Ischemic Acute Kidney Injury Research Articles

Proinflammatory P2Y14 receptor inhibition protects against ischemic acute kidney injury in mice.

Ischemic acute kidney injury (AKI), a complication that frequently occurs in hospital settings, is often associated with hemodynamic compromise, sepsis, cardiac surgery, or exposure to nephrotoxins. Here, using a murine renal ischemia/reperfusion injury (IRI) model, we show that intercalated cells (ICs) rapidly adopted a proinflammatory phenotype after IRI. Wwe demonstrate that during the early phase of AKI either blockade of the proinflammatory P2Y14 receptor located on the apical membrane of ICs or ablation of the gene encoding the P2Y14 receptor in ICs (a) inhibited IRI-induced increase of chemokine expression in ICs, (b) reduced neutrophil and monocyte renal infiltration, (c) reduced the extent of kidney dysfunction, and (d) attenuated proximal tubule damage. These observations indicate that the P2Y14 receptor participates in the very first inflammatory steps associated with ischemic AKI. In addition, we show that the concentration of the P2Y14 receptor ligand UDP-glucose (UDP-Glc) was higher in urine samples from intensive care unit patients who developed AKI compared with patients without AKI. In particular, we observed a strong correlation between UDP-Glc concentration and the development of AKI in cardiac surgery patients. Our study identifies the UDP-Glc/P2Y14 receptor axis as a potential target for the prevention and/or attenuation of ischemic AKI.

P0539NICOTIFLORIN ATTENUATES RENAL ISCHEMIA/REPERFUSION INJURY THROUGH REDUCING CELL APOPTOSIS

Abstract Background and Aims Renal ischemia/reperfusion (I/R) is the main cause for acute kidney injury, Nicotiflorin can ameliorate ischemia/reperfusion injury in other organs, just like in cerebral ischemic damage. Therefore, this article intends to explore whether Nicotiflorin has protective effects on renal tubular epithelial cell after ischemia-reperfusion. On the one hand, We use C57 mice to establish the Nicotiflorin group, DMSO group, AKI group, sham group and control group to investigate whether Nicotiflorin can ameliorate ischemia-reperfusion injury of kidney. In other hand, we use CCK8 to explore the optimal concentration of Nicotiflorin in renal tubular epithelial cells and find optimal hypoxia oxygenation time, in order to analysis the influence of Nicotiflorin. The results indicate that Nicotiflorin can alleviate ischemia-reperfusion injury by reducing apoptosis of renal tubular epithelial cells. Method In this study, we investigated the protective mechanism of Nicotiflorin on ischemic acute kidney injury by analyzing gene chip in patients with acute kidney injury and proving in vitro and in vivo experiments. The main methods are as follows: (1) Multiple nucleus ischemia-reperfusion model transcriptase data were selected from the NCBI GEO Datasets database and analyzed to screen out related proteins that may be involved in ischemia-reperfusion kidney injury; (2) The tertiary structure of Nicotiflorin and related proteins was obtained from the SWISS-MODEL database and the PubChem compound database. The molecular docking between protein and Nicotiflorin was performed using Autodock software, and the binding energy between Nicotiflorin and the selected protein was analyzed to determine Nicotiflorin binds to each other; (3) We set different groups, such as control group, sham group, AKI group, Nicotiflorin group and DMSO group in animals. The blood function was used to detect renal injury related function indicators 24 hours after modeling. Renal tissue samples were collected for real-time fluorescent RT-PCR, Western blotting and histopathological analysis; (4)Renal tubular epithelial cells were treated with different concentrations of Nicotiflorin, CCK8 was screened for the most appropriate concentration, and the hypoxic and reoxygenated cells were intervened at the concentration to explore the interaction between Nicotiflorin and the docking protein, and to observe the protective mechanism of Nicotiflorin on the kidney Results Conclusion Nicotiflorin binds to ATF3 and promotes the expression of Cyr61 through protein interactions to improve renal ischemia-reperfusion injury.

