Post Ischemia-reperfusion Injury Research Articles

Abstract 16879: Endothelial Prolyl Hydroxylase 3 Regulates Post-Ischemic Kidney Injury Through HIF Dependent Mechanisms

Introduction: Acute kidney injury (AKI) is highly prevalent clinical condition after cardiac surgeries and is associated with high morbidity and mortality in hospitalized patients. Ischemia reperfusion injury (IRI) is one of the major causes of AKI. Previously, we have reported a critical role for the endothelial oxygen sensor prolyl hydroxylase (PHD2) in ischemic AKI. Recently, we found that prolyl hydroxylase 3 (PHD3) shows robust increase in kidney endothelium after renal IRI. However, its role in ischemic AKI is unknown. Hypothesis: We hypothesized that endothelial PHD3 regulates post-ischemic kidney injury. Methods: To delete endothelial PHD3, VECadherin (Cdh5)-CreER transgenic mice were crossed to mice carrying conditional PHD3 allele, generating PHD3 iEC mice. To test the HIF-dependent role of PHD3, we generated Cdh5CreER; Phd3 flox/flox Arnt flox/flox mice ( PHD3ARNT iEC ). Inactivating ARNT (aryl hydrocarbon receptor nuclear translocator), also known as Hif-1β, blocks HIF signaling. Unilateral IRI was induced by renal artery clamping, and tamoxifen treatment was started at day 1 post IRI, followed by analysis at day 14. Results: Compared to Cre - controls, inactivation of Phd3 in endothelial cells (ECs) following IRI exacerbated kidney damage and inflammation, as indicated by increased expression of kidney injury molecule 1 ( Kim1 ) and profibrotic genes lysyl oxidase-like 2 ( Loxl2 ), transforming growth factor-beta 1 ( Tgfb1 ) , smooth muscle actin ( Acta2 ) and collagen deposition assessed by Picro Sirius red staining (n=6-7; p< 0.05). Post-ischemic inactivation of ARNT along with PHD3 ameliorated the effects of only PHD3 inactivation and normalized levels of Kim1 , Loxl2 , Tgfb1 and collagen deposition ( n=7; p< 0.05) . In vitro suppression of PHD3 in human primary EC using PHD3-siRNA showed significant upregulation of inflammatory molecules such as ICAM1 (Intercellular Adhesion Molecule 1), VCAM1 (Vascular Cell Adhesion Molecule 1), C-X-C motif chemokine ligand 9 and 10 ( CXCL9 and CXCL10 ), which were again blunted by ARNT- siRNA treatment (n= 3; p< 0.05). Conclusions: In summary, our data support a critical role for endothelial PHD3 in regulating post-ischemic kidney inflammation and fibrosis through HIF-dependent mechanisms.

Cardioprotective efficacy of ultrasound-targeted nitrofatty acid microbubbles in rat myocardial ischemia-reperfusion injury model

Over 1 million Americans suffer from acute myocardial infarction (AMI) annually. Although mortality from AMI has decreased, post-AMI congestive heart failure is increasing due to microvascular obstruction (MVO). MVO comprises mechanical obstruction, along with oxidate stress and inflammation. Currently available treatments for MVO are not consistently effective. Hence, we have been developing ultrasound (US)-targeted microbubble cavitation (UTMC) as a potential treatment for MVO. Nitro-fatty acids (NFA) are pleiotropic signaling molecules with broad anti-inflammatory actions, potentially beneficial for the treatment of MVO. NFA are amphipathic thus can seamlessly integrate into the phospholipid shell of MBs. Thus, we have constructed microbubbles with NFA (NFABs) to treat ischemia-reperfusion injury (IRI) with UTMC in the rat myocardial model. Left anterior descending (LAD) coronary artery was ligated for 30 min, allowing for IRI. After 15 min of reperfusion, UTMC + NFABs therapy was administered. Echocardiography measurements were recorded at baseline, during ligation and post-treatment. Left ventricular fractional shortening (%) was calculated and NFA concentration in cardiac tissue was determined. UTMC with NFABs exhibited promising efficacy in improving fractional shortening post IRI, and in targeted myocardial delivery of NFA. Studies assessing ejection fraction, myocardial area at risk, histopathology, inflammatory burden, oxidative stress and cytoprotective biomarkers are underway.

Cathepsin-K inhibition improves cardiac function post-Ischemia reperfusion injury

Cathepsin-K inhibition improves cardiac function post-Ischemia reperfusion injury

Serial Intravital 2‐photon Imaging Deciphers in Vivo Renal Myofibroblast Dynamics at the Fork of Acute Kidney Injury‐induced Tubular Repair and Nephron Loss

