Sham Kidneys Research Articles

Intestinal and Renal Adaptations to Changes of Dietary Phosphate Concentrations in Rat.

We have studied the role of the intestine, kidney, and several hormones when adapting to changes in dietary P concentration. Normal and parathyroidectomized (PTX) rats were fed pH-matched diets containing 0.1%, 0.6%, and 1.2% P concentrations. 32Pi uptake was determined in the jejunum and kidney cortex brush border membrane vesicles. Several hormone and ion concentrations were determined in the blood and urine of rats. Both jejunum and kidney cortex Pi transport was regulated with 5 d of chronic feeding of P diets in normal rats. Acute adaptation was determined by switching foods on day 6, which was only clearly observed in the kidney cortex of normal rats, with more statistical variability in the jejunum. However, no paradoxical increase of Pi uptake in the jejunum was reproduced after the acute switch to the 1.2% P diet. Pi uptake in the jejunum was parathyroid hormone (PTH)-independent, but in the kidney, the chronic adaptation was reduced, and no acute dietary adaptations were observed. The NaPi2a protein was more abundant in the PTX than the sham kidneys, but contrary to the modest or absent changes in Pi uptake adaptation, the transporter was similarly regulated by dietary P, as in the sham rats. PTH and fibroblast growth factor 23 (FGF23) were the only hormones regulated by all diet changes, even in fasting animals, which exhibited regulated Pi transport despite similar phosphatemia. Evidence of Pi appetite effects was also observed. In brief, our results show new characteristics of Pi adaptations, including a lack of correlation between Pi transport, NaPi2a expression, and PTH/FGF23 concentrations.

#3553 TUBULE CELL-DERIVED ANGIOPOIETIN-2 EXACERBATES INFLAMMATION AND IS DETRIMENTAL TO RENAL FIBROSIS

Abstract Background and Aims Our previous studies have shown that angiogenic growth factors are dysregulated in fibrotic kidneys. In murine models of progressive renal disease, angiopoietin-2 (Angpt2) was upregulated in injured tubule epithelial cells. We hypothesize that overexpression of Angpt2 in tubule epithelial cells would exacerbate renal fibrosis and inflammation. Method The unilateral ureteral obstruction (UUO) and unilateral ischemia-reperfusion injury (UIRI) are used as the murine model of chronic kidney disease. We use the tetracycline/doxycycline-controlled Tet-on system to induce tubule cell-specific overexpression of Angpt2 before the induction of renal fibrosis. Transgenic mice carrying both Pax8-rtTA and pTetos-hAngpt2 genes (double transgenic mice) are used for Angpt2 overexpression. Littermates that inherit single or no transgene serve as experimental controls. Doxycycline is administered to adult (8 weeks) mice to overexpress Angpt2 in tubule epithelial cells. Results Doxycycline administration induce overexpression of human Anpgt2 in the tubule epithelial cells of both sham and fibrotic kidneys. Compared to littermate controls after the UUO and UIRI surgery, the expression of fibrosis-related genes, Col1a1, Col3a1, and Acta2 are higher in the kidneys of double transgenic mice. Deposition of picrosirius red-stained collagen fibrils is significantly higher in double transgenic mice. Inflammatory cell infiltration is also significantly higher in double transgenic mice. Conclusion Overall, these findings suggest that increased Angpt2 in tubule epithelial cells deteriorate renal fibrosis through detrimental inflammation. Further studies are necessary to explore the participating cellular components and pathogenic mechanisms.

Effects of interleukin-17A on liver and kidney injury and prognosis in septic mice

To explore the effect of interleukin-17A (IL-17A) on liver and kidney injury and prognosis in septic mice. A total of 84 SPF male C57BL/6 mice were randomly divided into sham operation group (Sham group), cecal ligation and puncture (CLP) induced sepsis model group (CLP group), and IL-17A intervention group. IL-17A intervention group were then divided into five subgroups according to the dose of IL-17A (0.25, 0.5, 1, 2, 4 μg). Mice in the IL-17A intervention group were intraperitoneally injected with the corresponding dose of IL-17A 100 μL immediately after surgery. The other groups were intraperitoneally injected with 100 μL phosphate buffer solution (PBS). The survival rate of mice was observed at 7 days, and peripheral blood and liver, kidney and spleen tissues were collected. According to the 7-day survival, another 18 mice were randomly divided into Sham group, CLP group, and 1 μg IL-17A intervention group. Peripheral blood samples were collected at 12 hours and 24 hours after CLP, and the mice were sacrificed to obtain liver, kidney, and spleen tissues. The behavior and abdominal cavity of each group were observed. The levels of peripheral blood liver and kidney function indexes and inflammatory factors were detected. The histopathological changes of liver and kidney were observed under light microscope. The peripheral blood and spleen tissues were inoculated in the medium, the number of bacterial colonies was calculated, and the bacterial migration of each group was evaluated in vitro. Except for the Sham group, the 7-day survival rate of mice in the 1 μg IL-17A intervention group was the highest (75.0%), so this condition was selected as the intervention condition for the subsequent study. Compared with Sham group, the liver and kidney functions of CLP group were significantly damaged at each time point after operation. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and serum creatinine (SCr) reached the peak at 24 hours after operation, and the liver and kidney pathological scores reached the peak at 7 days after operation, the levels of inflammatory cytokines interleukin (IL-17A, IL-6, IL-10) reached the peak at 12 hours after operation, and tumor necrosis factor-α (TNF-α) reached the peak at 24 hours after operation. In addition, a large number of bacteria proliferated in the peripheral blood and spleen, which reached the peak on day 7. Compared with the CLP group, exogenous administration of 1 μg IL-17A significantly delayed the rising trend of each index in the early stage of sepsis [24-hour ALT (U/L): 166.95±5.20 vs. 271.30±6.11, 24-hour AST (U/L): 599.42±7.25 vs. 1 013.27±3.37, 24-hour BUN (mg/L): 815.4±26.3 vs. 1 191.2±39.4, 24-hour SCr (μmol/L): 29.34±0.87 vs. 60.75±3.83, 7-day liver pathological score: 2.50 (2.00, 3.00) vs. 9.00 (8.50, 9.00), 7-day kidney pathological score: 1.00 (1.00, 2.00) vs. 5.00 (4.50, 5.00), 12-hour IL-17A (ng/L): 105.21±0.31 vs. 111.28±1.37, 12-hour IL-6 (ng/L): 83.22±1.01 vs. 108.88±0.99, 12-hour IL-10 (ng/L): 731.54±3.04 vs. 790.25±2.54, 24-hour TNF-α (μg/L): 454.67±0.66 vs. 576.18±0.76, 7-day peripheral blood colony count (CFU/mL): 600 (400, 600) vs. 4 200 (4 200, 4 300), 7-day spleen tissue colony count (CFU/g): 4 600 (4 400, 4 600) vs. 23 400 (23 200, 23 500), all P < 0.05]. Appropriate dose (1 μg) of exogenous IL-17A can reduce the lethal inflammatory response induced by CLP and improve the ability of bacterial clearance, thereby alleviating liver and kidney injury and improving the 7-day survival rate of septic mice.