P0522INHIBITING 15-PGDH PREVENTS ISCHEMIC RENAL INJURY BY A PGE2/EP4 SIGNALING PATHWAY MEDIATING VASODILATION, INCREASED RENAL BLOOD FLOW, AND INCREASED ADENOSINE/A2A RECEPTOR

Abstract Background and Aims We demonstrate the marked activity of SW033291, an inhibitor of 15-hydoxyprostaglandin dehydrogenase (15-PGDH), in preventing acute kidney injury (AKI) in a murine model of ischemia reperfusion injury (IRI). AKI due to ischemic injury represents a significant clinical problem. Prostaglandin E2 (PGE2) is vasodilator in the kidney, but is rapidly degraded in vivo due to catabolism by 15-PGDH. We investigated the potential of SW033291, a potent and specific 15-PGDH inhibitor, as prophylactic treatment for ischemic AKI. Method 10-week aged male C57/BL6 mice were randomly allocated to five groups (n=8 to 15 in each group): the sham-control group, sham-SW033291 group, IRI-vehicle group, IRI-indomethacin group and the IRI-SW033291 group. IRI were induced by clamping bilateral renal artery for 30 min followed by 24 hours of reperfusion. Vehicle, indomethacin, or SW033291 were intraperitoneally administered three times at 1 hour before, immediately after, and 12 hours after IRI. Renal function, histological changes, and renal blood flow were compared and the relevant parameters of oxidative stress and inflammation were detected. Results Prophylactic administration of SW033291 significantly increased renal tissue PGE2 levels and increased post-AKI renal blood flow and renal arteriole area. In parallel, prophylactic SW033291 decreased post-AKI histologic injury score and tubular apoptosis and markedly reduced biomarkers of renal injury that included BUN, creatinine, NGAL and KIM-1. Prophylactic SW033291 also reduced post-AKI induction of proinflammatory cytokines, high mobility group box 1 (HMGB1), and malondialdehyde (MDA). Protective effects of SW033291 were mediated by PGE2 signaling as they could be blocked by pharmacologic inhibition of PGE2 synthesis or of the EP4 type PGE2 receptor. Consistent with activation of PGE2 signaling, SW033291 induced renal levels of both EP4 and of cyclic adenosine monophosphate (cAMP), along with other vasodilatory effectors including AMP, adenosine, and the adenosine A2A receptor (A2A). Protective effects of SW0333291 could largely be achieved with a single prophylactic dose of the drug. Conclusion Inhibiting 15-PGDH may thus represent a novel strategy for prophylaxis of ischemic AKI in multiple clinical settings, including renal transplantation and cardiovascular surgery.

Protective effect of genistein in a rat model of ischemic acute kidney injury.

This study determined the possible anti-inflammatory and antioxidant renal protective effect of genistein, a soy isoflavone, against kidney damage and functional disorders following renal ischemia/reperfusion (I/R) in male rats. The animals were dedicated to five groups (n=7 per group): Sham, Sham+Geni (genistein, 15mg/kg in 1ml 1% DMSO, i.p.), Sham+DMSO (1ml 1% DMSO, i.p.), I/R (bilateral renal ischemia for 45min followed by 24h reperfusion), I/R+Geni (genistein, 15mg/kg). 24-h urine samples, blood and tissue samples of the kidney were collected at the end of 24h reperfusion period. Compared to sham, sham+Geni and sham+DMSO groups, IR injury (IRI) ended in kidney dysfunction (decreased creatinine clearance, and increased fractional excretion of sodium), increased levels of malondialdehyde, decreased activities of antioxidant enzymes (superoxide dismutase, gluthatione peroxidase, and catalase), increased gene expression levels of TLR4 (Toll-like receptor 4) and TNF-α (tumor necrosis factor-alpha), as well as histological damages in kidney tissue. Genistein administration decreased all the changes. Therefore, genistein apparently protects the kidney against IRI by mitigating both oxidative stress and inflammation. The antioxidant and anti-inflammatory properties of genistein probably exert important roles in improving functional disorders and offer renal protection against IRI.

Erythropoietin Ameliorates Ischemia/Reperfusion-Induced Acute Kidney Injury via Inflammasome Suppression in Mice.

Acute kidney injury (AKI) is the most common condition in hospitalized patients. As ischemia/reperfusion-induced AKI (IR-AKI) is as a major contributor to end-stage disease, an effective therapeutic intervention for IR-AKI is imperative. Erythropoietin (EPO) is a potent stimulator of erythroid progenitor cells and is significantly upregulated during hypoxia. Here, we investigated the renoprotective effects of EPO in an IR-AKI mouse model. Mice were assigned to sham, EPO only, and IR only groups, and the IR group was treated with EPO prior to injury. EPO was administered twice at 30 min prior to bilateral renal artery occlusion, and 5 min before reperfusion, with all mice sacrificed 24 h after IR-AKI. The serum was harvested for renal functional measurements. The kidneys were subjected to histological evaluation, and the biochemical changes associated with renal injury were assessed. EPO significantly attenuated the renal dysfunction associated with IR-AKI, as well as tissue injury. Apoptotic cell death and oxidative stress were significantly reduced in EPO-treated mice. Macrophage infiltration and expression of ICAM-1 and MCP-1 were also significantly reduced in EPO-treated mice. Furthermore, the expression of inflammasome-related factors (NLRP1, NLRP3, and caspase-1 cleavage), via the activation of the COX-2 and NF-κB signaling pathways were significantly reduced following EPO treatment. To our knowledge, this is the first study to demonstrate that inflammasome-mediated inflammation might be a potential target of EPO as a treatment for ischemic AKI.