Myofibroblasts were suggested to play a pivotal role in the transition of acute kidney injury (AKI) to chronic kidney disease, by engaging in progressive and self‐perpetuating tubulointerstitial fibrosis. Wound healing from AKI gives rise to myofibroblast‐activation from renal interstitial PDGFRβ‐cells. In this study, we exploited a novel model of partial ischemia reperfusion injury (IRI), to track dynamic PDGFRbeta‐cell interactions with injured and non‐injured tissue over time and addressed the question if and how myofibroblast‐derived renal interstitial fibrosis may progress and deteriorate non‐injured renal tissue after AKI.PDGFRβ‐CreERT2‐Salsa6F reporter mice identified PDGFRβ‐cells by tdTomato expression. IRI was performed by unilateral occlusion of only one branch of the left renal artery for 21 min, resulting in half of the left kidney being ischemic, while the other half remained non‐ischemic. This was followed by abdominal imaging window implantation for serial imaging of the same kidney cells in border areas for 2 to 3 weeks (N=5). To test for myofibroblast differentiation, we performed ex vivo staining for αSMA on the same tissue regions as previously imaged in vivo.IRI‐induced necrotic tubular cell death, as detected in vivo by Propidium Iodide (PI)‐staining, was only observed on day 0 (D0) post IRI. Proximal tubules (PTs) with intermediate necrotic injury (mean ± SD) (21±15% dead cells of total cell number per segment) underwent successful self‐repair within 7 days through dedifferentiation as indicated by shedding of the apical membrane (peaking at D2), followed by the temporal flattening of the epithelium. In highly injured PT segments (39±15% cell death), dedifferentiation was followed by epithelial vacuolization (starting at D4/D7), resulting in failed repair in 35% of the cases as indicated by collapsed and karyolitic tubules evident between D7 and D14.Tubule injury initiated pronounced PDGFRβ‐cell recruitment, which resulted in remarked enclosure of the remodeling epithelium. Thus, PDGFRβ‐cell recruitment peaked at D4, and D7 with a maximum coverage of PT cell surface of 29±6% and 44±14% for surviving tubules of initially low and high necrotic injury, respectively. In non‐surviving tubules, PDGFRβ‐cell recruitment peaked D7 with 63±18% of the remodeling PT cells covered. Ex vivo stainings for αSMA confirmed PDGFRβ‐cell differentiation to myofibroblasts. Importantly, regardless of respective tubule faith, PDGFRβ‐cell accumulation resolved over the period from D7‐D14/21 and did not progress towards non‐injured tubules. Further, ex vivo stainings at D14/21 indicated αSMA‐negativity of persistent and previously injury‐interacting PDGFRβ‐cells, indicating the reversibility of myofibroblast differentiation.Our data indicate locally restricted, injury‐dependent and reversible PDGFRβ‐cell accumulation and myofibroblast‐differentiation in ischemic tissue regions. There is no indication for the existence of self‐perpetuating interstitial fibrosis and deterioration of healthy nephrons. Furthermore, our data indicates the reversibility of myofibroblast‐differentiation upon completed repair programs.

Fatty acid amide hydrolase (FAAH) inhibition mitigates TGF‐β1‐induced fibrogenesis via the anandamide‐COX‐2‐dependent pathway

Chronic proximal tubular injury with subsequent fibrosis is the key factor in the transition of acute kidney injury (AKI) to chronic kidney disease (CKD). An increasing body of evidence has shown that endocannabinoids such as anandamide (AEA) and 2‐arachidonoylglycerol (2AG) and their cannabinoid receptors (CBs) play important roles in renal diseases. It remains unclear whether substrates or metabolites of AEA‐metabolizing enzyme FAAH participate in fibrogenesis of kidneys. Though AEA binds the CB1 receptor with high binding affinity, its in vivo pharmacological effects mediated by cannabinoid receptors are of short duration. Likely, active metabolites produced from the rapid catabolism of AEA exert physiological actions. We have previously showed that inhibition of FAAH protects against TGF‐β1‐induced damages via actions independent of CBs in renal tubular cells. Therefore, our aims are to study the effects of downstream metabolites derived from FAAH as well as AEA‐ cyclooxygenase‐2 (COX‐2) pathways on TGF‐β1‐induced renal fibrogenesis. To produce the AKI to CKD transition model, male wild type (WT) C57BL6 and Faah−/− mice were subjected to post ischemia‐reperfusion injury (PIR) by unilateral kidney ischemia‐reperfusion and contralateral nephrectomy 10 days after ischemia‐reperfusion. Our previous studies showed that Faah−/− mice protected against PIR‐induced renal fibrogenesis. Here, LC‐MS/MS assay demonstrated that PIR‐injured WT mice had a significant downregulation of renal AEA level, which was blocked in Faah−/−‐PIR mice (68% reduction in WT‐PIR versus 12% in Faah−/−‐PIR mice compared with WT‐sham group, p<0.001), whereas 2AG showed no difference between WT‐PIR and Faah−/−‐PIR mice (1.2‐fold versus 1.1‐fold increase, p>0.9). In addition, Faah−/− mice did not show difference in the levels of arachidonic acid (AA) (1.13‐fold to WT‐sham, p=0.25), the downstream metabolites of FAAH. Our previous in vitro studies showed that TGF‐β1‐induced profibrotic effects in HK‐2 cells (human kidney proximal tubular epithelial cells) were abolished after the treatment of FAAH inhibitor (PF‐04457845, PF, 1μM) in a CB2‐independent manner. In the current study, blockage of AEA‐COX‐2 derived prostamides pathway by a substrate‐selective COX‐2 inhibitor (LM‐4131, 300nM) reversed the decreased level of a profibrotic marker α‐smooth muscle actin (α‐SMA) caused by PF (1.9‐fold in TGF‐β1, 0.83‐fold in PF+TGF‐β1 and 2.88‐fold in PF+LM‐4131+TGF‐β1 compared to the control group, p<0.0001). Further, supplement of AA did not reverse the protection of FAAH inhibition against TGF‐β1‐induced expression of α‐SMA (2.53‐fold in TGF‐β1, 0.62‐fold in PF+TGF‐β1 versus 0.48‐fold in AA+PF+TGF‐β1 compared to the control group, p>0.99). The AEA‐COX‐2 metabolite, prostamide E2 mimicked the protective effect of FAAH inhibition against TGF‐β1‐induced α‐SMA expression (2.17‐fold in TGF‐β1 vs. 0.9‐fold in prostamide E2 +TGF‐β1 compared to the control group, p<0.0001). Overall, the present study suggests that inactivation of FAAH augments the actions of downstream metabolites of AEA‐COX‐2, which mediates the protection against TGF‐β1‐induced renal fibrogenesis.