IL-1R mediated renal sensory nerve activity drives hypertension in the DOCA-Salt mouse

Clinical trials of renal denervation (RDN) have shown unanticipated changes in non-renal sympathetic activity. It has been hypothesized that this is due to ablation of sympathoexcitatory afferent renal nerves, which are overactive under conditions of renal inflammation. This study sought to investigate the interaction of the inflammatory cytokine, IL-1, and afferent renal nerves in the pathogenesis of hypertension. To test this hypothesis, we compared the effect of afferent RDN (ARDN) to the administration of the IL-1R antagonist anakinra, on the pathogenesis DOCA-salt hypertension. In addition, the combination of ARDN and anakinra treatments in DOCA-salt mice was administered to assess the degree of mechanistic overlap. 10-week-old male C57BL6/J mice were implanted with radio-telemetry probes in the carotid artery for measurement of mean arterial pressure (MAP). DOCA was administered via subcutaneous implantation of a silicon pellet containing 50 mg of DOCA. Sham mice received a drug free silicon pellet. Uni-nephrectomy, pellet insertion, salt treatment, ARDN were all performed or started on the same day. ARDN was performed through peri-axonal application of capsaicin. IL-1R antagonist anakinra was used to suppress activation of IL-1R. Anakinra (75mg/kg) was delivered via intraperitoneal injection daily 10 days post induction of hypertension. Animals in the untreated or monotherapy groups received sham RDN and/or vehicle injections. Baseline MAP was measured 3 days prior to induction of DOCA-salt hypertension by radiotelemetry. MAP increased in untreated DOCA-salt mice by 42 ± 2 mmHg (n=7) from baseline in the final week of the study. In comparison, this response was attenuated by 42% by ARDN (+24 ± 2 mmHg, n=7). IL-1α and IL-1β were both increased in DOCA-salt kidneys (112.4 ± 19.2 pg/mg and 2.1 ± 0.4 pg/mg respectively) compared to sham kidneys (31.98 ± 4.4 pg/mg and 0.36 ± 0.06 pg/mg respectively). The DOCA-salt induced increase of MAP in mice treated with anakinra alone (+23 ± 3 mmHg, n=9), or the combination of ARDN and anakinra (+26 ± 3 mmHg, n=5) was similar to ARDN monotherapy (+24 ± 2 mmHg, n=7). All treatment groups showed statistically significant changes in MAP compared to the SHAM and Vehicle treated group with 2 Way ANOVA and Tukey test for multiple comparisons. The finding that ARDN and anakinra combination therapy provides no additional benefit beyond ARDN or anakinra alone, suggests a common mechanism. We propose that antagonism of IL-1R blocks IL-1 mediated activation of afferent renal nerves resulting in decreased central sympathetic tone and decreased MAP. Further investigation into the mechanisms of cytokine mediated sensory fiber activation in the kidney are the subject of ongoing studies. T32DK083250-01A1, R01HL116476 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Circ_0000181 regulates miR-667-5p/NLRC4 axis to promote pyroptosis progression in diabetic nephropathy

Our previous research demonstrated that NOD-like receptor family CARD domain-containing protein 4 (NLRC4) inflammasome was overexpressed in renal tissues of patients with diabetic nephropathy (DN). This study further investigated the effect of circRNAs-miRNAs interaction on NLRC4 and their potential mechanisms. DN mice models were first established using STZ. Then, pyroptosis related marker expression was detected using qPCR, western blot (WB), and immunohistochemistry analysis. After that, differentially expressed circRNAs, miRNAs, and mRNAs were investigated using next-generation sequencing. Additionally, the function and potential mechanism of circ_0000181 and miR-667-5p on pyroptosis were measured in vitro DN cell model using MTS, WB, and Enzyme-linked immunosorbent assay. There was an apparent elevation of NLRC4, Caspase1, IL-1β, and IL-18 levels in DN mice. The next-generation sequencing results revealed that there were 947 circRNAs and 390 miRNAs significantly different between the DN and sham kidney tissue, of which circ_0000181 and miR-667-5p had potential targeting effects with NLRC4. Dual-luciferase and functional rescue experiments demonstrated that circ_0000181 promoted NLRC4 inflammasome activation via competitive sponge of miR-667-5p, promoted the release of IL-1β and IL-18, and caused pyroptosis. Altogether, circ_0000181 regulates miR-667-5p/NLRC4 axis to promote pyroptosis progression in DN.

