Scutellarin alleviates renal ischemia-reperfusion injury by inhibiting the MAPK pathway and pro-inflammatory macrophage polarization.

Renal ischemia-reperfusion injury (IRI) is an integral process in renal transplantation, which results in compromised graft survival. Macrophages play an important role in both the early inflammatory period and late fibrotic period in response to IRI. In this study, we investigated whether scutellarin (SCU) could protect against renal IRI by regulating macrophage polarization. Mice were given SCU (5-50 mg/kg) by gavage 1 h earlier, followed by a unilateral renal IRI. Renal function and pathological injury were assessed 24 h after reperfusion. The results showed that administration of 50 mg/kg SCU significantly improved renal function and renal pathology in IRI mice. In addition, SCU alleviated IRI-induced apoptosis. Meanwhile, it reduced macrophage infiltration and inhibited pro-inflammatory macrophage polarization. Moreover, in RAW 264.7 cells and primary bone marrow-derived macrophages (BMDMs) exposed to SCU, we found that 150 µM SCU inhibited these cells to polarize to an inflammatory phenotype induced by lipopolysaccharide (LPS) and interferon-γ (IFN-γ). However, SCU has no influence on anti-inflammatory macrophage polarization in vivo and in vitro induced by in interleukin-4 (IL-4). Finally, we explored the effect of SCU on the activation of the mitogen-activated protein kinase (MAPK) pathway both in vivo and in vitro. We found that SCU suppressed the activation of the MAPK pathway, including the extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and p38. Our results demonstrated that SCU protects the kidney against IRI by inhibiting macrophage infiltration and polarization toward pro-inflammatory phenotype via the MAPK pathway, suggesting that SCU may be therapeutically important in treatment of IRI.

AST-120 alleviates renal ischemia-reperfusion injury by inhibiting HK2-mediated glycolysis.

OBJECTIVE: Renal ischemia/reperfusion injury (IRI) is a major cause of acute kidney injury (AKI), which is associated with high incidence and mortality. AST-120 is an oral carbonaceous adsorbent that can alleviate kidney damage. This study aimed to explore the effects of AST-120 on renal IRI and the molecular mechanism. METHODS: A renal IRI mouse model was established and administrated AST-120, and differentially expressed genes were screened using RNA sequencing. Renal function and pathology were analyzed in mice. Hypoxia/reoxygenation (H/R) cell model was generated, and glycolysis was evaluated by detecting lactate levels and Seahorse analysis. Histone lactylation was analyzed by western blotting, and its relationship with hexokinase 2 (HK2) was assessed using chromatin immunoprecipitation. RESULTS: The results showed that HK2 expression was increased after IRI, and AST-120 decreased HK2 expression. Knockout of HK2 attenuated renal IRI and inhibits glycolysis. AST-120 inhibited renal IRI in the presence of HK2 rather than HK2 absence. In proximal tubular cells, knockdown of HK2 suppressed glycolysis and H3K18 lactylation caused by H/R. H3K18 lactylation was enriched in HK2 promoter and upregulated HK2 levels. Rescue experiments revealed that lactate reversed IRI that suppressed by HK2 knockdown. CONCLUSIONS: In conclusion, AST-120 alleviates renal IRI via suppressing HK2-mediated glycolysis, which suppresses H3K18 lactylation and further reduces HK2 levels. This study proposes a novel mechanism by which AST-120 alleviates IRI.

Connexin32 gap junction channels deliver miR155-3p to mediate pyroptosis in renal ischemia-reperfusion injury.