Sevoflurane alleviates liver ischemia reperfusion injury through inactivation of the TRAF6/NF-κB signaling pathway

Purpose: To evaluate the role and mechanism of action of sevoflurane in liver ischemia reperfusion injury.Methods: Rats were pretreated with sevoflurane and then underwent liver ischemia followed by reperfusion to establish an animal model of liver ischemia reperfusion injury. Pathological changes in liver tissues were investigated by hematoxylin and eosin (H & E) staining, and serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined using a chemistryanalyzer. ELISA was used to determine the levels of myeloperoxidase (MPO), tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1β), IL-6, superoxide (SOD), malonaldehyde (MDA), catalase (CAT), and glutathione (GSH).Results: Pathological changes in liver tissue, including sinusoidal congestion, vacuole formation, and infiltration of inflammatory cells and lymphocytes, were identified in rats post-ischemia reperfusion injury. In addition, serum ALT and AST levels increased following ischemia reperfusion injury. However, administration of sevoflurane ameliorated the pathological liver damage and decreased the serum ALTand AST levels induced by ischemia reperfusion. Pro- inflammatory cytokines, such as MPO, TNF-α, IL- 1β, and IL-6 were upregulated in rats following ischemia reperfusion injury, and this upregulation was reversed by sevoflurane administration. Sevoflurane administration also attenuated the ischemia reperfusion-induced increase in MDA and decrease in SOD, CAT, and GSH. Ischemia reperfusionrepressed IκBα protein expression and promoted protein expression of TNF receptor associated factor 6 (TRAF6), phospho (p)-IκBα, and p-p65 in liver tissue. However, sevoflurane reversed the effect of ischemia reperfusion on IκBα, TRAF6, p-IκBα, and p-65 expression.Conclusion: Sevoflurane administration reduced pathological liver injury post-ischemia reperfusion bysuppressing the inflammatory response and oxidative stress through inactivation of the TRAF6/NF-κB pathway.

Properdin Deficiency Impairs Phagocytosis and Enhances Injury at Kidney Repair Phase Post Ischemia-Reperfusion.

Properdin, a positive regulator of complement alternative pathway, participates in renal ischemia–reperfusion (IR) injury and also acts as a pattern-recognition molecule affecting apoptotic T-cell clearance. However, the role of properdin in tubular epithelial cells (TECs) at the repair phase post IR injury is not well defined. This study revealed that properdin knockout (PKO) mice exhibited greater injury in renal function and histology than wild-type (WT) mice post 72-h IR, with more apoptotic cells and macrophages in tubular lumina, increased active caspase-3 and HMGB1, but better histological structure at 24 h. Raised erythropoietin receptor by IR was furthered by PKO and positively correlated with injury and repair markers. Properdin in WT kidneys was also upregulated by IR, while H2O2-increased properdin in TECs was reduced by its small-interfering RNA (siRNA), with raised HMGB1 and apoptosis. Moreover, the phagocytic ability of WT TECs, analyzed by pHrodo Escherichia coli bioparticles, was promoted by H2O2 but inhibited by PKO. These results were confirmed by counting phagocytosed H2O2-induced apoptotic TECs by in situ end labeling fragmented DNAs but not affected by additional serum with/without properdin. Taken together, PKO results in impaired phagocytosis at the repair phase post renal IR injury. Properdin locally produced by TECs plays crucial roles in optimizing damaged cells and regulating phagocytic ability of TECs to effectively clear apoptotic cells and reduce inflammation.

Inactivation of Endocannabinoid‐metabolizing Enzyme FAAH Represses TGF‐β1‐induced Epithelial‐mesenchymal Transition in Renal Proximal Tubular Epithelial Cells