Transcriptomic Profiling Reveals Left Ventricle and Kidney Pathology Resulting from Peritoneal Dialysis

Approximately 50% of deaths in ESRD patients are attributed to cardiovascular (CV) related pathology. Only 2.5% of ESRD patients receive a kidney transplant, while the rest rely on dialysis for survival. Peritoneal dialysis (PD) therapy has been increasingly utilized in recent years, yet there is a paucity in data characterizing the effects of PD on CV function. The goal of this study was to assess molecular changes in response to PD to understand how PD could impact prevalence of CV mortality in ESRD. We hypothesized that PD would not improve CKD‐related CV pathology despite providing support during renal insufficiency. To determine how PD effects renal and left ventricle (LV) structural and functional, organ morphology, and gene expression, 10‐week‐old male rats underwent 5/6Nx or Sham operation. Peritoneal catheters were implanted 6 weeks post‐surgery. PD was initiated 3 days later in half of the 5/6Nx and sham animals (15ml [Baxter PD‐2 2.5%] 1‐hour dwell 3x/day x 8 days) for n=6‐7 per surgical and treatment group. Echocardiography, blood collection, and urine collection were performed at baseline, 6, and 7‐weeks post‐surgery. Acute carotid blood pressure was measured week 7. Results were analyzed by two‐way ANOVA and significance determined at p&lt;0.05. Sequencing of RNA isolated from kidney and LV was performed (n=6 per surgical group and treatment). Statistical analysis of transcriptomic results compared transcript levels from rats that received PD and those that did not. Differential gene expression was determined using the R package DESeq2. The Benjamini‐Hochberg method was used to control FDR in differential expression analysis to provide an adjusted p‐value. Transcripts with an adjusted p‐value (q‐value) &lt;0.05 were identified as differentially expressed genes (DEGs) for subsequent pathway analysis, regardless of fold change. The list of DEGs was entered into Ingenuity Pathway Analysis (IPA) for subsequent analysis. PD had no effect on LV parameters or blood pressure in Sham or 5/6Nx rats. Blood urea nitrogen, serum creatine, SDMA and cholesterol were significantly higher in 5/6Nx rats compared to sham rats and PD attenuated that increase. Serum triglycerides were also increased in 5/6Nx compared to sham, but augmented further with PD. RNA sequencing yielded &gt;50 million reads (100 bp in length) per sample. The mapping rate for this dataset was &gt;96% for all samples. Transcripts were considered differentially expressed with an adjusted p‐value &lt;0.05. This identified numerous DEGs resulting from PD in sham LV, 5/6Nx LV, sham kidney and 5/6Nx kidney tissues (853, 162, 637, and 33 DEGs, respectively). IPA analysis predicted gene changes consistent with increased inflammatory responses in both the LV and kidney tissues from sham controls receiving PD, suggesting that dialysis alone may induce multi‐organ inflammatory signaling. The DEG pattern of enrichment in LV tissue from sham rats receiving PD indicated that the “Phagosome Formation“ pathway was activated, while the “EIF2 Signaling” pathway was highly enriched and activated in kidney tissue from sham rats receiving PD. In summary, our transcriptomic analysis identified a pro‐immune/inflammatory state resulting from PD alone, even in sham‐operated controls with normal renal function. The transcriptomic data align with our physiological measurements indicating PD did not diminish or improve cardiac function in sham or 5/6Nx rats, rather PD may augment inflammatory signaling to increase risk factors for CVD and cardiac mortality.

Afferent renal denervation attenuates DOCA‐salt hypertension in the mouse: Potential role of an IL‐1β afferent renal nerve interaction

Catheter based renal denervation (RDN) has emerged as a novel treatment for hypertension in patients. However, the mechanisms by which RDN lowers arterial pressure are unclear. We hypothesize that an interaction between afferent and efferent renal nerves and renal inflammation contributes to hypertension. Specifically, we hypothesized an interaction between afferent renal nerves and the inflammatory cytokine IL‐1β contributes to the pathogenesis of hypertension in the DOCA‐salt mouse. To test this hypothesis, we compared the effect of total RDN (TRDN), afferent RDN (ARDN) on pathogenesis DOCA‐salt hypertension to that observed in IL‐1R knockout mice. We also measured the effect of administration of the IL‐1R antagonist anakinra on arterial pressure in DOCA‐salt mice.10‐week‐old male C57BL6/J mice were implanted with radio‐telemetry probes in the carotid artery for measurement of arterial pressure. DOCA was administered via subcutaneous implantation of a silicon pellet containing 50 mg of DOCA. Sham mice received a drug free silicon pellet. Uni‐nephrectomy, pellet insertion, salt treatment, TRDN or ARDN were all performed on the same day. TRDN was performed through surgical ablation and peri‐axonal application of 10% phenol. ARDN was performed through peri‐axonal application of capsaicin. IL‐1R knockout mice and the IL‐1R antagonist anakinra were used in two separate groups to suppress activation of IL‐1R. Anakinra (75mg/kg) was delivered via intraperitoneal injection daily 10 days post induction of hypertension. Baseline arterial pressure was measured beginning 3 days prior to induction of DOCA‐salt hypertension by radiotelemetry. Separate cohorts of mice were generated for protein quantification and histology, with urine and kidneys harvested 21 days post induction of DOCA‐salt hypertension. Cytokine protein quantification was obtained through Multiplex ELISA. All values reported are mean + SEM.The mean arterial pressure (MAP) elevation in the sham treated DOCA‐salt mice (+43 ± 1 mmHg increase from baseline during the 3rd week of the study) was attenuated by ~40% by TRDN (+25.9 ± 4 mmHg) and by ~44% in ARDN DOCA‐salt mice (+24.1 ± 3 mmHg). Renal cytokines and chemokines were increased in DOCA‐salt compared to sham treated mice. IL‐1α and IL‐1β were both increased in DOCA‐salt kidneys (112.4 ± 19.2 pg/mg and 2.1 ± 0.4 pg/mg respectively) compared to sham kidneys (31.98 ± 4.4 pg/mg and 0.36 ± 0.06 pg/mg respectively). The DOCA‐salt induced increase in MAP in IL‐1R Knockout mice was similar to sham DOCA‐salt mice (+39.1 ± 1 mmHg). In contrast, Anakinra treatment attenuated the increase in MAP similar to TRDN and ARDN mice (+22.4 ± 4 mmHg). The difference in outcome between the knockout and antagonist group suggests compensation by other inflammatory factors. The comparable attenuation in the MAP response to DOCA‐salt ARDN and Anakinra treated groups is consistent with a common mechanism of action. We hypothesize that intrarenal IL‐1b activates sympathoexcitatory renal afferent nerves to increase MAP in DOCA‐salt mice. Future studies are needed to directly test this hypothesis.