New insights into tubular cell recovery after ischemic acute kidney injury

Acute kidney injury (AKI) is common, and depending on the severity and frequency of the insult, can contribute to the development of chronic kidney disease (CKD).1 Although the cellular and molecular mechanisms necessary for the restoration of kidney function after AKI remain to be elucidated, failed differentiation and maladaptive repair are now recognized as important contributors to the AKI-to-CKD transition. Several mechanisms are thought to be involved in this transition: fibrosis due to capillary rarefaction and tissue hypoxia, the infiltration of inflammatory cells that contribute to the repair process after AKI, and cell cycle arrest during AKI.

TCR+CD4-CD8- (double negative) T cells protect from cisplatin-induced renal epithelial cell apoptosis and acute kidney injury.

Acute kidney injury (AKI) due to cisplatin is a significant problem that limits its use as an effective chemotherapeutic agent. T cell receptor+CD4-CD8- double negative (DN) T cells constitute the major T cell population in the human and mouse kidney, express programmed cell death protein (PD)-1, and protect from ischemic AKI. However, the pathophysiological roles of DN T cells in cisplatin-induced AKI is unknown. In this study, wild-type mice were treated with cisplatin (30 mg/kg) or vehicle, and the effects on kidney DN T cell numbers and function were measured. In vitro experiments evaluated effects of kidney DN T cells on cisplatin-induced apoptosis and PD ligand 1 (PD-L1) in renal epithelial cells. Adoptive transfer experiments assessed the therapeutic potential of DN T cells during cisplatin-induced AKI. Our results show that kidney DN T cell population increased at 24 h and declined by 72 h after cisplatin treatment. Cisplatin treatment increased kidney DN T cell proliferation, apoptosis, CD69, and IL-10 expression, whereas CD62L, CD44, IL-17A, interferon-γ, and TNF-α were downregulated. Cisplatin treatment decreased both PD-1 and natural killer 1.1 subsets of kidney DN T cells with a pronounced effect on the PD-1 subset. In vitro kidney DN T cell coculture decreased cisplatin-induced apoptosis in kidney proximal tubular epithelial cells, increased Bcl-2, and decreased cleaved caspase 3 expression. Cisplatin-induced expression of PD ligand 1 was reduced in proximal tubular epithelial cells cocultured with DN T cells. Adoptive transfer of DN T cells attenuated kidney dysfunction and structural damage from cisplatin-induced AKI. These results demonstrate that kidney DN T cells respond rapidly and play a protective role during cisplatin-induced AKI.

Microbiome-Metabolome Signature of Acute Kidney Injury.

Intestinal microbiota play a considerable role in the host’s organism, broadly affecting its organs and tissues. The kidney can also be the target of the microbiome and its metabolites (especially short-chain fatty acids), which can influence renal tissue, both by direct action and through modulation of the immune response. This impact is crucial, especially during kidney injury, because the modulation of inflammation or reparative processes could affect the severity of the resulting damage or recovery of kidney function. In this study, we compared the composition of rat gut microbiota with its outcome, in experimental acute ischemic kidney injury and named the bacterial taxa that play putatively negative or positive roles in the progression of ischemic kidney injury. We investigated the link between serum creatinine, urea, and a number of metabolites (acylcarnitines and amino acids), and the relative abundance of various bacterial taxa in rat feces. Our analysis revealed an increase in levels of 32 acylcarnitines in serum, after renal ischemia/reperfusion and correlation with creatinine and urea, while levels of three amino acids (tyrosine, tryptophan, and proline) had decreased. We detected associations between bacterial abundance and metabolite levels, using a compositionality-aware approach—Rothia and Staphylococcus levels were positively associated with creatinine and urea levels, respectively. Our findings indicate that the gut microbial community contains specific members whose presence might ameliorate or, on the contrary, aggravate ischemic kidney injury. These bacterial taxa could present perspective targets for therapeutical interventions in kidney pathologies, including acute kidney injury.