Epithelial-mesenchymal transition (EMT) of proximal tubular epithelial cells plays a very important role in the development of renal fibrosis and progressive chronic kidney disease (CKD). No effective therapy for renal fibrosis currently exists. Endocannabinoid system has been described to be involved in renal fibrosis. Anandamide and 2-arachidonoylglycerol, two major endocannabinoids, have been shown to participate in the regulation of renal physiology via cannabinoid receptors (CB1 and CB2). CB1 and CB2 are demonstrated to also participate in CKD. However, the role of fatty acid amide hydrolase (FAAH), a major anandamide-metabolizing enzyme, in CKD still remains poorly understood. We previously reported that FAAH knockout mice showed improved renal function and kidney fibrosis following post ischemia-reperfusion injury. The present study tested the hypothesis that FAAH participates in TGF-β1–induced EMT in renal cells. In HK2 cells, a human proximal tubular epithelial cell line, treatment of FAAH inhibitor PF-04457845 significantly attenuated TGF-β1–induced expression of the mesenchymal marker α-smooth muscle actin (α-SMA, 1.6-fold in TGF-β1 vs. 0.7-fold in TGF-β1+PF to the control group, p<0.05) and tubular epithelial injury protein NGAL (2.1-fold in TGF-β1 vs. 0.95-fold in TGF-β1+PF to the control group, p<0.05). Similarly, siRNA-mediated knockdown of FAAH also showed reduction of TGF-β1–induced expression of ɑ-SMA (0.94-fold vs. 1.8-fold to Ctrl group, p<0.05) and NGAL (1.3-fold vs. 2.8-fold to Ctrl group, p<0.05). The immunocytochemistry staining results demonstrated that TGF-β1-induced loss of epithelial tight junction protein ZO-1 was restored by inhibition of FAAH (46% reduction vs. 17% reduction to the control group, p<0.05). Because CB2 has been shown to mediate renoprotection, HK-2 cells were treated with CB2 antagonist (SR144528) in combination with FAAH inhibitor (PF-04457845). Blockage of CB2 receptor by SR showed no effect on the reduction of α-SMA expression by FAAH inhibitor, suggesting that the protective effect of FAAH inhibition on tubular EMT is independent of CB2 receptor activation. Overall, the present data suggest that suppression of FAAH attenuates TGF-β1-induced EMT in renal proximal tubular epithelial cells. Thus, FAAH could be a new target for developing an effective treatment for CKD.

Effects of Quercetin on Cardiac Function in Pressure Overload and Postischemic Cardiac Injury in Rodents: a Systematic Review and Meta-Analysis.

Cardiac dysfunction can occur as a sequela of a state of prolonged pressure overload and postischemic injury. Flavonoids such as quercetin may be protective against cardiovascular disease. This study aimed to systematically assess the effects of quercetin on cardiac function in pressure overload and postischemia-reperfusion injury in rodents. A systematic search of the literature up to May 2020 was conducted in PubMed, Ovid Medline, EBSCOhost, Scopus, and the Cochrane Library to identify relevant published studies on quercetin and cardiac function using standardized criteria. Meta-analyses were performed on animal studies of pressure overload and ischemia-reperfusion (I/R) injury. The effects of quercetin on cardiac function in both models were qualitatively reported in 14 studies. The effects of quercetin in four pressure-overload model studies involving 73 rodents and eight I/R-injury model studies involving 120 rodents were quantitatively assessed by meta-analysis. Quercetin improved the overall cardiac function in both pressure overload (n = 4 studies, n = 73 rodents; SMD = - 1.50; 95% CI: - 2.66 to - 0.33; P < 0.05; I2 = 74.05%) and I/R injury (n = 8 studies, n = 120 rodents; SMD = - 1.81; 95% CI: - 3.05 to - 0.56; P < 0.01; I2 = 84.93%) models. The improvement was associated with amelioration in cardiac structure in the pressure-overload model and both systolic and diastolic functioning in the I/R-injury model. The present meta-analysis suggested that quercetin has beneficial effects for improving cardiac left ventricular dysfunction in both pressure-overload and I/R-injury models.

Cooling as an Adjunctive Therapy to Percutaneous Intervention in Acute Myocardial Infarction: COOL-MI InCor Trial.

Endovascular Therapeutic hypothermia (ETH) reduces the damage caused by postischemia reperfusion injury syndrome in cardiopulmonary arrest and has already established its role in patients with sudden death; however, its role in ST-segment elevation myocardial infarction (STEMI) remains controversial. The objectives of this study were to investigate the safety, feasibility, and 30-day efficacy of rapid induction of therapeutic hypothermia as adjunctive therapy to percutaneous coronary intervention (PCI) in patients with anterior and inferior STEMIs. This was a prospective, controlled, randomized, two-arm, prospective, interventional study of patients admitted to the emergency department within 6 hours of angina onset, with anterior or inferior STEMI eligible for PCI. Subjects were randomized to the hypothermia group (primary PCI+ETH) or to the control group (primary PCI) at a 4:1 ratio. The ETH was induced by 1 L cold saline (1-4°C) associated with the Proteus™ System, by cooling for at least 18 minutes before coronary reperfusion with a target temperature of 32°C ± 1°C. Maintenance of ETH was conducted for 1-3 hours, and active reheating was done at a rate of 1°C/h for 4 hours. Primary safety outcomes were the feasibility of ETH in the absence of (1) door-to-balloon (DTB) delay; (2) major adverse cardiac events (MACE) within 30 days after randomization. The primary outcomes of effectiveness were infarct size (IS) and left ventricular ejection fraction (LVEF) at 30 days. An as-treated statistical analysis was performed. Fifty patients were included: 35 (70%) randomized to the hypothermia group and 15 (30%) to the control group. The mean age was 58 ± 12 years; 78% were men; and associated diseases were 60% hypertension, 42% diabetes, and 72% dyslipidemia. The compromised myocardial wall was anterior in 38% and inferior in 62%, and the culprit vessels were left anterior descending artery (LAD) (40%), right coronary artery (38%), and left circumflex (18%). All 35 patients who attempted ETH (100%) had successful cooling, with a mean endovascular coronary reperfusion temperature of 33.1°C ± 0.9°C. The mean ischemic time was 375 ± 89.4 minutes in the hypothermia group and 359.5 ± 99.4 minutes in the control group. The mean DTB was 92.1 ± 20.5 minutes in the hypothermia group and 87 ± 24.4 minutes in the control group. The absolute difference of 5.1 minutes was not statistically significant (p = 0.509). The MACE rates were similar between both groups (21.7% vs. 20% respectively, p = 0.237). In the comparison between the hypothermia and control groups, no statistically significant differences were observed at 30 days between mean IS (13.9% ± 8% vs. 13.8% ± 10.8%, respectively, p = 0.801) and mean final LVEF (43.3% ± 11.2% vs. 48.3 ± 10.9%, respectively; p = 0.194). Hypothermia as an adjunctive therapy to primary PCI in STEMI is feasible and can be implemented without delay in coronary reperfusion. Hypothermia was safe regarding the incidence of MACE at 30 days. However, there was a higher incidence of arrhythmia and in-hospital infection in the hypothermia group, with no increase in mortality. Regarding efficacy, there was no difference in IS or LVEF at 30 days that would suggest additional myocardial protection with ETH. ClinicalTrials.gov: NCT02664194.

