Succinylation of Park 7 Activates a Protective Metabolic Response to Acute Kidney Injury.

Acute Kidney Injury (AKI) is extremely prevalent among hospitalizations and presents a significant risk for the development of chronic kidney disease and increased mortality. Ischemia caused by shock, trauma, and transplant are common causes of AKI. To attenuate ischemic AKI therapeutically, we need a better understanding of the physiological and cellular mechanisms underlying damage. Instances of ischemia are most damaging in Proximal Tubule Epithelial Cells (PTECs) where hypoxic signaling cascades, and perhaps more rapidly, posttranslational modifications (PTMs), act in concert to change cellular metabolism. Here we focus on the effects of the understudied PTM, lysine succinylation. We have previously shown a protective effect of protein hypersuccinylation on PTECs after depletion of the desuccinylase Sirtuin 5. General trends in the results suggested that hypersuccinylation led to upregulation of peroxisomal activity and was protective against kidney injury. Included in the list of changes was the Parkinson's-related deglycase Park7. There is little known about any links between peroxisome activity and Park7. In this study we show in vitro and in vivo that Park7 has a crucial role in protection from AKI, and upregulated peroxisome activity. These data in combination with published results of Park7's protective role in cardiovascular damage and chronic kidney disease lead us to hypothesize that succinylation of Park7 may ameliorate oxidative damage resulting from AKI and prevent disease progression. This novel mechanism provides a potential therapeutic mechanism that can be targeted.

Dietary dicarboxylic acids provide a non-storable alternative fat source that protects mice against obesity.

Dicarboxylic fatty acids are generated in the liver and kidney in a minor pathway called fatty acid ω-oxidation. The effects of consuming dicarboxylic fatty acids as an alternative source of dietary fat have not been explored. Here, we fed dodecanedioic acid, a 12-carbon dicarboxylic (DC12), to mice at 20% of daily caloric intake for nine weeks. DC12 increased metabolic rate, reduced body fat, reduced liver fat, and improved glucose tolerance. We observed DC12-specific breakdown products in liver, kidney, muscle, heart, and brain, indicating that oral DC12 escaped first-pass liver metabolism and was utilized by many tissues. In tissues expressing the "a" isoform of acyl-CoA oxidase-1 (ACOX1), a key peroxisomal fatty acid oxidation enzyme, DC12 was chain shortened to the TCA cycle intermediate succinyl-CoA. In tissues with low peroxisomal fatty acid oxidation capacity, DC12 was oxidized by mitochondria. In vitro, DC12 was catabolized even by adipose tissue and was not stored intracellularly. We conclude that DC12 and other dicarboxylic acids may be useful for combatting obesity and for treating metabolic disorders.

In utero exposure to maternal diabetes exacerbates dietary sodium intake-induced endothelial dysfunction by activating cyclooxygenase 2-derived prostanoids.

Prenatal exposure to maternal diabetes has been recognized as a significant cardiovascular risk factor, increasing the susceptibility to the emergence of conditions such as high blood pressure, atherosclerosis, and heart disease in later stages of life. However, it is unclear if offspring exposed to diabetes in utero have worse vascular outcomes on a high-salt (HS) diet. To test the hypothesis that in utero exposure to maternal diabetes predisposes to HS-induced vascular dysfunction, we treated adult male wild-type offspring (DM_Exp, 6 mo old) of diabetic Ins2+/C96Y mice (Akita mice) with HS (8% sodium chloride, 10 days) and analyzed endothelial function via wire myograph and cyclooxygenase (COX)-derived prostanoids pathway by ELISA, quantitative PCR, and immunochemistry. On a regular diet, DM_Exp mice did not manifest any vascular dysfunction, remodeling, or inflammation. However, HS increased aortic contractility to phenylephrine and induced endothelial dysfunction (analyzed by acetylcholine-induced endothelium-dependent relaxation), vascular hydrogen peroxide production, COX2 expression, and prostaglandin E2 (PGE2) overproduction. Interestingly, ex vivo antioxidant treatment (tempol) or COX1/2 (indomethacin) or COX2 (NS398) inhibitors improved or reverted the endothelial dysfunction in DM_Exp mice fed a HS diet. Finally, DM_Exp mice fed with HS exhibited greater circulating cytokines and chemokines accompanied by vascular inflammation. In summary, our findings indicate that prenatal exposure to maternal diabetes predisposes to HS-induced vascular dysfunction, primarily through the induction of oxidative stress and the generation of COX2-derived PGE2. This supports the concept that in utero exposure to maternal diabetes is a cardiovascular risk factor in adulthood.NEW & NOTEWORTHY Using a unique mouse model of prenatal exposure to maternal type 1 diabetes, our study demonstrates the novel observation that prenatal exposure to maternal diabetes results in a predisposition to high-salt (HS) dietary-induced vascular dysfunction and inflammation in adulthood. Mechanistically, we demonstrated that in utero exposure to maternal diabetes and HS intake induces vascular oxidative stress, cyclooxygenase-derived prostaglandin E2, and inflammation.