OBJECTIVES: To explore whether the gap junction (GJ) composed by connexin32(Cx32) mediated pyroptosis in renal ischemia-reperfusion(I/R) injury via transmitting miR155-3p, with aim to provide new strategies for the prevention and treatment of acute kidney injury (AKI) after renal I/R. METHODS: 8-10 weeks of male C57BL/ 6 wild-type mice and Cx32 knockdown mice were divided into two groups respectively: control group and renal I/R group. MCC950 (50 mg/kg. ip.) was used to inhibit NLRP3 in vivo. Human kidney tubular epithelial cells (HK - 2) and rat kidney tubular epithelial cells (NRK-52E) were divided into high-density group and low-density group, and treated with hypoxia reoxygenation (H/R) to mimic I/R. The siRNA and plasmid of Cx32, mimic and inhibitor of miR155-3p were transfected into HK - 2 cells respectively. Kidney pathological and functional injuries were measured. Western Blot and immunofluorescent staining were used to observe the expression of NLRP3, GSDMD, GSDMD-N, IL - 18, and mature IL-18. The secretion of IL-18 and IL-1ß in serum, kidney tissue and cells supernatant were detected by enzyme-linked immuno sorbent assay (ELISA) kit, and the expression of NLPR3 and miR155-3p were detected by RT-qPCR and fluorescence in situ hybridization (FISH). RESULTS: Tubular pyroptosis were found to promote AKI after I/R in vivo and Cx32-GJ regulated pyroptosis by affecting the expression of miR155-3p after renal I/R injury. In vitro, H/R could lead to pyroptosis in HK-2 and NRK-52E cells. When the GJ channels were not formed, and Cx32 was inhibited or knockdown, the expression of miR155-3p was significantly reduced and the pyroptosis was obviously inhibited, leading to the reduction of injury and the increase of survival rate. Moreover, regulating the level of miR155-3p could affect survival rate and pyroptosis in vitro after H/R. CONCLUSIONS: The GJ channels composed of Cx32 regulated tubular pyroptosis in renal I/R injury by transmitting miR155-3p. Inhibition of Cx32 could reduce the level of miR155-3p further to inhibit pyroptosis, leading to alleviation of renal I/R injury which provided a new strategy for preventing the occurrence of AKI. Video Abstract.

Multi-Omics Integrated Analysis of the Protective Effect of EZH2 Inhibition in Mice with Renal Ischemia-Reperfusion Injury.

INTRODUCTION: Acute kidney injury (AKI) is a common clinical syndrome associated with high morbidity and mortality. Inhibition of the methyltransferase enhancer of zeste homolog 2 (EZH2) by its inhibitor 3-deazaneplanocin A (3-DZNeP) exerts renal benefits in acute renal ischemia-reperfusion injury (IRI). However, the underlying mechanisms are not completely known. This study aimed to elucidate the pathological mechanism of EZH2 in renal IRI by combination of multi-omics analysis and expression profiling in a public clinical cohort. METHODS: In this study, C57BL/6 J mice were used to establish the AKI model, which were treated with 3-DZNeP for 24 h. Kidney samples were collected for RNA-seq analysis, which was combined with publicly available EZH2 chromatin immunoprecipitation sequencing (ChIP-seq) data of mouse embryonic stem cell for a joint analysis to identify differentially expressed genes. Several selected differentially expressed genes were verified by quantitative PCR. Finally, single-nucleus sequencing data and expression profiling in public clinical datasets were used to confirm the negative correlation of the selected genes with EZH2 expression. RESULTS: 3-DZNeP treatment significantly improved renal pathology and function in IRI mice. Through RNA-seq analysis combined with EZH2 ChIP-seq database, 162 differentially expressed genes were found, which might be involved in EZH2-mediated pathology in IRI kidneys. Four differential expressed genes (Scd1, Cidea, Ghr, and Kl) related to lipid metabolism or cell growth were selected based on Gene Ontology and Kyoto Encyclopedia of Genes and Genome enrichment analysis, which were validated by quantitative PCR. Data from single-nucleus RNA sequencing revealed the negative correlation of these four genes with Ezh2 expression in different subpopulations of proximal tubular cells in IRI mice in a different pattern. Finally, the negative correlation of these four genes with EZH2 expression was confirmed in patients with AKI in two clinical datasets. CONCLUSIONS: Our study indicates that Scd1, Cidea, Ghr, and Kl are downstream genes regulated by EZH2 in AKI. Upregulation of EZH2 in AKI inhibits the expression of these four genes in a different population of proximal tubular cells to minimize normal physiological function and promote acute or chronic cell injuries following AKI.