MP44-10 HIGH MOLECULAR WEIGHT HYALURONAN ATTENUATES SCARRING IN ACUTE KIDNEY INJURY

You have accessJournal of UrologyPediatric Urology II (MP44)1 Sep 2021MP44-10 HIGH MOLECULAR WEIGHT HYALURONAN ATTENUATES SCARRING IN ACUTE KIDNEY INJURY Pratik Kanabur, Aditya Kaul, Scott Manson, Swathi Balaji, Paul Austin, Sandeep Keswani, and Xinyi Wang Pratik KanaburPratik Kanabur More articles by this author , Aditya KaulAditya Kaul More articles by this author , Scott MansonScott Manson More articles by this author , Swathi BalajiSwathi Balaji More articles by this author , Paul AustinPaul Austin More articles by this author , Sandeep KeswaniSandeep Keswani More articles by this author , and Xinyi WangXinyi Wang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002065.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Renal fibrosis induces morbidity in pediatric patients, which can persist into adulthood. Hyaluronan (HA), a major extracellular matrix component, regulates fibrosis based on its molecular weight (MW). We have reported that IL-10 upregulates high MW-HA synthesis via fibroblasts to attenuate fibrosis in a chronic kidney disease model. However, the effect of high MW-HA in acute kidney injury is unknown. Our data suggests that HA is upregulated in epithelial tubular cells in an acute kidney injury model. We hypothesize that tubular cell-specific high MW-HA reduces fibrosis caused by acute kidney injury. METHODS: We performed unilateral ischemia/reperfusion(I/R) on C57BL/6J male mice treated with and without IL-10 and also with and without 4-methylumbelliferone(4MU), an HA inhibitor. I/R or sham kidneys were collected at Day 3, 7, and 14 for the levels of HA synthases(HAS1-3) and hyaluronidases(HYAL1-2) by qPCR, total HA by ELISA, and fibrosis outcome and tubular cell proliferation by immunohistochemistry. In vitro studies were also done with human proximal tubular HK-2 cells, which were used to determine the effect of IL-10 on HA enzymes with/without TGFβ, which mimics the fibrotic phenotype. ANOVA and Student’s t-test were used as appropriate with statistical significance defined as p<0.05. RESULTS: I/R significantly increases HA production with the peak of enzyme production at day 3. With IL-10 treatment, we observe upregulated total HA at D3&D7, increased tubular cell proliferation, and reduced fibrosis. These effects however are not observed when HA is inhibited by 4-MU. In vitro, TGFβ significantly alters expression of HAS1&3 and HYAL1 compared to control, and IL-10 treatment increases HA and restores HAS1 level compared to control (all p<0.05) CONCLUSIONS: Tubular-specific HA plays a protective role in response to acute renal injury, and the upregulation of HA by IL-10 can attenuate fibrosis. We identified a previously unreported role for high MW-HA that occurs in response to kidney injury. We also elucidated the mechanisms underlying HA-attenuated fibrosis to inspire novel therapeutics. Source of Funding: N/A © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e796-e796 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Pratik Kanabur More articles by this author Aditya Kaul More articles by this author Scott Manson More articles by this author Swathi Balaji More articles by this author Paul Austin More articles by this author Sandeep Keswani More articles by this author Xinyi Wang More articles by this author Expand All Advertisement Loading ...

Targeting P2RX1 alleviates renal ischemia/reperfusion injury by preserving mitochondrial dynamics

Renal ischemia/reperfusion injury (IRI) is the major cause of acute kidney injury. However, mechanisms underlying the sudden loss in kidney function and tissue injury remain to be fully elucidated. Here, we performed RNA sequencing to systematically compare the transcriptome differences between IR injured kidneys and sham kidneys. We observed that mitochondrial dynamics was destructed in renal IRI. Expression of mitochondrial fusion-associated genes was reduced, whereas expression of mitochondrial fission-related genes was increased in renal IRI, and these findings were further confirmed by mitochondrial morphological observations. By screening 19 purinergic receptors, we noticed that P2RX1 expression was markedly upregulated in renal IRI. RNA sequencing and mitochondrial morphological observations revealed that mitochondrial dynamics was preserved in P2RX1 genetic knockout (P2rx1-/-) mice. Neutrophil extracellular traps (NETs) were reported to be essential for tissue injury in renal IRI, but the detailed mechanism remained unclear. In the present study, we found that P2RX1 favored the formation of neutrophil extracellular traps (NETs) in IRI, and NETs was essential for the impairment of mitochondrial dynamics. Mechanistically, P2RX1-involved metabolic interaction between platelets and neutrophils supported NETs formation. Activation of P2RX1 promoted platelets ATP release, which subsequently contributed to neutrophil glycolytic metabolism and NETs generation.

