afferent fiber stimulation both conferred kidney protection from ischemia-reperfusion injury. We identified the C1 neurons-sympathetic nervous system-splenic nerve-spleen-kidney axis as the downstream pathway of vagus afferent fiber stimulation. Our study provides a map of the neural circuits important for kidney protection induced by VNS, which is critical for the safe and effective clinical application of VNS for protection from acute kidney injury.

IL-6-mediated hepatocyte production is the primary source of plasma and urine neutrophil gelatinase-associated lipocalin during acute kidney injury.

Neutrophil gelatinase associated lipocalin (NGAL, Lcn2) is the most widely studied biomarker of acute kidney injury (AKI). Previous studies have demonstrated that NGAL is produced by the kidney and released into the urine and plasma. Consequently, NGAL is currently considered a tubule specific injury marker of AKI. However, the utility of NGAL to predict AKI has been variable suggesting that other mechanisms of production are present. IL-6 is a proinflammatory cytokine increased in plasma by two hours of AKI and mediates distant organ effects. Herein, we investigated the role of IL-6 in renal and extra-renal NGAL production. Wild type mice with ischemic AKI had increased plasma IL-6, increased hepatic NGAL mRNA, increased plasma NGAL, and increased urine NGAL; all reduced in IL-6 knockout mice. Intravenous IL-6 in normal mice increased hepatic NGAL mRNA, plasma NGAL and urine NGAL. In mice with hepatocyte specific NGAL deletion (Lcn2hep-/-) and ischemic AKI, hepatic NGAL mRNA was absent, and plasma and urine NGAL were reduced. Since urine NGAL levels appear to be dependent on plasma levels, the renal handling of circulating NGAL was examined using recombinant human NGAL. After intravenous recombinant human NGAL administration to mice, human NGAL in mouse urine was detected by ELISA during proximal tubular dysfunction, but not in pre-renal azotemia. Thus, during AKI, IL-6 mediates hepatic NGAL production, hepatocytes are the primary source of plasma and urine NGAL, and plasma NGAL appears in the urine during proximal tubule dysfunction. Hence, our data change the paradigm by which NGAL should be interpreted as a biomarker of AKI.

The Extracellular Matrix Receptor Discoidin Domain Receptor 1 Regulates Collagen Transcription by Translocating to the Nucleus.

BACKGROUND: The discoidin domain receptor 1 (DDR1) is activated by collagens, upregulated in injured and fibrotic kidneys, and contributes to fibrosis by regulating extracellular matrix production, but how DDR1 controls fibrosis is poorly understood. DDR1 is a receptor tyrosine kinase (RTK). RTKs can translocate to the nucleus via a nuclear localization sequence (NLS) present on the receptor itself or a ligand it is bound to. In the nucleus, RTKs regulate gene expression by binding chromatin directly or by interacting with transcription factors. METHODS: To determine whether DDR1 translocates to the nucleus and whether this event is mediated by collagen-induced DDR1 activation, we generated renal cells expressing wild-type or mutant forms of DDR1 no longer able to bind collagen. Then, we determined the location of the DDR1 upon collagen stimulation. Using both biochemical assays and immunofluorescence, we analyzed the steps involved in DDR1 nuclear translocation. RESULTS: We show that although DDR1 and its natural ligand, collagen, lack an NLS, DDR1 is present in the nucleus of injured human and mouse kidney proximal tubules. We show that DDR1 nuclear translocation requires collagen-mediated receptor activation and interaction of DDR1 with SEC61B, a component of the Sec61 translocon, and nonmuscle myosin IIA and ß-actin. Once in the nucleus, DDR1 binds to chromatin to increase the transcription of collagen IV, a major collagen upregulated in fibrosis. CONCLUSIONS: These findings reveal a novel mechanism whereby activated DDR1 translates to the nucleus to regulate synthesis of profibrotic molecules.