Protective effects of pioglitazone in renal ischemia-reperfusion injury (RIRI): focus on oxidative stress and inflammation.

BACKGROUND: Renal ischemia-reperfusion injury (RIRI) is a critical phenomenon that compromises renal function and is the most serious health concern related to acute kidney injury (AKI). Pioglitazone (Pio) is a known agonist of peroxisome proliferator-activated receptor-gamma (PPAR-γ). PPAR-γ is a nuclear receptor that regulates genes involved in inflammation, metabolism, and cellular differentiation. Activation of PPAR-γ is associated with antiinflammatory and antioxidant effects, which are relevant to the pathophysiology of RIRI. This study aimed to investigate the protective effects of Pio in RIRI, focusing on oxidative stress and inflammation. METHODS: We conducted a comprehensive literature search using electronic databases, including PubMed, ScienceDirect, Web of Science, Scopus, and Google Scholar. RESULTS: The results of this study demonstrated that Pio has antioxidant, anti-inflammatory, and anti-apoptotic activities that counteract the consequences of RIRI. The study also discussed the underlying mechanisms, including the modulation of various pathways such as TNF-α, NF-κB signaling systems, STAT3 pathway, KIM-1 and NGAL pathways, AMPK phosphorylation, and autophagy flux. Additionally, the study presented a summary of various animal studies that support the potential protective effects of Pio in RIRI. CONCLUSION: Our findings suggest that Pio could protect the kidneys from RIRI by improving antioxidant capacity and decreasing inflammation. Therefore, these findings support the potential of Pio as a therapeutic strategy for preventing RIRI in different clinical conditions.

Comparing open and robot-assisted partial nephrectomy - a single institution report.

BACKGROUND: Open partial nephrectomy (OPN) has previously been considered the gold standard procedure for treatment of T1 localized renal tumors. After introduction of robot assisted partial nephrectomy (RAPN) as an alternative method to OPN, OPN was gradually abandoned at our department. The aim of the study was to retrospectively compare the results of patients treated with either OPN or RAPN for suspected renal carcinoma. METHODS: Patients who underwent either open or robotic assisted partial nephrectomy between January 1st 2010 and December 31st 2020 were retrospectively included in the study. Each tumor subjected to surgery was scored preoperatively by the RENAL nephrometry score. Complications within 30 days were assessed according to the Clavien-Dindo classification system. RESULTS: A total of 197 patients who underwent partial nephrectomy were identified; 75 were subjected to OPN and 122 were treated with RAPN. There were no significant differences between the groups with respect to age (OPN: 63 years ± 11, RAPN: 62 years ± 10), gender (OPN: 71/29%, RAPN: 67/33%), body mass index (OPN: 28 ± 5, RAPN: 28 ± 5), ASA score (OPN: 2.4 ± 0.6, RAPN: 2.2 ± 0.5), or nephrometry score (OPN: 6.6 ± 1.7, RAPN: 6.9 ± 1.7, p = 0.2). The operative time was significantly shorter in the OPN group (81 min) compared to the RAPN group (144.5 min, p < 0.001). Mean perioperative blood loss was 227 ± 162 ml in the OPN group compared to 189 ± 152 ml in the RAPN group (p = 0.1). Mean length of stay was shorter in the RAPN group (3 days) compared to the OPN group (6, days, p < 0.001). Positive surgical margin rate was significantly higher in the OPN group (21.6%) compared to the RAPN group (4.2%, p < 0.001). There were no differences in the number of Clavien-Dindo graded complications between the groups (p = 0.6). CONCLUSIONS: The introduction of RAPN at our department resulted in shorter length of stay and fewer positive surgical margins, without increasing complications.

Protective effect of small extracellular vesicles (EVs) derived from ACE2-modified human umbilical cord mesenchymal stem cells against renal ischemia-reperfusion injury.