LncRNA GAS5 promotes apoptosis as a competing endogenous RNA for miR-21 via thrombospondin 1 in ischemic AKI

Mounting evidence has indicated that long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) played important roles in renal ischemia/reperfusion (I/R) injury. However, the involvement of lncRNA growth arrest specific 5 (GAS5) in acute kidney injury (AKI) remained largely unexplored. This study aimed to determine possible mechanisms of GAS5 in the renal I/R process. We found that GAS5, noticeably upregulated by renal I/R injury, was further suppressed by delayed IPC while knockdown of miR-21 in vivo before IPC could significantly increased the GAS5 levels. Concurrently, TSP-1 was negatively regulated by miR-21 in vivo and vitro. Additionally, Reciprocal repression of GAS5 and miR-21 was identified. Knockdown of miR-21 in H6R0.5 treated HK-2 cells promoted apoptosis. Co-transfection of miR-21 mimic and pcDNA-GAS5 or pcDNA-Vector were performed, results of which showed that inhibition of miR-21 on TSP-1 could be rescued by overexpression of GAS5. This study suggested that GAS5 facilitated apoptosis by competitively sponging miR-21, which negatively regulated TSP-1 in renal I/R injury. This novel regulatory axis could act as a therapeutic target for AKI in the future.

Ischemia‐induced vascular congestion of the ascending vasa recta precedes congestion of the peritubular capillaries in the renal medulla

Vascular congestion, or the aggregation of red blood cells in the microvasculature of the renal medulla is a prominent finding in ischemic acute kidney injury (AKI). Studies in rodent models of ischemia‐reperfusion injury (IRI) have suggested that vascular congestion occurs during the ischemic period and worsens following reperfusion, however, the location(s) in which congestion originates during ischemia have not been defined. We hypothesized that ‘vascular congestion first occurs in the medullary vasa recta (VR) capillaries during the ischemic period, prior to congestion of peritubular (PT) capillaries during reperfusion’. To test this hypothesis, a 45‐minute warm bilateral ischemia was performed in male WKY rats (10 wks old). Rats were randomized to 0, 1, 2, 6, 10, or 24 hour(s) of reperfusion (n=5/group). Congestion of the cortical PT capillaries, medullary VR and PT capillaries, and inner medulla (IM) was scored (blinded; scale: 0–5, 0=0% congestion, 5=100% congestion) in histological sections. To localize congestion within the VR, immunofluorescence of neural‐glial antigen 2 (NG2) was assessed. Medullary VR congestion score was 4.5 (90%) at time 0 (no reperfusion) and remained elevated at 1–10 hours of reperfusion. At 24 hours of reperfusion, VR congestion had significantly decreased to 30% (pANOVA= 0.004, post hoc p=0.002). Medullary PT congestion score was 0.5 (10%) at time 0 and was significantly less than the VR congestion at time 0 (90%) (pINTERACTION=0.004, post hoc p<0.0001). Non‐quantitative analysis of NG2 staining, localized congestion to the ascending VR. At time 0, IM congestion score was 0.5 (10%). IM congestion increased to 4 (80%) by 2 hours of reperfusion (pANOVA=0.022, post hoc p=0.019) and remained elevated through 10 hours of reperfusion, however by 24 hours of reperfusion IM congestion had decreased. Congestion of the cortical PT capillary congestion was significantly greater at time 0 and had an average score of 2.5 (50%) (pANOVA=0.0005). Following 1, 2, or 6 hour reperfusion, cortical PT congestion decreased to less than 10%, and with 10 or 24 hour reperfusion there was no cortical congestion. It has been reported that congestion occurs during ischemia and increases following reperfusion, however the localization of medullary congestion over the initial 24 hours of reperfusion remains unknown. Importantly, our data provides the first evidence that medullary congestion originates in the VR capillaries during the ischemic period and is evident prior to congestion in the PT capillaries during reperfusion. Pericytes are contractile cells which surround the descending vasa recta (DVR). Using a pericyte marker, NG2, we found that congestion was not in NG2‐positive DVR but was localized to the ascending VR (NG2‐negative). Vascular congestion of the renal medulla has been reported to be the primary factor contributing to non‐recovery from ischemic AKI. Understanding how, and where, congestion occurs in the renal medulla is critical to find therapeutic targets for congestion in order to improve recovery from ischemic AKI. We speculate that medullary congestion of AVR may drive blood into the PT capillaries during reperfusion where it then becomes trapped which may prolong ischemia and worsen injury.Support or Funding InformationNIH PO1HL134604 and DK099548

Effects of Jwagwieum on Renal Dysfunction in Ischemia/Reperfusion‐induced Acute Renal Failure in Rats