Substantial downregulation of mitochondrial and peroxisomal proteins during acute kidney injury revealed by data-independent acquisition proteomics.

Acute kidney injury (AKI) manifests as a major health concern, particularly for the elderly. Understanding AKI-related proteome changes is critical for prevention and development of novel therapeutics to recover kidney function and to mitigate the susceptibility for recurrent AKI or development of chronic kidney disease. In this study, mouse kidneys were subjected to ischemia-reperfusion injury, and the contralateral kidneys remained uninjured to enable comparison and assess injury-induced changes in the kidney proteome. A ZenoTOF 7600 mass spectrometer was optimized for data-independent acquisition (DIA) to achieve comprehensive protein identification and quantification. Short microflow gradients and the generation of a deep kidney-specific spectral library allowed for high-throughput, comprehensive protein quantification. Upon AKI, the kidney proteome was completely remodeled, and over half of the 3945 quantified protein groups changed significantly. Downregulated proteins in the injured kidney were involved in energy production, including numerous peroxisomal matrix proteins that function in fatty acid oxidation, such as ACOX1, CAT, EHHADH, ACOT4, ACOT8, and Scp2. Injured kidneys exhibited severely damaged tissues and injury markers. The comprehensive and sensitive kidney-specific DIA-MS assays feature high-throughput analytical capabilities to achieve deep coverage of the kidney proteome, and will serve as useful tools for developing novel therapeutics to remediate kidney function.

Dicarboxylic Acid Dietary Supplementation Protects against AKI.

SIGNIFICANCE STATEMENT: In this study, we demonstrate that a common, low-cost compound known as octanedioic acid (DC 8 ) can protect mice from kidney damage typically caused by ischemia-reperfusion injury or the chemotherapy drug cisplatin. This compound seems to enhance peroxisomal activity, which is responsible for breaking down fats, without adversely affecting mitochondrial function. DC 8 is not only affordable and easy to administer but also effective. These encouraging findings suggest that DC 8 could potentially be used to assist patients who are at risk of experiencing this type of kidney damage. BACKGROUND: Proximal tubules are rich in peroxisomes, which are damaged during AKI. Previous studies demonstrated that increasing peroxisomal fatty acid oxidation (FAO) is renoprotective, but no therapy has emerged to leverage this mechanism. METHODS: Mice were fed with either a control diet or a diet enriched with dicarboxylic acids, which are peroxisome-specific FAO substrates, then subjected to either ischemia-reperfusion injury-AKI or cisplatin-AKI models. Biochemical, histologic, genetic, and proteomic analyses were performed. RESULTS: Both octanedioic acid (DC 8 ) and dodecanedioic acid (DC 12 ) prevented the rise of AKI markers in mice that were exposed to renal injury. Proteomics analysis demonstrated that DC 8 preserved the peroxisomal and mitochondrial proteomes while inducing extensive remodeling of the lysine succinylome. This latter finding indicates that DC 8 is chain shortened to the anaplerotic substrate succinate and that peroxisomal FAO was increased by DC 8 . CONCLUSIONS: DC 8 supplementation protects kidney mitochondria and peroxisomes and increases peroxisomal FAO, thereby protecting against AKI.

Delayed graft function post renal transplantation: a review on animal models and therapeutics.

The incidence of end-stage renal disease (ESRD) has been increasing worldwide. Its treatment involves renal replacement therapy, either by dialyses or renal transplantation from a living or deceased donor. Although the initial mortality rates for patients on dialysis are comparable with kidney transplant recipients, the quality of life and long-term prognosis are greatly improved in transplanted patients. However, there is a large gap between availability and need for donor kidneys. This has led to the increase in the use of expanded kidney donor criteria. Allograft dysfunction immediately after transplant sets it up for many complications, such as acute rejection and shorter allograft survival. Delayed graft function (DGF) is one of the immediate posttransplant insults to the kidney allograft, which is increasing in prevalence due to efforts to maximize the available donor pool for kidneys and use of expanded kidney donor criteria. In this review, we discuss the risk factors for DGF, its implications for long-term allograft survival, animal models of DGF, and the therapeutic options currently under evaluation for prevention and management of DGF.