Restoration of afferent arteriolar autoregulatory behavior in ischemia-reperfusion injury in rat kidneys.

Renal autoregulation is critical in maintaining stable renal blood flow (RBF) and glomerular filtration rate (GFR). Renal ischemia-reperfusion (IR)-induced kidney injury is characterized by reduced RBF and GFR. The mechanisms contributing to renal microvascular dysfunction in IR have not been fully determined. We hypothesized that increased reactive oxygen species (ROS) contributed to impaired renal autoregulatory capability in IR rats. Afferent arteriolar autoregulatory behavior was assessed using the blood-perfused juxtamedullary nephron preparation. IR was induced by 60 min of bilateral renal artery occlusion followed by 24 h of reperfusion. Afferent arterioles from sham rats exhibited normal autoregulatory behavior. Stepwise increases in perfusion pressure caused pressure-dependent vasoconstriction to 65 ± 3% of baseline diameter (13.2 ± 0.4 μm) at 170 mmHg. In contrast, pressure-mediated vasoconstriction was markedly attenuated in IR rats. Baseline diameter averaged 11.7 ± 0.5 µm and remained between 90% and 101% of baseline over 65-170 mmHg, indicating impaired autoregulatory function. Acute antioxidant administration (tempol or apocynin) to IR kidneys for 20 min increased baseline diameter and improved autoregulatory capability, such that the pressure-diameter profiles were indistinguishable from those of sham kidneys. Furthermore, the addition of polyethylene glycol superoxide dismutase or polyethylene glycol-catalase to the perfusate blood also restored afferent arteriolar autoregulatory responsiveness in IR rats, indicating the involvement of superoxide and/or hydrogen peroxide. IR elevated mRNA expression of NADPH oxidase subunits and monocyte chemoattractant protein-1 in renal tissue homogenates, and this was prevented by tempol pretreatment. These results suggest that ROS accumulation, likely involving superoxide and/or hydrogen peroxide, impairs renal autoregulation in IR rats in a reversible fashion.NEW & NOTEWORTHY Renal ischemia-reperfusion (IR) leads to renal microvascular dysfunction manifested by impaired afferent arteriolar autoregulatory efficiency. Acute administration of scavengers of reactive oxygen species, polyethylene glycol-superoxide dismutase, or polyethylene glycol-catalase following renal IR restored afferent arteriolar autoregulatory capability in IR rats, indicating that renal IR led to reversible impairment of afferent arteriolar autoregulatory capability. Intervention with antioxidant treatment following IR may improve outcomes in patients by preserving renovascular autoregulatory function and potentially preventing the progression to chronic kidney disease after acute kidney injury.

Deletion of Alox15 improves kidney dysfunction and inhibits fibrosis by increased PGD2 in the kidney

BackgroundLipid-metabolizing enzymes and their metabolites affect inflammation and fibrosis, but their roles in chronic kidney disease (CKD) have not been completely understood.MethodsTo clarify their role in CKD, we measured the mRNA levels of major lipid-metabolizing enzymes in 5/6 nephrectomized (Nx) kidneys of C57BL/6 J mice. Mediator lipidomics was performed to reveal lipid profiles of CKD kidneys.ResultsIn 5/6 Nx kidneys, both mRNA and protein levels of Alox15 were higher when compared with those in sham kidneys. With respect to in situ hybridization, the mRNA level of Alox15 was higher in renal tubules of 5/6 Nx kidneys. To examine the role of Alox15 in CKD pathogenesis, we performed 5/6 Nx on Alox15−/− mice. Alox15−/− CKD mice exhibited better renal functions than wild-type mice. Interstitial fibrosis was also inhibited in Alox15−/− CKD mice. Mediator lipidomics revealed that Alox15−/− CKD mouse kidneys had significantly higher levels of PGD2 than the control. To investigate the effects of PGD2 on renal fibrosis, we administered PGD2 to TGF-β1-stimulated NRK-52E cells and HK-2 cells, which lead to a dose-dependent suppression of type I collagen and αSMA in both cell lines.ConclusionIncreased PGD2 in Alox15−/− CKD mouse kidneys could inhibit fibrosis, thereby resulting in CKD improvement. Thus, Alox15 inhibition and PGD2 administration may be novel therapeutic targets for CKD.

The dysregulation of metabolic pathways and induction of the pentose phosphate pathway in renal ischaemia-reperfusion injury.