Jwagwieum ( , JGE), a Korean traditional medicine, has been used for treatment of kidney‐yin deficient ( ) syndrome. However, there is no pharmacological study of JGE on ischemia/reperfusion‐induced acute renal failure (ARF). Therefore, the objective of this study was to evaluate whether JGE ameliorates renal function and its related mechanism in acute kidney injury‐induced by ischemia/reperfusion in rats. In this experiment, 5‐week‐old male Sprague Dawley (SD) rats were used under 12 h light and dark cycle. After a week of adaptation, they were randomly divided into following groups. 1) Cont. (Control), 2) ischemia/reperfusion injury (ARF), 3) ARF + JGE100 (100 mg/kg/day), 4) ARF + JGE200 (200 mg/kg/day). The ARF group was subjected to reperfusion after inserting a clip into the renal artery for 45 min. ARF + JGE100 group was orally administered with JGE 100 mg/kg/day after ischemia/reperfusion surgery, and ARF + JGE200 group was orally administered with JGE 200 mg/kg/day for 4 days, respectively. As a result, JGE suppressed the increase of kidney size in the acute renal failure animal model and alleviated polyuria symptoms. In addition, solute‐free water reabsorption (TcH2O) and creatinine clearance (Ccr), indicators of glomerular filtration rate, were significantly increased in JGE group compared with those of ARF rats. In ARF group, blood urea nitrogen (BUN)/creatinine (Cr) and lactate dehydrogenase (LDH) were significantly increased compared to the control group, respectively, while they were significantly decreased in JGE group. The present results also showed that JGE improved the morphological aspects of renal tissue and ameliorated fibrosis in ARF group. Inflammation is one of the main causes of ischemic acute kidney injury and was inhibited by JGE, which was confirmed by the NLRP3 inflammasome decline.The present results showed that JGE has protective effects against inflammatory process via inhibition of NLRP3 signal pathway, which is associated with improvement of renal function in rats with ARF. These results suggest that JGE has a therapeutic effect on acute kidney injury‐induced by ischemia/reperfusion in rats.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2017R1A5A2015805)(2015M3A9A5030620)(2017R1A2B4009884).

Norepinephrine‐Alpha 2 Adrenergic Receptor Axis is Critical to Long‐Term Sequelae of Ischemic Acute Kidney Injury

ObjectiveUpregulated levels of kidney norepinephrine (NE) have been recognized as a risk factor of development and progression of chronic kidney disease. Our reports showed renal denervation can prevent renal fibrosis and inflammation in renal fibrogenesis models through inhibition of NE‐alpha 2 adrenergic receptors (α2‐ARs) axis. However, the molecular mechanism by which NE‐α2‐ARs axis elicits long‐term sequelae of acute kidney injury (AKI) remains to be elucidated.MethodsBased on our published data, we hypothesized that NE‐α2‐ARs axis mediates its effect through macrophages recruited into ischemic kidneys. In this study, we investigated whether in vivo and in vitro NE‐α2‐AR axis regulates macrophage recruitment, activation and differentiation during the development of chronic kidney disease after ischemia/reperfusion injury (IRI).ResultsRenal bilateral IRI resulted in severe kidney fibrosis in medullary region over 2 weeks with enhanced tissue NE level, persistent tubular injury and massive macrophage infiltration. Inhibition of α2A‐ and/or α2C‐AR subtypes by pharmacological or genetic intervention prevented macrophage accumulation with downregulation of M1 and M2 macrophage markers, reduced levels of macrophage‐released pro‐inflammatory and/or fibrogenic cytokines, resulting in suppression of persistent renal tubular injury and fibrosis progression. In in vitro study using Raw264.7 cells, pharmacological inhibition of α2‐AR blocked macrophage migration to proximal tubule cells exposed to NE. In addition, we demonstrated that NE results in upregulation of both M1 and M2 macrophages.ConclusionsCollectively, NE‐α2‐ARs axis induces long‐term sequelae of ischemic AKI through macrophage recruitment and subsequent activation. Our data suggest that targeting NE‐α2‐ARs axis can be a potential therapeutic strategy to prevent long‐term sequelae of ischemic AKI.Support or Funding InformationThis work was supported by NIH grants DK‐116987, DK‐120533 and American Heart Association (AHA) Grant in Aid 15GRNT25080031 (BJP), AHA postdoctoral fellowship Grant 15POST25130003 (HSJ), and grants (NRF‐2016R1C1B2012080 and NRF‐2019R1F1A1041410) from the National Research Foundation of Korea (JK).