Metformin preconditioning protects against myocardial stunning and preserves protein translation in a mouse model of cardiac arrest.

Cardiac arrest (CA) causes high mortality due to multi-system organ damage attributable to ischemia-reperfusion injury. Recent work in our group found that among diabetic patients who experienced cardiac arrest, those taking metformin had less evidence of cardiac and renal damage after cardiac arrest when compared to those not taking metformin. Based on these observations, we hypothesized that metformin's protective effects in the heart were mediated by AMPK signaling, and that AMPK signaling could be targeted as a therapeutic strategy following resuscitation from CA. The current study investigates metformin interventions on cardiac and renal outcomes in a non-diabetic CA mouse model. We found that two weeks of metformin pretreatment protects against reduced ejection fraction and reduces kidney ischemia-reperfusion injury at 24 h post-arrest. This cardiac and renal protection depends on AMPK signaling, as demonstrated by outcomes in mice pretreated with the AMPK activator AICAR or metformin plus the AMPK inhibitor compound C. At this 24-h time point, heart gene expression analysis showed that metformin pretreatment caused changes supporting autophagy, antioxidant response, and protein translation. Further investigation found associated improvements in mitochondrial structure and markers of autophagy. Notably, Western analysis indicated that protein synthesis was preserved in arrest hearts of animals pretreated with metformin. The AMPK activation-mediated preservation of protein synthesis was also observed in a hypoxia/reoxygenation cell culture model. Despite the positive impacts of pretreatment in vivo and in vitro, metformin did not preserve ejection fraction when deployed at resuscitation. Taken together, we propose that metformin's in vivo cardiac preservation occurs through AMPK activation, requires adaptation before arrest, and is associated with preserved protein translation.

Genetically engineering endothelial niche in human kidney organoids enables multilineage maturation, vascularization and de novo cell types.

Vascularization plays a critical role in organ maturation and cell type development. Drug discovery, organ mimicry, and ultimately transplantation in a clinical setting thereby hinges on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcome this hurdle by combining an inducible ETS translocation variant 2 (ETV2) human induced pluripotent stem cell (iPSC) line, which directs endothelial fate, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive vascularization by endothelial cells with an identity most closely related to endogenous kidney endothelia. Vascularized organoids also show increased maturation of nephron structures including more mature podocytes with improved marker expression, foot process interdigitation, an associated fenestrated endothelium, and the presence of renin+ cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Furthermore, this approach is orthogonal to native tissue differentiation paths, hence readily adaptable to other organoid systems and thus has the potential for a broad impact on basic and translational organoid studies.

Substantial Downregulation of Mitochondrial and Peroxisomal Proteins during Acute Kidney Injury revealed by Data-Independent Acquisition Proteomics.

Acute kidney injury (AKI) manifests as a major health concern, particularly for the elderly. Understanding AKI-related proteome changes is critical for prevention and development of novel therapeutics to recover kidney function and to mitigate the susceptibility for recurrent AKI or development of chronic kidney disease. In this study, mouse kidneys were subjected to ischemia-reperfusion injury, and the contralateral kidneys remained uninjured to enable comparison and assess injury-induced changes in the kidney proteome. A fast-acquisition rate ZenoTOF 7600 mass spectrometer was introduced for data-independent acquisition (DIA) for comprehensive protein identification and quantification. Short microflow gradients and the generation of a deep kidney-specific spectral library allowed for high-throughput, comprehensive protein quantification. Upon AKI, the kidney proteome was completely remodeled, and over half of the 3,945 quantified protein groups changed significantly. Downregulated proteins in the injured kidney were involved in energy production, including numerous peroxisomal matrix proteins that function in fatty acid oxidation, such as ACOX1, CAT, EHHADH, ACOT4, ACOT8, and Scp2. Injured mice exhibited severely declined health. The comprehensive and sensitive kidney-specific DIA assays highlighted here feature high-throughput analytical capabilities to achieve deep coverage of the kidney proteome and will serve as useful tools for developing novel therapeutics to remediate kidney function.