AIM: Acute kidney injury is a severe disease that is closely associated with substantial morbidity and mortality. The most common cause of AKI is renal ischemia-reperfusion injury. Mesenchymal stem cells (MSCs) have previously been shown to have renoprotective effects. However, extracellular vesicles secreted by MSCs are thought to be the key for the therapeutic effects of MSCs. This study investigated whether small EVs derived from ACE2-modified human umbilical cord MSCs could alleviate RIRI and explored their underlying molecular mechanisms METHODS: A lentivirus carrying an ACE2 overexpression vector was constructed and used to infect MSCs. The small EVs were isolated from MSC-conditioned medium by ultracentrifugation. HK-2 cells were cocultured with MSC-ACE2-EVs and subjected to hypoxia/reoxygenation injury. MSCs-ACE2-EVs were injected into RIRI mice. Biochemical and morphological characteristics were assessed, and the levels of inflammatory-related factors, oxidative stress products, and apoptosis in HK-2 cells and kidney tissues were assessed RESULTS: In vitro, MSC-ACE2-EVs had stronger anti-inflammatory, antioxidative stress, and antiapoptotic effects in HK-2 cells subjected to H/R than MSC-NC-EVs. In vivo, MSC-ACE2-EVs could target the injured kidney, reduce blood creatinine and urea nitrogen levels, and protect the kidney from I/R, and this effect may have been related to the activation of the Nrf2/HO-1 signalling pathway CONCLUSION: Taken together, our results demonstrated the anti-inflammatory, antioxidative stress, and antiapoptotic effects of MSC-ACE2-EVs, which protected against I/R injury in vitro and vivo. MSC-ACE2-EVs may be therapeutic agents for RIRI.

The roles of TRPC6 in renal tubular disorders: a narrative review.

The transient receptor potential canonical 6 (TRPC6) channel, a nonselective cation channel that allows the passage of Ca2+, plays an important role in renal diseases. TRPC6 is activated by Ca2+ influx, oxidative stress, and mechanical stress. Studies have shown that in addition to glomerular diseases, TRPC6 can contribute to renal tubular disorders, such as acute kidney injury, renal interstitial fibrosis, and renal cell carcinoma (RCC). However, the tubule-specific physiological functions of TRPC6 have not yet been elucidated. Its pathophysiological role in ischemia/reperfusion (I/R) injury is debatable. Thus, TRPC6 may have dual roles in I/R injury. TRPC6 induces renal fibrosis and immune cell infiltration in a unilateral ureteral obstruction (UUO) mouse model. Additionally, TRPC6 overexpression may modify G2 phase transition, thus altering the DNA damage checkpoint, which can cause genomic instability and RCC tumorigenesis and can control the proliferation of RCC cells. This review highlights the importance of TRPC6 in various conditions of the renal tubular system. To better understand certain renal disorders and ultimately identify new therapeutic targets to improve patient care, the pathophysiology of TRPC6 must be clarified.

Nanoparticles transfected with plasmid-encoded lncRNA-OIP5-AS1 inhibit renal ischemia-reperfusion injury in mice via the miR-410-3p/Nrf2 axis.

Nanostructures composed of liposomes and polydopamine (PDA) have demonstrated efficacy as carriers for delivering plasmids, effectively alleviating renal cell carcinoma. However, their role in acute kidney injury (AKI) remains unclear. This study aimed to investigate the effects of the plasmid-encoded lncRNA-OIP5-AS1@PDA nanoparticles (POP-NPs) on renal ischemia/reperfusion (RI/R) injury and explore the underlying mechanisms. RI/R or OGD/R models were established in mice and HK-2 cells, respectively. In vivo, vector or POP-NPs were administered (10 nmol, IV) 48 h after RI/R treatment. In the RI/R mouse model, the OIP5-AS1 and Nrf2/HO-1 expressions were down-regulated, while miR-410-3p expression was upregulated. POP-NPs treatment effectively reversed RI/R-induced renal tissue injury, restoring altered levels of blood urea nitrogen, creatinine, malondialdehyde, inflammatory factors (IL-8, IL-6, TNF-α), ROS, apoptosis, miR-410-3p, as well as the suppressed expression of SOD and Nrf2/HO-1 in the model mice. Similar results were obtained in cell models treated with POP-NPs. Additionally, miR-410-3p mimics could reverse the effects of POP-NPs on cellular models, partially counteracted by Nrf2 agonists. The binding relationship between OIP5-AS1 and miR-410-3p, alongside miR-410-3p and Nrf2, has been substantiated by dual-luciferase reporter and RNA pull-down assays. The study revealed that POP-NPs can attenuate RI/R-induced injury through miR-410-3p/Nrf2 axis. These findings lay the groundwork for future targeted therapeutic approaches utilizing nanoparticles for RI/R-induced AKI.