Selective nanoparticle-mediated targeting of renal tubular Toll-like receptor 9 attenuates ischemic acute kidney injury

We developed an innovative therapy for ischemic acute kidney injury with discerning kidney-targeted delivery of a selective Toll-like receptor 9 (TLR9) antagonist in mice subjected to renal ischemia reperfusion injury. Our previous studies showed that mice deficient in renal proximal tubular TLR9 were protected against renal ischemia reperfusion injury demonstrating a critical role for renal proximal tubular TLR9 in generating ischemic acute kidney injury. Herein, we used 300-400 nm polymer-based mesoscale nanoparticles that localize to the renal tubules after intravenous injection. Mice were subjected to sham surgery or 30 minutes renal ischemia and reperfusion injury after receiving mesoscale nanoparticles encapsulated with a selective TLR9 antagonist (unmethylated CpG oligonucleotide ODN2088) or mesoscale nanoparticles encapsulating a negative control oligonucleotide. Mice treated with the encapsulated TLR9 antagonist either six hours before renal ischemia, at the time of reperfusion or 1.5 hours after reperfusion were protected against ischemic acute kidney injury. The ODN2088-encapsulated nanoparticles attenuated renal tubular necrosis, inflammation, decreased proinflammatory cytokine synthesis. neutrophil and macrophage infiltration and apoptosis, decreased DNA fragmentation and caspase 3/8 activation when compared to the negative control nanoparticle treated mice. Taken together, our studies further suggest that renal proximal tubular TLR9 activation exacerbates ischemic acute kidney injury by promoting renal tubular inflammation, apoptosis and necrosis after ischemia reperfusion. Thus, our studies suggest a potential promising therapy for ischemic acute kidney injury with selective kidney tubular targeting of TLR9 using mesoscale nanoparticle-based drug delivery.

P2X4 receptor exacerbates ischemic AKI and induces renal proximal tubular NLRP3 inflammasome signaling.

We tested the hypothesis that the P2X4 purinergic receptor (P2X4) exacerbates ischemic acute kidney injury (AKI) by promoting renal tubular inflammation after ischemia and reperfusion (IR). Supporting this, P2X4-deficient (KO) mice were protected against ischemic AKI with significantly attenuated renal tubular necrosis, inflammation, and apoptosis when compared to P2X4 wild-type (WT) mice subjected to renal IR. Furthermore, WT mice treated with P2X4 allosteric agonist ivermectin had exacerbated renal IR injury whereas P2X4 WT mice treated with a selective P2X4 antagonist (5-BDBD) were protected against ischemic AKI. Mechanistically, induction of kidney NLRP3 inflammasome signaling after renal IR was significantly attenuated in P2X4 KO mice. A P2 agonist ATPγS increased NLRP3 inflammasome signaling (NLRP3 and caspase 1 induction and IL-1β processing) in isolated renal proximal tubule cells from WT mice whereas these increases were absent in renal proximal tubules isolated from P2X4 KO mice. Moreover, 5-BDBD attenuated ATPγS induced NLRP3 inflammasome induction in renal proximal tubules from WT mice. Finally, P2X4 agonist ivermectin induced NLRP3 inflammasome and pro-inflammatory cytokines in cultured human proximal tubule cells. Taken together, our studies suggest that renal proximal tubular P2X4 activation exacerbates ischemic AKI and promotes NLRP3 inflammasome signaling.

CD4+ T Cell-Derived NGAL Modifies the Outcome of Ischemic Acute Kidney Injury.

CD4+ T cells mediate the pathogenesis of ischemic and nephrotoxic acute kidney injury (AKI). However, the underlying mechanisms of CD4+ T cell-mediated pathogenesis are largely unknown. We therefore conducted unbiased RNA-sequencing to discover novel mechanistic pathways of kidney CD4+ T cells after ischemia compared with normal mouse kidney. Unexpectedly, the lipocalin-2 (Lcn2) gene, which encodes neutrophil gelatinase-associated lipocalin (NGAL) had the highest fold increase (∼60). The NGAL increase in CD4+ T cells during AKI was confirmed at the mRNA level with quantitative real-time PCR and at the protein level with ELISA. NGAL is a potential biomarker for the early detection of AKI and has multiple potential biological functions. However, the role of NGAL produced by CD4+ T cells is not known. We found that ischemic AKI in NGAL knockout (KO) mice had worse renal outcomes compared with wild-type (WT) mice. Adoptive transfer of NGAL-deficient CD4+ T cells from NGAL KO mice into CD4 KO or WT mice led to worse renal function than transfer of WT CD4+ T cells. In vitro-simulated ischemia/reperfusion showed that NGAL-deficient CD4+ T cells express higher levels of IFN-γ mRNA compared with WT CD4+ T cells. In vitro differentiation of naive CD4+ T cells to Th17, Th1, and Th2 cells led to significant increase in Lcn2 expression. Human kidney CD4+ T cell NGAL also increased significantly after ischemia. These results demonstrate an important role for CD4+ T cell NGAL as a mechanism by which CD4+ T cells mediate AKI and extend the importance of NGAL in AKI beyond diagnostics.