P0722VITAMIN D-DEFICIENT RATS RECOVER RENAL ALTERATIONS AFTER VITAMIN D REPLACEMENT FOLLOWING ISCHEMIA-REPERFUSION INJURY

Abstract Background and Aims Several studies have been linking vitamin D deficiency (VDD) as a non-traditional risk factor to acute kidney injury (AKI). It has been shown that VDD associated with AKI potentiates the injury and may accelerate the progression of kidney disease. Conversely, some lines of evidence have been describing that vitamin D sufficiency can be considered as a renal protective factor. Thus, we aimed to verify the effect of vitamin D replacement (R) on the renal disease progression post ischemia-reperfusion injury (IRI) in vitamin D deficient rats. Method We performed bilateral 45 min IRI on day 30 in all animal groups. Male Wistar rats were randomized into three groups: 1- IRI (fed a standard diet for 120 days); 2- VDD+IRI (fed a vitamin D-free diet for 120 days); and 3- VDD+IRI+R (fed a vitamin D-free diet for 30 days and just after IRI, on day 31, we reintroduced the standard diet for more 90 days). We evaluated inulin clearance (Cin); mean arterial pressure (MAP); renal blood flow (RBF); plasma levels of Vitamin D2+D3 [25(OH)D] and Parathormone (PTH) by ELISA. In addition, we performed histomorphometrical studies for CD3+ cells, fibronectin and vimentin as well as the fractional interstitial area (FIA). Moreover, we run qPCR studies and immunoblotted for vitamin D receptor (VDR) and Klotho. All the results are described in table 1. Results Vitamin D replacement restored the plasma levels of 25(OH)D and PTH in VDD+IRI+R group. Also, VDD+IRI+R presented an improvement of renal function and hemodynamic parameters. In addition, we observed more evident alterations in tubulointerstitial compartment, featuring interstitial expansion (renal fibrosis and inflammatory cell infiltrates) and fibronectin and vimentin expression in VDD+IRI rats. These alterations were recovered by vitamin D replacement. Concerning VDR and Klotho data, our results revealed a decreased expression of these targets in VDD+IRI group. On the other hand, vitamin D replacement retrieved the expression of VDR and Klotho. Conclusion Our study suggests that vitamin D replacement improved the recovery of renal function, hemodynamics, inflammatory and morphological alterations. In addition, vitamin D replacement was able to reestablish the expression of VDR and Klotho in IRI-AKI associated with vitamin D deficiency. Thus, it is recommendable that vitamin D levels should be observed as well as its reposition should be taken into account in renal patients.

Erythropoietin Derived Peptide Improved Endoplasmic Reticulum Stress and Ischemia-Reperfusion Related Cellular and Renal Injury.

Ischemia-reperfusion (IR) injury often affects transplant and native kidneys alike. IR injury is one of the main causes of acute kidney injury (AKI) and further associated with the progression of chronic kidney disease. Our previous study revealed the renoprotection of erythropoietin derived cyclic helix-B surface peptide (CHBP) against IR injury. However, the precise role and underlying mechanism of endoplasmic reticulum stress (ERS) in the injury and the renoprotection induced by IR or CHBP, respectively, have not been fully defined. This study using mouse kidney epithelial cells (TCMK-1) revealed that the level of CHOP (a key marker of ERS), PERK, and JNK (regulators of CHOP) was gradually increased by the prolonged time of hydrogen peroxide (H2O2) stimulation. In addition, CHOP mRNA and protein were significantly reduced by small interfering RNA (siRNA) target CHOP, as were apoptotic and inflammatory mediator caspase-3 and HMGB-1, and early apoptosis. Furthermore, CHOP mRNA was correlated positively with PERK protein, active caspase-3, HMGB-1 and apoptosis, but negatively with cell viability in vitro, while CHOP protein was also correlated positively with the level of tubulointerstitial damage and active caspase-3 protein in vivo. Finally, CHBP improved the viability of TCMK-1 cells subjected to H2O2 stimulation time-dependently, with reduced level of CHOP mRNA. CHBP also inhibited the increase of CHOP protein, not only in TCMK-1 cells, but also in the IR injury kidneys at 2 weeks. Moreover, CHBP reduced the expression of PERK mRNA and protein, JNK and HMGB-1 protein, as well as early and later apoptosis. In addition, raised CHOP at 12 h post IR injury might be an early time window for intervention. In conclusion, the differential role of ERS and CHBP in IR-related injury was proved in mouse TCMK-1 cells and kidneys, in which the mechanistic signaling pathway was associated with CHOP/PERK/JNK, HMGB-1/caspase-3, and apoptosis. CHOP might be a potential biomarker and CHBP might be therapeutic drug for IR-induced AKI.