High-fat diet-induced mitochondrial dysfunction is associated with loss of protection from ischemic preconditioning in renal ischemia reperfusion.

Consumption of high-fat diet (HFD) promotes mitochondrial dysfunction and the latter act as a critical factor in determining the severity of ischemia-reperfusion (IR) injury in different cell types. Ischemic preconditioning (IPC), a well-known protocol that render IR protection in kidney works via mitochondria. In the present study, we evaluated how HFD kidney with underlying mitochondrial changes respond to precondition protocol after IR induction. Wistar male rats were used in this study and were divided into two groups: SD (standard diet; n = 18) and HFD (high-fat diet; n = 18), which were further subdivided into sham, ischemia-reperfusion, and precondition groups at the end of the dietary regimen. Blood biochemistry, renal injury marker, creatinine clearance (CrCl), mitochondrial quality (fission, fusion, and phagy), mitochondrial function via ETC enzyme activities and respiration, and signalling pathway were analysed. Sixteen weeks of HFD administration to the rat deteriorated the renal mitochondrial health measured via 10% decline in mitochondrial respiration index ADP/O (in GM), reduced mitochondrial copy number (55%), biogenesis (56%), low bioenergetics potential (19% complex I + III and 15% complex II + III), increased oxidative stress, and reduced expression of mitochondrial fusion genes compared with SD rats. IR procedure in HFD rat kidney inflicted significant mitochondrial dysfunction and further deteriorated copy number along with impaired mitophagy and mitochondrial dynamics. IPC could effectively ameliorate the renal ischemia injury in normal rat but failed to provide similar kind of protection in HFD rat kidney. Even though the IR-associated mitochondrial dysfunction in both normal and HFD rats were similar, the magnitude of overall dysfunction and corresponding renal injury and compromised physiology was high in HFD rats. This observation was further confirmed via in vitro protein translation assay in isolated mitochondria from normal and HFD rat kidney that showed significantly reduction in the response ability of mitochondria in HFD. In conclusion, the deteriorated mitochondrial function and its quality along with low mitochondrial copy number and downregulation of mitochondrial dynamic gene exhibited by HFD rat kidney augments the sensitivity of renal tissue towards the IR injury which leads to the compromised protective ability by ischemic preconditioning.

Impaired renal ischemia reperfusion recovery after bilateral renal artery ligation in rats treated with adenine: role of renal mitochondria.

Vascular calcification (VC) and ischemia reperfusion (IR) injury is characterised to have mitochondrial dysfunction. However, the impact of dysfunctional mitochondria associated with vascular calcified rat kidney challenged to IR is not explored and is addressed in the present study. Male Wistar rats were treated with adenine for 20 days to induce chronic kidney dysfunction and VC. After 63 days, renal IR protocol was performed with subsequent recovery for 24 h and 7 days. Various mitochondrial parameters and biochemical assays were performed to assess kidney function, IR injury and its recovery. Adenine-induced rats with VC, decreased creatinine clearance (CrCl), and severe tissue injury demonstrated an increase in renal tissue damage and decreased CrCl after 24 h of IR (CrCl in ml: IR-0.220.02, VC-IR-0.050.01). Incidentally, the 24 h IR pathology in kidney was similar in both VC-IR and normal rat IR. But, the magnitude of dysfunction was higher with VC-IR due to pre-existing basal tissue alterations. We found severed deterioration in mitochondrial quantity and quality supported by low bioenergetic function in both VC basal tissue and IR challenged sample. However, post 7 days of IR, unlike normal rat IR, VC rat IR did not improve CrCl and corresponding mitochondrial damage in terms of quantity and its function were observed. Based on the above findings, we conclude that IR in VC rat adversely affect the post-surgical recovery, mainly due to the ineffective renal mitochondrial functional restoration from the surgery.