Targets for Renal Carcinoma Growth Control Identified by Screening FOXD1 Cell Proliferation Pathways.

Clinical association studies suggest that FOXD1 is a determinant of patient outcome in clear cell renal cell carcinoma (ccRCC), and laboratory investigations have defined a role for this transcription factor in controlling the growth of tumors through regulation of the G2/M cell cycle transition. We hypothesized that the identification of pathways downstream of FOXD1 may define candidates for pharmacological modulation to suppress the G2/M transition in ccRCC. We developed an analysis pipeline that utilizes RNA sequencing, transcription factor binding site analysis, and phenotype validation to identify candidate effectors downstream from FOXD1. Compounds that modulate candidate pathways were tested for their ability to cause growth delay at G2/M. Three targets were identified: FOXM1, PME1, and TMEM167A, which were targeted by compounds FDI-6, AMZ-30, and silibinin, respectively. A 3D ccRCC tumor replica model was used to investigate the effects of these compounds on the growth of primary cells from five patients. While silibinin reduced 3D growth in a subset of tumor replicas, FDI-6 reduced growth in all. This study identifies tractable pathways to target G2/M transition and inhibit ccRCC growth, demonstrates the applicability of these strategies across patient tumor replicas, and provides a platform for individualized patient testing of compounds that inhibit tumor growth.

Role of ERK signaling in bladder urothelium in response to cyclophosphamide injury.

Mice with inducible urothelial deletion of fibroblast growth factor receptor 2 (ShhCreERT2;Fgfr2Fl/Fl ) injured with cyclophosphamide had aberrant basal cell endoreplication and poor regeneration. The endoreplication correlated with an absence of phosphorylated (activated) ERK expression in urothelium. We assessed whether inhibiting ERK activity phenocopied the urothelial defects in injured Fgfr2 mutant mice. We co-administered cyclophosphamide and an ERK inhibitor (ERKi) systemically in mice and assessed general histology and immunofluorescence for various markers post injury. Since AKT also signals downstream of FGFR2, we assessed effects of an AKT inhibitor (AKTi) on cyclophosphamide injury. ERK knockdown did not affect urothelial injury or proliferation 24 h after cyclophosphamide. Conversely, ERK inhibition led to larger basal cell nuclei, more submucosal hemorrhage and attenuated uroplakin staining 3 days after injury versus vehicle-treated mice. Compared to vehicle-treated mice, ERKi-treated mice had a trend for more Ki67+ urothelial cells and had statistically fewer phospho-Histone H3+ cells normalized to Ki67 and higher basal cell DNA content, consistent with endoreplication 3 days after injury. Ten days after injury, ERKi-treated mice still had signs of poor urothelial regeneration with absent or aberrant expression of differentiation markers and ectopic lumenal expression of keratin 14 (basal progenitor marker). Co-administration of the AKTi led to no apparent urothelial defects 3 days after cyclophosphamide. Thus, ERK knockdown (but not AKT knockdown) leads to urothelial regenerative responses after cyclophosphamide reminiscent of Fgfr2 mutant mice. Together, it appears that FGFR2 acts through ERK to prevent aberrant urothelial basal cell endoreplication and ensure normal regeneration after cyclophosphamide.

Stress-Induced Changes in Trophic Factor Expression in the Rodent Urinary Bladder: Possible Links With Angiogenesis.

PURPOSE: Substantive evidence supports a role of chronic stress in the development, maintenance, and even enhancement of functional bladder disorders such as interstitial cystitis/bladder pain syndrome (IC/BPS). Increased urinary frequency and bladder hyperalgesia have been reported in rodents exposed to a chronic stress paradigm. Here, we utilized a water avoidance stress (WAS) model in rodents to investigate the effect of chronic stress on vascular perfusion and angiogenesis. METHODS: Female Wistar-Kyoto rats were exposed to WAS for 10 consecutive days. Bladder neck tissues were analyzed by western immunoblot for vascular endothelial growth factor (VEGF) and nerve growth factor precursor (proNGF). Vascular perfusion was assessed by fluorescent microangiography followed by Hypoxyprobe testing to identify regions of tissue hypoxia. RESULTS: The expression of VEGF and proNGF in the bladder neck mucosa was significantly higher in the WAS rats than in the controls. There was a trend toward increased vascular perfusion, but without a statistically significant difference from the control group. The WAS rats displayed a 1.6-fold increase in perfusion. Additionally, a greater abundance of vessels was observed in the WAS rats, most notably in the microvasculature. CONCLUSION: These findings show that chronic psychological stress induces factors that can lead to increased microvasculature formation, especially around the bladder neck, the region that contains most nociceptive bladder afferents. These findings may indicate a link between angiogenesis and other inflammatory factors that contribute to structural changes and pain in IC/BPS.