Progenitor/Stem Cell Delivery by Suprarenal Aorta Route in Acute Kidney Injury

Progenitor/stem cell-based kidney regenerative strategies are a key step towards the development of novel therapeutic regimens for kidney disease treatment. However, the route of cell delivery, e.g., intravenous, intra-arterial, or intra-parenchymal, may affect the efficiency for kidney repair in different models of acute and chronic injury. Here, we describe a protocol of intra-aorta progenitor/stem cell injection in rats following either acute ischemia-reperfusion injury or acute proteinuria induced by puromycin aminonucleoside (PAN) – the experimental prototype of human minimal change disease and early stages of focal and segmental glomerulosclerosis. Vascular clips were applied across both renal pedicles for 35 min, or a single dose of PAN was injected via intra-peritoneal route, respectively. Subsequently, 2 x 106 stem cells [green fluorescent protein (GFP)-labeled c-Kit+ progenitor/stem cells or GFP-mesenchymal stem cells] or saline were injected into the suprarenal aorta, above the renal arteries, after application of a vascular clip to the abdominal aorta below the renal arteries. This approach contributed to engraftment rates of ∼10% at day 8 post ischemia-reperfusion injury, when c-Kit+ progenitor/stem cells were injected, which accelerated kidney recovery. Similar rates of engraftment were found after PAN-induced podocyte damage at day 21. With practice and gentle surgical technique, 100% of the rats could be injected successfully, and, in the week following injection, ∼ 85% of the injected rats will recover completely. Given the similarities in mammals, much of the data obtained from intra-arterial delivery of progenitor/stem cells in rodents can be tested in translational research and clinical trials with endovascular catheters in humans.

515-P: Mitochondrial AKT1 in Renal Tubules Is a Novel Modulator for the Development of Glomerulosclerosis and Subsequent Renal Failure

Diabetes increases the risk of renal ischemia/reperfusion injury (IRI). Recent evidence suggests that tubular dysfunction is associated with glomerular impairment, but its causal relationship is unclear. Renal tubules have abundant mitochondria; Akt is translocated into mitochondria upon IRI. To test the hypothesis that tubular mitochondrial Akt1 (mito-Akt) plays a protective role during IRI and subsequent renal failure, we have generated novel renal tubule-specific transgenic mice harboring inducible mitochondria-targeting dominant negative Akt1 (KMDAKT) or constitutively active Akt1 (KMCAKT) with the Cre-lox system. After KMDAKT mice were induced with tamoxifen (T) and subjected to IRI, histological analysis showed increased tubule injury index Jablonski score (p&amp;lt;0.04) and greater renal fibrosis as compared to the corn-oil (CO) injected mice (p&amp;lt;0.02). Kidney Injury Molecule-1 (KIM1), PAS-positive glomerulosclerosis, and serum creatinine were all increased (p&amp;lt;0.01) in the T-KMDAKT mice when compared to the CO mice 45-days post IRI. Renal inflammatory markers, including IL-1B, were also increased, suggesting inflammation played a role. Kaplan-Meier analysis showed much lower survival in the T-KAMDAKT mice post IRI than the CO mice (p&amp;lt;0.01). To study whether augmentation of tubular mito-Akt can protect against renal IRI, T-KMCAKT and CO-KMCAKT were subjected to renal IRI.T-KMCAKT mice showed an improved Jablonski score (p&amp;lt;0.02), less renal fibrosis (p&amp;lt;0.04), and lower KIM1 expression (p&amp;lt;0.02). T-KMCAKT mice exhibited less glomerulosclerosis and better survival (p&amp;lt;0.01). Summary: 1] tubular mito-Aktcritically modulated the outcomes of kidney IRI beyond renal tubules, 2] tubular mitochondria dysfunction causally triggered development of glomerulosclerosis during acute and chronic kidney failure, and 3] renal tubular mito-Akt, which is activated by insulin, may represent a novel therapeutic target for diabetic nephropathy. Disclosure H.Y. Lin: None. Y. Chen: None. A. Ta: None. H. Lee: None. Y. Chen: None. P.H. Wang: None.

CXCL16/ROCK1 signaling pathway exacerbates acute kidney injury induced by ischemia-reperfusion

Renal ischemia-reperfusion injury (IRI) is a leading cause of acute kidney injury (AKI) resulting in an abrupt deterioration of kidney function. CXC chemokine ligand 16 (CXCL16) contributes significantly to the pathogenesis of renal injury. However, the signaling pathway mechanisms of CXCL16 in IRI-induced AKI remains obscured. In this study, we examined the role of the CXCL16/ Rho Associated Coiled-Coil Containing Protein Kinase-1 (ROCK1) signaling pathway in AKI induced by IRI. In vivo, CXCL16 was induced markedly after IRI. Mice treated with anti-CXCL16 antibody displayed less severe renal dysfunction and tubular injury in response to IRI compared with vehicle-treated mice. Inhibition of CXCL16 substantially reduced apoptotic cells and suppresses caspase-3 activation in the kidneys of mice following IRI. Additionally, CXCL16 inhibition profoundly decreased the production of TNF-α, IL-1β and IL-6 in the kidneys of mice post IRI. Furthermore, the level of ROCK1 protein was upregulated in the kidney in response to IRI, an effect that was abolished by CXCL16 inhibitor. Finally, treatment with Y-27632 (a ROCK1 inhibitor) attenuated deterioration of renal function and tubular damage of mice after IRI. Administration of Y-27632 ameliorated apoptosis in the IRI-treated kidneys of mice. In injured HK-2 cells, CXCL16 activated ROCK1 resulting in the upregulation of caspase-3 protein and pro-inflammatory molecules, which was abolished by Y-27632. In summary, our findings demonstrate that CXCL16/ROCK1 signaling pathway may play an important role in the pathogenesis of IRI-induced AKI.