Denervation aggravates renal ischemia reperfusion injury via BMAL1-mediated Nrf2/ARE pathway.

AIM: To explore the change of clock gene rhythm under renal denervation (RDN) and its effect on renal function and oxidative stress during renal ischemia-reperfusion (IR) injury. METHOD: C57/BL6 mice were randomly divided into 4 groups at daytime 7 A M (zeitgeber time [ZT] 0) or at nighttime 7 P M (ZT12) in respectively: Sham (S) group, RDN group, IR group and RDN + IR (DIR) group. Renal pathological and functional changes were assessed by H&E staining, and serum creatinine, urea nitrogen and neutrophil gelatinase-associated lipocalin levels. Renal oxidative stress was detected by SOD and MDA levels, and renal inflammation was measured by IL-6, IL-17 A F and TNF-ɑ levels. BMAL1, CLOCK, Nrf2 and HO-1 mRNA and protein expressions were tested by qPCR and Western Blot. RESULT: Compared with S groups, the rhythm of BMAL1, CLOCK and Nrf2 genes in the kidney were disordered in RDN groups, while renal pathological and functional indexes did not change significantly. Compared with IR groups, renal pathological and functional indexes were significantly higher in the DIR groups, as well as oxidative stress and inflammation in renal tissues. The nocturnal IR injury in the RDN kidney was the worst while the BMAL1, Nrf2 and HO-1 expressions were the highest. In DIR groups, renal injury was aggravated after the Brusatol treatment, but there was no significant improvement after the t-BHQ treatment at night, which might be consistent with the changes of Nrf2 and HO-1 protein expressions. CONCLUSION: RDN lead to the disruption of BMAL1-mediated Nrf2 rhythm accumulation in the kidney, which reduced the renal ability to resist oxidative stress and inflammation, due to the impaired effect of activating Nrf2/ARE pathway in renal IR injury at nighttime.

TRPC6 inhibits renal tubular epithelial cell pyroptosis through regulating zinc influx and alleviates renal ischemia-reperfusion injury.

Canonical transient receptor potential-6 (TRPC6) has been reported to be involved in cell damage after ischemia/reperfusion (I/R) injury in target organs. While the effect and of TRPC6 on pyroptosis in renal I/R injury remain unclear. In our study, we first established the renal I/R mouse model and oxygen-glucose deprivation and re-oxygenation (OGD/R) cell model, and investigated the impacts of TRPC6 on the pyroptosis-related proteins using CCK-8, western blot, ELISA, and immunofluorescence probes. Besides, we also explored the mechanism of TRPC6 in pyroptosis of renal tubular epithelial cells through A20 knockdown or overexpression and zinc chloride (ZnCl2 ) or a zinc ion chelator (TPEN) treatment. Our results indicated that I/R injury could cause downregulation of TRPC6 both in vivo and in vitro. In the I/R injury murine model, TRPC6 inhibition exacerbated tissue damage and upregulated NLRP3, ASC, caspase-1, IL-18, and IL-1ß, which could be alleviated by the administration of ZnCl2 . In the OGD/R cell model, inhibitor of TRPC6 (SAR7334) reduced zinc ion influx, aggravated cell death and upregulated pyroptosis-related protein. The pyroptosis phenotype also could be alleviated by ZnCl2 and intensified by TPEN. Overexpression of A20 reduced the expression of pyroptosis-related protein, increased cell viability in the sh-TRPC6 and TPEN-treated OGD/R cell models, while A20 deficiency impaired the protective effect of zinc ion. Therefore, our results indicate that TRPC6 could promote zinc ion influx in renal tubular epithelial cells, thereby upregulating intracellular A20, inhibiting the activation of inflammasome NLRP3, and ultimately attenuating renal I/R injury.