MiR-214 represses mitofusin-2 to promote renal tubular apoptosis in ischemic acute kidney injury

Disruption of mitochondrial dynamics is an important pathogenic event in both acute and chronic kidney diseases, but the underlying mechanism remains poorly understood. Here, we report the regulation of mitofusin-2 (Mfn2; a key mitochondrial fusion protein) by microRNA-214 (miR-214) in renal ischemia-reperfusion that contributes to mitochondrial fragmentation, renal tubular cell death, and ischemic acute kidney injury (AKI). miR-214 was induced, whereas Mfn2 expression was decreased, in mouse ischemic AKI and cultured rat kidney proximal tubular cells (RPTCs) following ATP depletion treatment. Overexpression of miR-214 decreased Mfn2. Conversely, inhibition of miR-214 with anti-miR-214 prevented Mfn2 downregulation in RPTCs following ATP depletion. Anti-miR-214 further ameliorated mitochondrial fragmentation and apoptosis, whereas overexpression of miR-214 increased apoptosis, in ATP-depleted RPTCs. To test regulation in vivo, we established a mouse model with miR-214 specifically deleted from kidney proximal tubular cells (PT-miR-214-/-). Compared with wild-type mice, PT-miR-214-/- mice had less severe tissue damage, fewer apoptotic cells, and better renal function after ischemic AKI. miR-214 induction in ischemic AKI was suppressed in PT-miR-214-/- mice, accompanied by partial preservation of Mfn2 in kidneys. These results unveil the miR-214/Mfn2 axis that contributes to the disruption of mitochondrial dynamics and tubular cell death in ischemic AKI, offering new therapeutic targets.

Pum2-Mff axis fine-tunes mitochondrial quality control in acute ischemic kidney injury.

Mitochondrial fission factor (Mff) has been demonstrated to play a role in the activation of mitochondrial cleavage and mitochondrial death, denoting its role in the regulation of mitochondrial quality control. Recent evidence suggested that the mRNA translation of Mff is under the negative regulation by the RNA-binding protein Pumilio2 (Pum2). This study was designed to examine the role of Pum2 and Mff in the governance of mitochondrial quality control in a murine model of acute ischemic kidney injury. Our results indicated that genetic deletion of Mff overtly attenuated ischemic acute kidney injury (AKI)-induced renal failure through inhibition of pro-inflammatory response, tubular oxidative stress, and ultimately cell death in the kidney. Furthermore, Mff inhibition effectively preserved mitochondrial homeostasis through amelioration of mitochondrial mitosis, restoration of Sirt1/3 expression, and boost of mitochondrial respiration. Western blot analysis revealed that levels of Pum2 were significantly downregulated by ischemic AKI, inversely coinciding with levels of Mff. Overexpression of Pum2 reduced ischemic AKI-mediated Mff upregulation and offered protection on renal tubules through modulation of mitochondrial quality control. Taken together, our data have unveiled the molecular mechanism of the Pum2-Mff axis in mitochondrial quality control in a mouse model of ischemic AKI. These data indicated the therapeutic potential of Pum2 activation and Mff inhibition in the management of ischemic AKI.

Metabolic effects of RUBCN/Rubicon deficiency in kidney proximal tubular epithelial cells