Lipid accumulation is associated with various forms of acute renal injury; however, the causative factors and pathways underpinning this lipid accumulation have not been thoroughly investigated. In this study, we performed lipidomic profiling of renal tissue following ischaemia–reperfusion injury (IRI). We identified a significant accumulation of cholesterol and specific phospholipids and sphingolipids in kidneys 24 h after IRI. In light of these findings, we hypothesised that pathways involved in lipid metabolism may also be altered. Through the analysis of published microarray data, generated from sham and ischaemic kidneys, we identified nephron‐specific metabolic pathways affected by IRI and validated these findings in ischaemic renal tissue. In silico analysis revealed the downregulation of several energy and lipid metabolism pathways, including mitochondrial fatty acid beta‐oxidation (FAO), peroxisomal lipid metabolism, fatty acid (FA) metabolism, and glycolysis. The pentose phosphate pathway (PPP), which is fuelled by glycolysis, was the only metabolic pathway that was upregulated 24 h following IRI. In this study, we describe the effect of renal IRI on metabolic pathways and how this contributes to lipid accumulation. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Cold Saline Perfusion before Ischemia-Reperfusion Is Harmful to the Kidney and Is Associated with the Loss of Ezrin, a Cytoskeletal Protein, in Rats

Background: Organ protection for transplantation is perfusion with ice-cold preservation solutions, although saline is also used in animal experiments and living donor transplantations. However, ice-cold perfusion can contribute to initial graft injury. Our aim was to test if cytoskeletal damage of parenchymal cells is caused by saline itself or by the ice-cold solution. Methods: F344 rat kidneys were flushed with cold (4 °C) saline, ischemic and sham kidneys were not perfused. In a separate set, F344 kidneys were flushed with saline or preservation solution at 4 or 15 °C. Ischemia time was 30 min. Results: Renal injury was significantly more severe following cold ischemia (CI) than after ischemia-reperfusion without flushing (ischemia/reperfusion (I/R)). Functional and morphologic damage was accompanied by severe loss of ezrin from glomerular and tubular epithelial cells after CI. Moreover, saline caused serious injury independently from its temperature, while the perfusion solution was more beneficial, especially at 4 °C. Conclusions: Flushing the kidney with ice-cold saline can cause more severe injury than ischemia-reperfusion at body temperature even during a short (30 min) ischemia. Saline perfusion can prolong recovery from ischemia in kidney transplantation, which can be prevented by using preservation solutions.

Abstract 361: The Augmentation of Oxygen-sensitive Generation of Oxygen Radicals in Mitochondria and the Attenuation of Hyperoxia-induced Oxidative Stress After Cardiac Arrest

Introduction: Hyperoxia can induce oxidative stress resulting in organ injuries after cardiac arrest (CA). Mitochondrial reactive oxygen species (ROS) are key to understanding its mechanism, however the augmentation of oxygen-sensitive ROS generation caused by CA ( Fig 1 ) has not been well described. Methods: Rats were assigned into: 1) sham with normoxia, 30% O2; 2) sham with hyperoxia, 100% O2; 3) CA with normoxia; and 4) CA with hyperoxia (n=15 for each group). CA was induced by 10-minute asphyxia and CPR was delivered. Neurological deficit score (NDS), histological lung injury score (LIS), survival time were obtained from the CA groups. Carbonyl protein as an oxidative stress indicator was measured at 2 hrs after resuscitation. H2O2 generation from isolated mitochondria were measured ex vivo at a normoxic and hyperoxic condition set up with nitrogen and air saturated medium. Results: Between the CA groups, the normoxia group had a higher 48hr-survival rate (77%), lower NDS (359±140), and lower LIS (4.3±2.9) compared to those (28%, 452±85, 14±2, respectively) of the hyperoxia group (p&lt;0.01, p&lt;0.05, and p&lt;0.01, respectively). In the brain, lung, and kidney, CA augmented the hyperoxia-induced increase in carbonyl protein (absolute change: sham brain, 0.18 nmol/mg protein vs. CA brain, 0.61 nmol/mg protein; sham lung, 0.29 nmol/mg protein vs. CA lung, 0.72 nmol/mg protein; sham kidney, 0.04 nmol/mg protein vs. CA kidney, 0.55 nmol/mg protein, respectively). In the brain and kidney isolated mitochondria, CA augmented the hyperoxia-induced increase in H2O2 (absolute change: sham brain, 18 pmol/mg protein vs. CA brain, 60 pmol/mg protein; sham kidney, 16 pmol/mg protein vs. CA kidney, 45 pmol/mg protein, respectively). Conclusions: The normoxic therapy may attenuate organ damage and improve survival of CA. The reduction of augmented mitochondrial ROS generation, which is oxygen-sensitive, would be an important mechanism and therapeutic target of post-CA care.

Abstract P034: Vasculature Remodeling In Two Models Of Reduced Renal Mass

Remnant kidney models of chronic kidney disease include the 1/3 nephrectomy (1/3Nx), and 5/6 nephrectomy rats. The 1/3Nx remnant kidney spontaneously atrophies with time, and the 5/6Nx hypertrophies. This disparate remodeling must occur with commensurate vascular changes, examination of which may reveal important molecular pathways and mechanisms underlying changes in renal function. The first step toward realizing this, and the aim of this study, was to characterize the renal vasculature from 1/3Nx, 5/6Nx, and sham Nx rats. Methods: The vasculature was examined in rats 10 weeks following surgery. To-date, these renal vasculatures have been quantified by (1) assessment of vascular density by analysis of bisected, renal vascular casts, and (2) renal blood flow doppler ultrasound. Data: Avg±SEM. Results: 1/3Nx remnant kidney atrophied and 5/6Nx hypertrophied (cross-sectional area: sham n=9; 241±7. 5/6Nx, n=9; 247±8. 1/3Nx, n=12; 132±11: mm 2 ). Despite this, kidney height (intact kidney long axis) to width (short axis) ratio was similar between the remnant models (5/6Nx; 0.8±0.04. 1/3Nx; 0.9±0.04). Sham kidney vascular density was significantly greater than that of 1/3Nx remnant kidneys, which exceeded that of 5/6Nx rats. Cortex vascular density was significantly different between all groups (sham; 95±2%. 1/3Nx; 57±9%. 5/6Nx; 25±11%). The statistical mode of the arterial blood flow distribution was not different between the groups, but mode venous flow was (sham; 100±6. 1/3Nx; 118±9. 5/6Nx; 136±8. Relative units.). Homogeneity of flow was quantified by expressing flow-direction boundaries as a percent of flow area. Sham flow was significantly more homogenous (1.8±0.2) than 1/3Nx (3.7±0.5), and 5/6Nx rats (3.9±0.3). Conclusion: These data demonstrate changes in vascular anatomy and function in both remnant kidney models. Interrogation of anatomical changes and molecular pathways may reveal novel treatment approaches.