The Extracellular Matrix Environment of Clear Cell Renal Cell Carcinoma Determines Cancer Associated Fibroblast Growth.

Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer and is often caused by mutations in the oxygen-sensing machinery of kidney epithelial cells. Due to its pseudo-hypoxic state, ccRCC recruits extensive vasculature and other stromal components. Conventional cell culture methods provide poor representation of stromal cell types in primary cultures of ccRCC, and we hypothesized that mimicking the extracellular environment of the tumor would promote growth of both tumor and stromal cells. We employed proteomics to identify the components of ccRCC extracellular matrix (ECM) and found that in contrast to healthy kidney cortex, laminin, collagen IV, and entactin/nidogen are minor contributors. Instead, the ccRCC ECM is composed largely of collagen VI, fibronectin, and tenascin C. Analysis of single cell expression data indicates that cancer-associated fibroblasts are a major source of tumor ECM production. Tumor cells as well as stromal cells bind efficiently to a nine-component ECM blend characteristic of ccRCC. Primary patient-derived tumor cells bind the nine-component blend efficiently, allowing to us to establish mixed primary cultures of tumor cells and stromal cells. These miniature patient-specific replicas are conducive to microscopy and can be used to analyze interactions between cells in a model tumor microenvironment.

Varenicline limits ischemia reperfusion injury following testicular torsion in mice.

BACKGROUND: Torsion of the spermatic cord and the resulting testicular ischemia leads to the production of inflammatory cytokines and cell death due to impaired aerobic metabolism. Following reperfusion of the testis, a robust innate inflammatory response furthers tissue injury due to the production of reactive oxygen species and disruption of normal capillary function. Blunting the innate immune response with antioxidants, anti-inflammatory medications and targeted genetic interventions reduces long term testicular injury in animal models of torsion, however these approaches have limited clinical applicability. Mediated via α7 nACh receptors, the cholinergic anti-inflammatory pathway limits NFKB signaling and prevents renal fibrosis following warm renal ischemia. We identified varenicline as an FDA approved α7 nAChR agonist and hypothesized that varenicline administration would decrease long-term testicular atrophy and fibrosis in a murine model of testicular torsion. METHODS: Using an established model, unilateral testicular torsion was induced in mature male CD1 mice by rotating the right testicle 720° for 2 h. In the treatment group, 4 doses of varenicline (1mg/grm) were administered via intraperitoneal injection every 12 h, with the first dose given 1 h after the creation of testicular torsion. The acute inflammatory response was evaluated 48 h following reperfusion of the testis. Long term outcomes were evaluated 30 days following testicular perfusion. RESULTS: 48 h following reperfusion, the testis of animals treated with varenicline demonstrated a significant reduction in the inflammatory response as measured by the acute immune cell infiltrate, myeloperoxidase activity, concentration of reduced glutathione and expression of downstream NF-KB targets. 30 days following reperfusion, animals treated with varenicline, demonstrated decreased testicular atrophy (Summary Figure), fibrosis and expression of pro-fibrotic genes. CONCLUSION: Activation of a central immunosuppressive cascade with varenicline after the onset of testicular torsion reduces ischemia reperfusion injury and prevents long term testicular atrophy and fibrosis. Further studies are needed to define the optimum dose and varenicline administration regimen. Our results suggest that varenicline offers a novel, FDA approved, adjunct to the current management of testicular torsion.

Glycolysis Inhibition Induces Functional and Metabolic Exhaustion of CD4+ T Cells in Type 1 Diabetes.