ABSTRACT Macroautophagy/autophagy is a lysosomal degradation system which plays a protective role against kidney injury. RUBCN/Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein) inhibits the fusion of autophagosomes and lysosomes. However, its physiological role in kidney proximal tubular epithelial cells (PTECs) remains uncertain. In the current study, we analyzed the phenotype of newly generated PTEC-specific rubcn-deficient (KO) mice. Additionally, we investigated the role of RUBCN in lipid metabolism using isolated rubcn-deficient PTECs. Although KO mice exhibited sustained high autophagic flux in PTECs, they were not protected from acute ischemic kidney injury. Unexpectedly, KO mice exhibited hallmark features of metabolic syndrome accompanied by expanded lysosomes containing multi-layered phospholipids in PTECs. RUBCN deficiency in cultured PTECs promoted the mobilization of phospholipids from cellular membranes to lysosomes via enhanced autophagy. Treatment of KO PTECs with oleic acid accelerated fatty acids transfer to mitochondria. Furthermore, KO PTECs promoted massive triglyceride accumulation in hepatocytes (BNL-CL2 cells) co-cultured in transwell, suggesting accelerated fatty acids efflux from the PTECs contributes to the metabolic syndrome in KO mice. This study shows that sustained high autophagic flux by RUBCN deficiency in PTECs leads to metabolic syndrome concomitantly with an accelerated mobilization of phospholipids from cellular membranes to lysosomes. Abbreviations: ABC: ATP binding cassette; ACADM: acyl-CoA dehydrogenase medium chain; ACTB: actin, beta; ATG: autophagy related; AUC: area under the curve; Baf: bafilomycin A1; BAT: brown adipose tissue; BODIPY: boron-dipyrromethene; BSA: bovine serum albumin; BW: body weight; CAT: chloramphenicol acetyltransferase; CM: complete medium; CPT1A: carnitine palmitoyltransferase 1a, liver; CQ: chloroquine; CTRL: control; EGFP: enhanced green fluorescent protein; CTSD: cathepsin D; EAT: epididymal adipose tissue; EGFR: epidermal growth factor receptor; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; FA: fatty acid; FBS: fetal bovine serum; GTT: glucose tolerance test; HE: hematoxylin and eosin; HFD: high-fat diet; I/R: ischemia-reperfusion; ITT: insulin tolerance test; KAP: kidney androgen regulated protein; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LD: lipid droplet; LRP2: low density lipoprotein receptor related protein 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MAT: mesenteric adipose tissue; MS: mass spectrometry; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NDRG1: N-myc downstream regulated 1; NDUFB5: NADH:ubiquinone oxidoreductase subunit B5; NEFA: non-esterified fatty acid; OA: oleic acid; OCT: optimal cutting temperature; ORO: Oil Red O; PAS: Periodic-acid Schiff; PFA: paraformaldehyde; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PPARA: peroxisome proliferator activated receptor alpha; PPARGC1A: PPARG coactivator 1 alpha; PTEC: proximal tubular epithelial cell; RAB7A: RAB7A, member RAS oncogene family; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase B1; RT: reverse transcription; RUBCN: rubicon autophagy regulator; SAT: subcutaneous adipose tissue; SFC: supercritical fluid chromatography; SQSTM1: sequestosome 1; SREBF1: sterol regulatory element binding transcription factor 1; SV-40: simian virus-40; TFEB: transcription factor EB; TG: triglyceride; TS: tissue specific; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; UN: urea nitrogen; UQCRB: ubiquinol-cytochrome c reductase binding protein; UVRAG: UV radiation resistance associated; VPS: vacuolar protein sorting; WAT: white adipose tissue.

КРУПНООЧАГОВЫЙ ИНФАРКТ МИОКАРДА И ОСТРОЕ ИШЕМИЧЕСКОЕ ПОВРЕЖДЕНИЕ ПОЧЕК

The aim of the study was to investigate specific clinical manifestations, homeostasis indices and parameters of the cardiovascular system in patients with acute ST-elevation myocardial infarction (STEMI) and acute ischemic kidney injury. Methods. 173 patients with STEMI participated in the study. The study group consisted of 111 patients with acute ischemic kidney injury associated with myocardial infarction (MI); 62 patients with MI and normal kidney function were enrolled in the comparison group. Clinical, anthropometric, laboratory, and instrumental diagnostic methods were used. Results. Compared with patients of the MI and normal kidney function group, those with MI and acute ischemic kidney injury had a higher average heart rate, required more prolonged vasopressor and/or inotropic therapy, and more frequently developed tachyarrhythmias with adverse prognostic impact and postinfarction aneurysms. The study revealed that patients of the MI and acute ischemic kidney injury group demonstrated more severe dilatation of the left ventricle (LV), more pronounced reduction in myocardial LV contractility according to echocardiography results; they developed multivessel coronary artery disease more frequently. Furthermore, patients of this group had a higher incidence of infarction-associated artery damage located in the proximal segments of major coronary arteries and more frequently developed thrombotic occlusion in the infarction-affected artery. Patients with MI and acute ischemic kidney injury had higher levels of inflammatory, myocardial necrosis, hemostasis and neurohormonal activation markers. Higher concentration of neutrophil gelatinase-associated lipocalin (uNGAL) was observed in patients with MI and acute ischemic kidney injury; moreover, in 14,1% of patients belonging to this group, elevated levels of this marker preceded the diagnostically significant increase in creatinine concentration and decrease in glomerular filtration rate. Conclusion. In patients with MI, the development of acute ischemic kidney injury was accompanied by more severe clinical manifestations, prognostically adverse indicators of early LV remodeling and coronary arteries disease, enhanced inflammatory processes and neuroendocrine system activity, as well as by elevated levels of myocardial necrosis and blood coagulation activity markers. The present study suggests applying uNGAL as an early marker of acute ischemic kidney injury in patients with MI.

Effects of acute arginine treatment on vascular endothelial dysfunction associated with ischemic acute kidney injury.

Effects of acute arginine treatment on vascular endothelial dysfunction associated with ischemic acute kidney injury.