ERK1/2 Activation Following Renal Injury Is Required For Kidney Injury Molecule‐1 Production

BackgroundBecause substantial damage can occur before detection of acute kidney injury (AKI) new early predictor biomarkers are needed, such as kidney injury molecule‐1 (KIM‐1). KIM‐1 is a transmembrane glycoprotein that is expressed at very low levels in healthy kidney and is highly upregulated during injury. KIM‐1 is shed during injury and can be detected in blood and urine. While KIM‐1 has been studied in various models of AKI, the regulatory mechanisms leading to KIM‐1 upregulation remains limited. Here, we explored the role of ERK1/2 activation on KIM‐1 expression following toxicant exposure to proximal tubule cells and renal ischemia reperfusion injury (IRI) and LPS‐induced sepsis in mice.MethodsMouse kidney proximal tubule cells (TK) were treated with varying concentrations of hydroxyurea (HU), hydrogen peroxide (H2O2), and tert‐butyl‐hydroperoxide (TBHP) with and without pretreatment with the MEK1/2 inhibitor trametinib (10 nM). Trametinib (1mg/kg) was administered 1 hr before renal IRI to C57Bl/6 mice and were euthanized at 3 hr and 24 hr. TLR4 KO and WT mice underwent 3 hr renal IRI or septic AKI for 18 hr using LPS (10 mg/kg) injection. Signaling pathways were explored using RT‐qPCR and immunoblot analysis and kidney function was measured using BUN and serum creatinine.ResultsHU, H2O2, and TBHP exposure to TK cells for 24 hr caused ERK1/2 phosphorylation and upregulated KIM‐1 mRNA. Pretreatment with trametinib prevented KIM‐1 up‐regulation and ERK1/2 phosphorylation at 24 hr. At 3 hr post renal IRI KIM‐1 mRNA increased 4‐fold while pretreatment with trametinib resulted in a 2‐fold increase in KIM‐1 mRNA. Serum creatinine increased 4‐fold 3 hr after IRI and was attenuated by trametinib pretreatment. Cortical KIM‐1 mRNA increased 600‐fold 24 hr after IRI and was reduced 66% using trametinib in IRI. KIM‐1 protein was observed at 24 hr but not at 3 hr post IRI. Trametinib decreased KIM‐1 protein in the kidney 24 hr post IRI. TLR4 KO mice subjected to IRI exhibited increased KIM‐1 mRNA and ERK1/2 phosphorylation after 3 hr. In contrast, TLR4 KO mice exposed to LPS did not exhibit increased KIM‐1 mRNA or protein, nor activated ERK1/2 after 18 hr. WT mice treated with LPS had increased KIM‐1 mRNA and protein and ERK1/2 phosphorylation.ConclusionWe have linked pathophysiological ERK1/2 phosphorylation to the regulation of KIM‐1 within the kidney cortex. ERK1/2 phosphorylation and KIM‐1 mRNA increased following toxicant exposure in TK cells, IRI in mice, and LPS in mice, and trametinib attenuated these increases. LPS‐induced AKI required TLR4 for ERK1/2 activation and subsequent upregulation of KIM‐1. In contrast, IRI mice did not require TLR4 for ERK1/2 activation. These results demonstrate that ERK1/2 is a key initiator of renal injury.Support or Funding InformationGM084147 ‐ RGSF31DK105782 ‐ JBC

The effects of glomerular and tubular renal progenitors and derived extracellular vesicles on recovery from acute kidney injury

BackgroundMesenchymal stromal cells (MSCs) and renal stem/progenitors improve the recovery of acute kidney injury (AKI) mainly through the release of paracrine mediators including the extracellular vesicles (EVs). Several studies have reported the existence of a resident population of MSCs within the glomeruli (Gl-MSCs). However, their contribution towards kidney repair still remains to be elucidated. The aim of the present study was to evaluate whether Gl-MSCs and Gl-MSC-EVs promote the recovery of AKI induced by ischemia-reperfusion injury (IRI) in SCID mice. Moreover, the effects of Gl-MSCs and Gl-MSC-EVs were compared with those of CD133+ progenitor cells isolated from human tubules of the renal cortical tissue (T-CD133+ cells) and their EVs (T-CD133+-EVs).MethodsIRI was performed in mice by clamping the left renal pedicle for 35 minutes together with a right nephrectomy. Immediately after reperfusion, the animals were divided in different groups to be treated with: Gl-MSCs, T-CD133+ cells, Gl-MSC-EVs, T-CD133+-EVs or vehicle. To assess the role of vesicular RNA, EVs were either isolated by floating to avoid contamination of non-vesicles-associated RNA or treated with a high dose of RNase. Mice were sacrificed 48 hours after surgery.ResultsGl-MSCs, and Gl-MSC-EVs both ameliorate kidney function and reduce the ischemic damage post IRI by activating tubular epithelial cell proliferation. Furthermore, T-CD133+ cells, but not their EVs, also significantly contributed to the renal recovery after IRI compared to the controls. Floating EVs were effective while RNase-inactivated EVs were ineffective. Analysis of the EV miRnome revealed that Gl-MSC-EVs selectively expressed a group of miRNAs, compared to EVs derived from fibroblasts, which were biologically ineffective in IRI.ConclusionsIn this study, we demonstrate that Gl-MSCs may contribute in the recovery of mice with AKI induced by IRI primarily through the release of EVs.