In Type 1 Diabetes (T1D), CD4+ T cells initiate autoimmune attack of pancreatic islet ß cells. Importantly, bioenergetic programs dictate T cell function, with specific pathways required for progression through the T cell lifecycle. During activation, CD4+ T cells undergo metabolic reprogramming to the less efficient aerobic glycolysis, similarly to highly proliferative cancer cells. In an effort to limit tumor growth in cancer, use of glycolytic inhibitors have been successfully employed in preclinical and clinical studies. This strategy has also been utilized to suppress T cell responses in autoimmune diseases like Systemic Lupus Erythematosus (SLE), Multiple Sclerosis (MS), and Rheumatoid Arthritis (RA). However, modulating T cell metabolism in the context of T1D has remained an understudied therapeutic opportunity. In this study, we utilized the small molecule PFK15, a competitive inhibitor of the rate limiting glycolysis enzyme 6-phosphofructo-2-kinase/fructose-2,6- biphosphatase 3 (PFKFB3). Our results confirmed PFK15 inhibited glycolysis utilization by diabetogenic CD4+ T cells and reduced T cell responses to ß cell antigen in vitro. In an adoptive transfer model of T1D, PFK15 treatment delayed diabetes onset, with 57% of animals remaining euglycemic at the end of the study period. Protection was due to induction of a hyporesponsive T cell phenotype, characterized by increased and sustained expression of the checkpoint molecules PD-1 and LAG-3 and downstream functional and metabolic exhaustion. Glycolysis inhibition terminally exhausted diabetogenic CD4+ T cells, which was irreversible through restimulation or checkpoint blockade in vitro and in vivo. In sum, our results demonstrate a novel therapeutic strategy to control aberrant T cell responses by exploiting the metabolic reprogramming of these cells during T1D. Moreover, the data presented here highlight a key role for nutrient availability in fueling T cell function and has implications in our understanding of T cell biology in chronic infection, cancer, and autoimmunity.

Development of a mechanically matched silk scaffolded 3D clear cell renal cell carcinoma model.

Development of a 3D, biomaterials-based model for clear cell renal cell carcinoma (ccRCC) would be advantageous for understanding disease progression in vitro. This study demonstrated the development of lyophilized silk scaffolds that mechanically match the experimentally determined Young's modulus for ex vivo ccRCC samples and normal kidney tissue. Scaffolds fabricated from silk solutions ranging from 3 to 12% (w/v) were evaluated through mechanical testing. Following mechanical characterization of ccRCC samples, it was demonstrated that 6% silk scaffolds mechanically matched ccRCC samples. No impact of pathological grade and stage on the calculated ccRCC modulus was observed and all tumors evaluated mechanically matched the 6% silk scaffold formulation. Stratifying tissue specimens based upon histological observations (e.g. evidence of high levels of collagen deposition) resulted in no significant differences between groups. To investigate the impact of a mechanically matched culturing environment on in vitro ccRCC disease characteristics a model ccRCC cell line, 786-O, was utilized. Scaffolded 786-O cells demonstrated increased lipid droplet accumulation, a hallmark of ccRCC, compared to standard two-dimensional (2D) culture conditions. Additionally, scaffolded 786-O cells demonstrated increased expression of genes associated with ccRCC aggressiveness (ex. VEGFA, TNF, and IL-6) or immune markers under investigation as therapeutic targets (ex. PDL1, CTLA4). Comparison with 786-O cells grown on non-mechanically matched scaffolds demonstrated that these improved ccRCC characteristics were driven by scaffold modulus. Overall, our findings support the use of silk scaffolds in replicating physiologic tumor behavior for clear cell renal cell carcinoma and provide a platform for investigating disease progression.

Endothelial-Derived miR-17∼92 Promotes Angiogenesis to Protect against Renal Ischemia-Reperfusion Injury.

BACKGROUND: Damage to the renal microvasculature is a hallmark of renal ischemia-reperfusion injury (IRI)-mediated AKI. The miR-17∼92 miRNA cluster (encoding miR-17, -18a, -19a, -20a, -19b-1, and -92a-1) regulates angiogenesis in multiple settings, but no definitive role in renal endothelium during AKI pathogenesis has been established. METHODS: Antibodies bound to magnetic beads were utilized to selectively enrich for renal endothelial cells from mice. Endothelial-specific miR-17∼92 knockout (miR-17∼92endo-/- ) mice were generated and given renal IRI. Mice were monitored for the development of AKI using serum chemistries and histology and for renal blood flow using magnetic resonance imaging (MRI) and laser Doppler imaging. Mice were treated with miRNA mimics during renal IRI, and therapeutic efficacies were evaluated. RESULTS: miR-17, -18a, -20a, -19b, and pri-miR-17∼92 are dynamically regulated in renal endothelial cells after renal IRI. miR-17∼92endo-/- exacerbates renal IRI in male and female mice. Specifically, miR-17∼92endo-/- promotes renal tubular injury, reduces renal blood flow, promotes microvascular rarefaction, increases renal oxidative stress, and promotes macrophage infiltration to injured kidneys. The potent antiangiogenic factor thrombospondin 1 (TSP1) is highly expressed in renal endothelium in miR-17∼92endo-/- after renal IRI and is a target of miR-18a and miR-19a/b. miR-17∼92 is critical in the angiogenic response after renal IRI, which treatment with miR-18a and miR-19b mimics can mitigate. CONCLUSIONS: These data suggest that endothelial-derived miR-17∼92 stimulates a reparative response in damaged renal vasculature during renal IRI by regulating angiogenic pathways.