P0734THE DELETION OF AKT1 PROMOTES THE RENAL FIBROSIS IN THE MURINE MODEL OF UNILATERAL URETERAL OBSTRUCTION

Abstract Background and Aims Previous studies have found the increased Akt activity in experimental renal fibrosis. We investigated the role of Akt1, one of the three Akt isoforms, in renal fibrosis using the murine model of unilateral ureteral obstruction (UUO). Method In vivo, we subjected the wild type and Akt1−/− mice to UUO. In vitro, gene silencing of Akt1 was achieved using the short hairpin RNA delivered by the lentiviral vector in immortalized human proximal tubular cells (HK2 cells) and rat kidney fibroblasts (NRK-49F cells). Results In immunohistochemical stain, the expression of Akt1 was significantly higher in obstructed kidneys of wild type mice compared with control sham kidneys and increased gradually as UUO progressed. The fibronectin, type I collagen, and heat shock protein 47 (HSP47) were markedly more expressed in obstructed kidneys of Akt1−/− mice than in those of the wild type mice. Transforming growth factor β1 (TGFβ1) was highly induced within 1 day of UUO in obstructed kidneys of Akt1−/− mice and the expression of TGFβ1 was significantly higher in the Akt1−/− mice than in the wild type mice as UUO progressed. Western blot showed that silencing of Akt1 increased the expression of TGFβ1, which was enhanced by angiotensin II stimulation in HK2 cells, but not in NRK-49F cells. Immunohistochemical stain demonstrated that the expression of cleaved caspase-3 in renal tubules was significantly higher in the Akt1−/− mice than in the wild type mice. Western blot showed that silencing of Akt1 increased the expression of cleaved caspase-3 in HK2 cells, but not in NRK-49F cells. Conclusion Our findings suggest that the deletion of Akt1 might contribute to tubulointerstitial fibrosis and tubular apoptosis via TGFβ1 induction. We also showed that TGFβ1 upregulation by genetic deletion of Akt1 is associated with activation of STAT3 independently of the TGFβ1/Smad signaling pathway.

SPARC: Renal Denervation Attenuates DOCA‐salt Hypertension in the Mouse

Hypertension affects approximately one third of the adult population in the United States. Catheter based renal denervation (RDN) has emerged as a novel treatment for hypertension in patients. However, the mechanisms by which RDN lowers arterial pressure has not been fully determined. We hypothesize that an interaction between renal nerves (afferent and efferent) and renal inflammation contributes to hypertension. To test this hypothesis, we utilized a model of hypertension in the mouse produced by the combination of uni‐nephrectomy with deoxycorticosterone acetate (DOCA) and a high salt diet (DOCA‐salt). 10‐week‐old male C57BL6/J mice were implanted with radio‐telemetry probes in the carotid artery for measurement of arterial pressure. DOCA was administered via subcutaneous implantation of a silicon pellet containing 50 mg of DOCA. Sham mice received a drug free silicon pellet. Uni‐nephrectomy, pellet insertion, salt treatment, and RDN were all performed or started on the same day. RDN was performed through surgical ablation and peri‐axonal application of 10% phenol. Baseline arterial pressure was measured 3 days prior to induction of DOCA‐salt hypertension by radiotelemetry. Separate cohorts of mice were generated for protein quantification and histology, with urine and kidneys harvested 21 days post induction of DOCA‐salt hypertension. Cytokine protein quantification was obtained through Multiplex ELISA. All errors reported are SEM. Following 7 days of DOCA‐salt, mean arterial pressure (MAP) increased +44.8 ± 3.2 mmHg from baseline (153.1± 1.8 mmHg day 7 absolute value), comparted to −1.5 ± 5.5 mmHg in sham mice (105.6 ± 0.5 mmHg day 7 absolute value). RDN reduced this increase by more than 50% in RDN‐DOCA‐salt mice (+20.5 ± 5.0 mmHg delta, 131.6 ± 8.1 mmHg absolute). Renal and urinary cytokines and chemokines were increased in DOCA‐salt compared to sham treated mice. For example, renal MCP‐5 was 24.7 ± 7.2 pg/mg in DOCA‐salt mice compared to 5.0 ± 0.6 pg/mg in sham treated mice. Similarly, urinary MCP‐5 was significantly higher in in DOCA‐salt mice (12.4 ± 5.1 pg/mg) compared to sham mice (2.1 ± 1.1 pg/mg). IL‐1α and IL‐1β were both increased in DOCA‐salt kidneys (112.4 ± 19.2 pg/mg and 2.1 ± 0.4 pg/mg respectively) compared to sham kidneys (31.98 ± 4.4 pg/mg and 0.36 ± 0.06 pg/mg respectively). We also measured, by immunohistochemistry, an increase expression of the IL‐1 receptor in kidneys of DOCA‐salt mice compared to sham controls. We hypothesize that renal cytokines activate renal afferent nerves and ultimately elevate arterial pressure by activation of the sympathetic nervous system. Future studies will test this hypothesis by examining whether elimination of cytokine generated afferent nerve activity attenuates hypertension in DOCA‐salt mice.Support or Funding InformationNIH: R01HL116476, MnOPT: T32DK083250–01A1, SPARC: 5U01DK116320‐03

Regulation of Innate Lymphoid Cells in Acute Kidney Injury: Crosstalk between Cannabidiol and GILZ.