Metabolomics assessment reveals oxidative stress and altered energy production in the heart after ischemic acute kidney injury in mice.

Acute kidney injury (AKI) is a systemic disease associated with widespread effects on distant organs, including the heart. Normal cardiac function is dependent on constant ATP generation, and the preferred method of energy production is via oxidative phosphorylation. Following direct ischemic cardiac injury, the cardiac metabolome is characterized by inadequate oxidative phosphorylation, increased oxidative stress, and increased alternate energy utilization. We assessed the impact of ischemic AKI on the metabolomics profile in the heart. Ischemic AKI was induced by 22 minutes of renal pedicle clamping, and 124 metabolites were measured in the heart at 4 hours, 24 hours, and 7 days post-procedure. Forty-one percent of measured metabolites were affected, with the most prominent changes observed 24 hours post-AKI. The post-AKI cardiac metabolome was characterized by amino acid depletion, increased oxidative stress, and evidence of alternative energy production, including a shift to anaerobic forms of energy production. These metabolomic effects were associated with significant cardiac ATP depletion and with echocardiographic evidence of diastolic dysfunction. In the kidney, metabolomics analysis revealed shifts suggestive of energy depletion and oxidative stress, which were reflected systemically in the plasma. This is the first study to examine the cardiac metabolome after AKI, and demonstrates that effects of ischemic AKI on the heart are akin to the effects of direct ischemic cardiac injury.

Pyridoxamine reduces postinjury fibrosis and improves functional recovery after acute kidney injury.

Acute kidney injury (AKI) is a common and independent risk factor for death and chronic kidney disease (CKD). Despite promising preclinical data, there is no evidence that antioxidants reduce the severity of injury, increase recovery, or prevent CKD in patients with AKI. Pyridoxamine (PM) is a structural analog of vitamin B6 that interferes with oxidative macromolecular damage via a number of different mechanisms and is in a phase 3 clinical efficacy trial to delay CKD progression in patients with diabetic kidney disease. Because oxidative stress is implicated as one of the main drivers of renal injury after AKI, the ability of PM to interfere with multiple aspects of oxidative damage may be favorable for AKI treatment. In these studies we therefore evaluated PM treatment in a mouse model of AKI. Pretreatment with PM caused a dose-dependent reduction in acute tubular injury, long-term postinjury fibrosis, as well as improved functional recovery after ischemia-reperfusion AKI (IR-AKI). This was associated with a dose-dependent reduction in the oxidative stress marker isofuran-to-F2-isoprostane ratio, indicating that PM reduces renal oxidative damage post-AKI. PM also reduced postinjury fibrosis when administered 24 h after the initiating injury, but this was not associated with improvement in functional recovery after IR-AKI. This is the first report showing that treatment with PM reduces short- and long-term injury, fibrosis, and renal functional recovery after IR-AKI. These preclinical findings suggest that PM, which has a favorable clinical safety profile, holds therapeutic promise for AKI and, most importantly, for prevention of adverse long-term outcomes after AKI.

Bridging translation for acute kidney injury with better preclinical modeling of human disease.

The current lack of effective therapeutics for patients with acute kidney injury (AKI) represents an important and unmet medical need. Given the importance of the clinical problem, it is time for us to take a few steps back and reexamine current practices. The focus of this review is to explore the extent to which failure of therapeutic translation from animal studies to human studies stems from deficiencies in the preclinical models of AKI. We will evaluate whether the preclinical models of AKI that are commonly used recapitulate the known pathophysiologies of AKI that are being modeled in humans, focusing on four common scenarios that are studied in clinical therapeutic intervention trials: cardiac surgery-induced AKI; contrast-induced AKI; cisplatin-induced AKI; and sepsis associated AKI. Based on our observations, we have identified a number of common limitations in current preclinical modeling of AKI that could be addressed. In the long term, we suggest that progress in developing better preclinical models of AKI will depend on developing a better understanding of human AKI. To this this end, we suggest that there is a need to develop greater in-depth molecular analyses of kidney biopsy tissues coupled with improved clinical and molecular classification of patients with AKI.