Sirtuins play critical and diverse roles in acute kidney injury.

Acute kidney injury (AKI) is an extremely common medical affliction affecting both adult and pediatric patients resulting from hypoxic, nephrotoxic, and septic insults affecting approximately 20% of all hospital patients and up to 50% of patients in the intensive care unit. There are currently no therapeutics for patients who suffer AKI. Much recent work has focused on designing and implementing therapeutics for AKI. This review focuses on a family of enzymes known as sirtuins that play critical roles in regulating many cellular and biological functions. There are 7 mammalian sirtuins (SIRT1-7) that play roles in regulating the acylation of a wide variety of pathways. Furthermore, all but one of the mammalian sirtuins have been shown to play critical roles in mediating AKI based on preclinical studies. These diverse enzymes show exciting potential for therapeutic manipulation. This review will focus on the specific roles of each of the investigated sirtuins and the potential for manipulation of the various sirtuins and their effector pathways in mediating kidney injury.

A novel ultrasound-guided mouse model of sudden cardiac arrest.

AIM: Mouse models of sudden cardiac arrest are limited by challenges with surgical technique and obtaining reliable venous access. To overcome this limitation, we sought to develop a simplified method in the mouse that uses ultrasound-guided injection of potassium chloride directly into the heart. METHODS: Potassium chloride was delivered directly into the left ventricular cavity under ultrasound guidance in intubated mice, resulting in immediate asystole. Mice were resuscitated with injection of epinephrine and manual chest compressions and evaluated for survival, body temperature, cardiac function, kidney damage, and diffuse tissue injury. RESULTS: The direct injection sudden cardiac arrest model causes rapid asystole with high surgical survival rates and short surgical duration. Sudden cardiac arrest mice with 8-min of asystole have significant cardiac dysfunction at 24 hours and high lethality within the first seven days, where after cardiac function begins to improve. Sudden cardiac arrest mice have secondary organ damage, including significant kidney injury but no significant change to neurologic function. CONCLUSIONS: Ultrasound-guided direct injection of potassium chloride allows for rapid and reliable cardiac arrest in the mouse that mirrors human pathology without the need for intravenous access. This technique will improve investigators' ability to study the mechanisms underlying post-arrest changes in a mouse model.

Impaired mitochondrial medium-chain fatty acid oxidation drives periportal macrovesicular steatosis in sirtuin-5 knockout mice.

Medium-chain triglycerides (MCT), containing C8-C12 fatty acids, are used to treat several pediatric disorders and are widely consumed as a nutritional supplement. Here, we investigated the role of the sirtuin deacylase Sirt5 in MCT metabolism by feeding Sirt5 knockout mice (Sirt5KO) high-fat diets containing either C8/C10 fatty acids or coconut oil, which is rich in C12, for five weeks. Coconut oil, but not C8/C10 feeding, induced periportal macrovesicular steatosis in Sirt5KO mice. 14C-C12 degradation was significantly reduced in Sirt5KO liver. This decrease was localized to the mitochondrial ß-oxidation pathway, as Sirt5KO mice exhibited no change in peroxisomal C12 ß-oxidation. Endoplasmic reticulum ω-oxidation, a minor fatty acid degradation pathway known to be stimulated by C12 accumulation, was increased in Sirt5KO liver. Mice lacking another mitochondrial C12 oxidation enzyme, long-chain acyl-CoA dehydrogenase (LCAD), also developed periportal macrovesicular steatosis when fed coconut oil, confirming that defective mitochondrial C12 oxidation is sufficient to induce the steatosis phenotype. Sirt5KO liver exhibited normal LCAD activity but reduced mitochondrial acyl-CoA synthetase activity with C12. These studies reveal a role for Sirt5 in regulating the hepatic response to MCT and may shed light into the pathogenesis of periportal steatosis, a hallmark of human pediatric non-alcoholic fatty liver disease.