Low T3 State Is Correlated with Cardiac Mitochondrial Impairments after Ischemia Reperfusion Injury: Evidence from a Proteomic Approach.

Mitochondria are major determinants of cell fate in ischemia/reperfusion injury (IR) and common effectors of cardio-protective strategies in cardiac ischemic disease. Thyroid hormone homeostasis critically affects mitochondrial function and energy production. Since a low T3 state (LT3S) is frequently observed in the post infarction setting, the study was aimed to investigate the relationship between 72 h post IR T3 levels and both the cardiac function and the mitochondrial proteome in a rat model of IR. The low T3 group exhibits the most compromised cardiac performance along with the worst mitochondrial activity. Accordingly, our results show a different remodeling of the mitochondrial proteome in the presence or absence of a LT3S, with alterations in groups of proteins that play a key role in energy metabolism, quality control and regulation of cell death pathways. Overall, our findings highlight a relationship between LT3S in the early post IR and poor cardiac and mitochondrial outcomes, and suggest a potential implication of thyroid hormone in the cardio-protection and tissue remodeling in ischemic disease.

Induction of Autologous Bone-Marrow Stem Cells by Low-Level Laser Therapy Has Beneficial Effects on the Kidneys Post-Ischemia-Reperfusion Injury in the Rat

Acute renal failure has a 50% - 80% mortality rate. Currently, treatment options for this life-threatening disease are limited. Low-level laser therapy (LLLT) has been found to modulate biological activity. The aim of the present study was to investigate the possible beneficial effects of laser application to stem cells in the bone marrow, on the kidneys of rats that had undergone ischemia-reperfusion injury (IRI). IRI was induced by occlusion of the renal artery to 3- and 7-month-old rats for 15 or 30 minutes. In an additional experiment IRI was applied to both kidneys for 20 min each in 2-3-month-old rats. Rats were then divided randomly into two groups of control and laser-treated. Laser therapy (Ga-Al-As 810 nm, 200 mW output for 2 min) was applied to the bone marrow 1 and 7 days post-IRI to the kidneys, and rats were sacrificed 2 weeks later. Histomorphometry and immunohistochemistry were performed on kidney sections and blood markers for kidney function. Quantitative histomorphometric analysis revealed a reduction in dilatation of the renal tubules, restored structural integrity of the renal tubules, and reduced necrosis in the laser-treated rats as compared to the control, non-laser-irradiated group. C-kit positive cell density in kidneys post-IRI and laser-treatment was significantly (p = 0.015) 3.2-fold higher compared to the control group. Creatinine and blood urea nitrogen content were significantly lower in the laser-treated rats as compared to control. It is concluded that LLLT application to the bone marrow (BM) causes a significant increase in the density of mesenchymal stem cells in the kidneys post-IRI, probably by induction of stem cells in the BM, which subsequently migrate to the IRI kidney, significantly reducing the pathological features of the kidney and increasing kidney function post IRI.

HMGB1 preconditioning protects against kidney ischemia reperfusion injury (P2126)

Abstract High-mobility group box1 (HMGB1) is an endogenous ligand for TLR4. We have reported that HMGB1 mediates kidney ischemia reperfusion injury (IRI) via TLR4. We aimed to determine: 1) the potential of preconditioning with rHMGB1 to block kidney IRI; 2) whether this effect is dependent upon TLR4; 3) whether this involves down-regulation of TLR signaling. Methods: Wild-type (WT) and TLR4-/- mice were receive rHMGB1 (20 ug/animal) or saline by i.p. injection 2 hours before 21 minute kidney ischemia. Results: WT mice pre-treated with rHMGB1 were protected against kidney IRI, with lower serum creatinine, less tubular damage, less tubulo-interstitial neutrophil, macrophage and CD4+ cell infiltration and less tubular epithelial cell apoptosis versus controls at day 1 and/or 5 post IRI. mRNA expression of cytokines IL6 &amp; TNFa and chemokines CXCL2 &amp; CCL2 in the kidney were significantly reduced by rHMGB1 pre-treatment at day 5. TLR4-/- mice were protected against kidney IRI, with no additional protection afforded by pre-administration of rHMGB1. The protective effect of rHMGB1 preconditioning on WT kidney involved Siglec-10, a negative regulator of HMGB1-TLR4 activation, which is induced through NF-kB signalling (p&amp;lt;0.05 versus control). Conclusion: HMGB1 preconditioning affords significant protection from kidney IRI by negatively regulating TLR4 signaling through Siglec-10 and NF-kB signalling, indicating the therapeutic potential.