Delayed treatment with PTBA analogs reduces postinjury renal fibrosis after kidney injury.

No therapies have been shown to accelerate recovery or prevent fibrosis after acute kidney injury (AKI). In part, this is because most therapeutic candidates have to be given at the time of injury and the diagnosis of AKI is usually made too late for drugs to be efficacious. Strategies to enhance post-AKI repair represent an attractive approach to address this. Using a phenotypic screen in zebrafish, we identified 4-(phenylthio)butanoic acid (PTBA), which promotes proliferation of embryonic kidney progenitor cells (EKPCs), and the PTBA methyl ester UPHD25, which also increases postinjury repair in ischemia-reperfusion and aristolochic acid-induced AKI in mice. In these studies, a new panel of PTBA analogs was evaluated. Initial screening was performed in zebrafish EKPC assays followed by survival assays in a gentamicin-induced AKI larvae zebrafish model. Using this approach, we identified UPHD186, which in contrast to UPHD25, accelerates recovery and reduces fibrosis when administered several days after ischemia-reperfusion AKI and reduces fibrosis after unilateral ureteric obstruction in mice. UPHD25 and 186 are efficiently metabolized to the active analog PTBA in liver and kidney microsome assays, indicating both compounds may act as PTBA prodrugs in vivo. UPHD186 persists longer in the circulation than UPHD25, suggesting that sustained levels of UPHD186 may increase efficacy by acting as a reservoir for renal metabolism to PTBA. These findings validate use of zebrafish EKPC and AKI assays as a drug discovery strategy for molecules that reduce fibrosis in multiple AKI models and can be administered days after initiation of injury.

Retinoic Acid Signaling Coordinates Macrophage-Dependent Injury and Repair after AKI.

Retinoic acid (RA) has been used therapeutically to reduce injury and fibrosis in models of AKI, but little is known about the regulation of this pathway and what role it has in regulating injury and repair after AKI. In these studies, we show that RA signaling is activated in mouse and zebrafish models of AKI, and that these responses limit the extent of injury and promote normal repair. These effects were mediated through a novel mechanism by which RA signaling coordinated the dynamic equilibrium of inflammatory M1 spectrum versus alternatively activated M2 spectrum macrophages. Our data suggest that locally synthesized RA represses proinflammatory macrophages, thereby reducing macrophage-dependent injury post-AKI, and activates RA signaling in injured tubular epithelium, which in turn promotes alternatively activated M2 spectrum macrophages. Because RA signaling has an essential role in kidney development but is repressed in the adult, these findings provide evidence of an embryonic signaling pathway that is reactivated after AKI and involved in reducing injury and enhancing repair.

Ischemia-reperfusion model of acute kidney injury and post injury fibrosis in mice.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

Histone deacetylase inhibitor enhances recovery after AKI.

At present, there are no effective therapies to ameliorate injury, accelerate recovery, or prevent postinjury fibrosis after AKI. Here, we sought to identify candidate compounds that accelerate recovery after AKI by screening for small molecules that increase proliferation of renal progenitor cells in zebrafish embryos. One compound identified from this screen was the histone deacetylase inhibitor methyl-4-(phenylthio)butanoate, which we subsequently administered to zebrafish larvae and mice 24-48 hours after inducing AKI. In zebrafish, treatment with the compound increased larval survival and proliferation of renal tubular epithelial cells. In mice, treatment accelerated recovery, reduced postinjury tubular atrophy and interstitial fibrosis, and increased the regenerative capacity of actively cycling renal tubular cells by decreasing the number of cells in G2/M arrest. These data suggest that accelerating recovery may be a viable approach to treating AKI and provide proof of concept that a screen in zebrafish embryos can identify therapeutic candidates for kidney injury.