Innate lymphoid cells (ILCs) have emerged as largely tissue-resident archetypal cells of the immune system. We tested the hypotheses that renal ischemia-reperfusion injury (IRI) is a contributing factor to polarization of ILCs and that glucocorticoid-induced leucine zipper (GILZ) and cannabidiol regulate them in this condition. Mice subjected to unilateral renal IRI were treated with the following agents before restoration of renal blood flow: cannabidiol, DMSO, transactivator of transcription- (TAT-) GILZ, or the TAT peptide. Thereafter, kidney cells were prepared for flow cytometry analyses. Sham kidneys treated with either cannabidiol or TAT-GILZ displayed similar frequencies of each subset of ILCs compared to DMSO or TAT, respectively. Renal IRI increased ILC1s and ILC3s but reduced ILC2s compared to the sham group. Cannabidiol or TAT-GILZ treatment of IRI kidneys reversed this pattern as evidenced by reduced ILC1s and ILC3s but increased ILC2s compared to their DMSO- or TAT-treated counterparts. While TAT-GILZ treatment did not significantly affect cells positive for cannabinoid receptors subtype 2 (CB2+), cannabidiol treatment increased frequency of both CB2+ and GILZ-positive (GILZ+) cells of IRI kidneys. Subsequent studies showed that IRI reduced GILZ+ subsets of ILCs, an effect less marked for ILC2s. Treatment with cannabidiol increased frequencies of each subset of GILZ+ ILCs, but the effect was more marked for ILC2s. Indeed, cannabidiol treatment increased CB2+ GILZ+ ILC2s. Collectively, the results indicate that both cannabidiol and GILZ regulate ILC frequency and phenotype, in acute kidney injury, and that the effects of cannabidiol likely relate to modulation of endogenous GILZ.

The contralateral kidney presents with impaired mitochondrial functions and disrupted redox homeostasis after 14 days of unilateral ureteral obstruction in mice.

In unilateral ureteral obstruction (UUO), both oxidative stress and mitochondrial dysfunction are related to cell death. The aim of this study has been to characterize profiles of enzyme antioxidant activities and mitochondrial functioning of the contralateral (CL) compared to UUO and Sham (false-operated) kidneys of Balb/c mice. Kidneys were resected 14 days after obstruction for immunohistochemical and cortical mitochondrial functioning assays. Antioxidant enzymes activities were investigated in mitochondria and cytosol. Oxygen consumption (QO2) and formation of O2 reactive species (ROS) were assessed with pyruvate plus malate or succinate as the respiratory substrates. QO2 decreased in CL and UUO in all states using substrates for complex II, whereas it was affected only in UUO when substrates for complex I were used. Progressive decrease in mitochondrial ROS formation–in the forward and reverse pathway at complex I–correlates well with the inhibition of QO2 and, therefore, with decreased electron transfer at the level of complexes upstream of cytochrome c oxidase. CL and UUO transmembrane potential responses to ADP were impaired with succinate. Intense Ca2+-induced swelling was elicited in CL and UUO mitochondria. Important and selective differences exist in CL antioxidant enzymes with respect to either Sham or UUO kidneys: CL kidneys had increased mitochondrial glutathione peroxidase and cytosolic catalase activities, indicative of compensatory responses in the face of an early altered ROS homeostasis (as detected by 4-hydroxynonenal), and of a significant tendency to apoptosis. In CL and UUO, upregulation of nuclear (erythroid-derived 2)-like 2 transcription factor (Nrf2), as well as of cytoplasmic and nuclear Kelch-like ECH-associated protein 1 (Keap1) in opposition to decreased heme oxygenase-1 (HO-1), suggest impairment of the Nrf2/Keap1/HO-1 system. It is concluded that chronic obstruction impairs mitochondrial function in CL and UUO, preferentially affecting complex II.

Cold Storage Increases Albumin and Advanced Glycation-End Product-Albumin Levels in Kidney Transplants: A Possible Cause for Exacerbated Renal Damage.

Background.Prolonged cold storage (CS) of kidneys is associated with poor renal outcome after transplantation (Tx). We recently showed that in rats (Lewis), proteasome and renal function were severely compromised in kidney transplants subjected to CS (CS/Tx) as compared with those without CS exposure (autotransplanted [ATx]).Methods.Evaluation of whole-kidney extracts from our rat kidney transplant model showed a subset of proteins induced after CS/Tx when compared with ATx or sham groups; this study examined those proteins using mass spectrometry, western blotting, immunoprecipitation, and immunohistochemistry.Results.Mass spectrometry identified basal albumin levels in sham kidney extracts; western blots and immunohistochemistry confirmed this. Western blotting showed exceptionally higher albumin levels in both soluble and insoluble fractions of CS/Tx renal extracts when compared with ATx and sham groups. Surprisingly, levels of advanced glycation-end products (AGE) were higher in CS/Tx renal extracts. Furthermore, immunoprecipitation of albumin followed by western blotting for AGE revealed AGE-albumin in all 3 extracts; its levels were highest in CS/Tx extracts. Immunohistochemistry analysis of kidney sections revealed higher albumin or AGE levels in the CS/Tx group, and the protein was detected all over (within glomeruli, and intratubular and extratubular compartments) when compared with ATx and sham groups, which show confinement of these proteins to the extratubular compartment and within glomeruli. As expected, kidneys of the ATx group showed evidence of more macrophages, which was exacerbated in the CS/Tx group.Conclusions.These results suggested that CS/Tx increased AGE-albumin, which was correlated with increased inflammation and renal damage.