Empagliflozin improves renal ischemia-reperfusion injury by reducing inflammation and enhancing mitochondrial fusion through AMPK-OPA1 pathway promotion.

BACKGROUND: Renal ischemia-reperfusion injury (IRI) is one reason for renal transplantation failure. Recent studies have shown that mitochondrial dynamics is closely related to IRI, and that inhibition or reversal of mitochondrial division protects organs against IRI. Optic atrophy protein 1 (OPA1), an important factor in mitochondrial fusion, has been shown to be upregulated by sodium-glucose cotransporter 2 inhibitor (SGLT2i). Also, the antiinflammatory effects of SGLT2i have been demonstrated in renal cells. Thus, we hypothesized that empagliflozin could prevent IRI through inhibiting mitochondrial division and reducing inflammation. METHODS: Using hematoxylin-eosin staining, enzyme linked immunosorbent assay (ELISA), flow cytometry, immunofluorescent staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining, real-time PCR, RNA-sequencing, and western blot, we analyzed renal tubular tissue from in vivo and in vitro experiments. RESULTS: Through animal experiments and sequencing analysis, we first confirmed the protection against IRI and the regulation of mitochondrial dynamics-related factors and inflammatory factors by empagliflozin pretreatment. Then, through hypoxia/reoxygenation (H/R) cellular experiments, we confirmed that empagliflozin could inhibit mitochondrial shortening and division and upregulate OPA1 in human renal tubular epithelial cell line (HK-2) cells. Subsequently, we knocked down OPA1, and mitochondrial division and shortening were observed, which could be alleviated by empagliflozin treatment. Combined with the previous results, we concluded that OPA1 downregulation leads to mitochondrial division and shortening, and empagliflozin can alleviate the condition by upregulating OPA1. We further explored the pathway through which empagliflozin functions. Related studies have shown the activation of AMPK pathway by empagliflozin and the close correlation between the AMPK pathway and OPA1. In our study, we blocked the AMPK pathway, and OPA1 upregulation by empagliflozin was not observed, thus demonstrating the dependence of empagliflozin on the AMPK pathway. CONCLUSION: The results indicated that empagliflozin could prevent or alleviate renal IRI through antiinflammatory effects and the AMPK-OPA1 pathway. Ischemia-reperfusion injury is an inevitable challenge in organ transplantation. It is necessary to develop a new therapeutic strategy for IRI prevention in addition to refining the transplantation process. In this study, we confirmed the preventive and protective effects of empagliflozin in renal ischemia-reperfusion injury. Based on these findings, empagliflozin is promising to be a preventive agent for renal ischemia-reperfusion injury and can be applied for preemptive administration in kidney transplantation.

MicroRNA-192-5p downregulates Fat Mass and Obesity-associated Protein to aggravate renal ischemia/reperfusion injury.

Acute kidney injury (AKI) is a common disorder without effective therapy yet. Renal ischemia/reperfusion (I/R) injury is a common cause of AKI. MicroRNA miR-192-5p has been previously reported to be upregulated in AKI models. However, its functional role in renal I/R injury is not fully understood. This study aimed to investigate the effects and the underlying mechanism of miR-192-5p in renal I/R progression. Hypoxia/reoxygenation (H/R)-induced cell injury model in HK-2 cells and I/R-induced renal injury model in mice were established in this study. Cell counting kit-8 assay was performed to determine cell viability. Quantitative real-time PCR and western blot analysis were performed to detect gene expressions. Hematoxylin-eosin and periodic acid-Schiff staining were performed to observe the histopathological changes. Enzyme-linked immunosorbent assay was performed to detect the kidney markers' expression. In vivo and in vitro results showed that miR-192-5p was up-regulated in the I/R-induced mice model and H/R-induced cell model, and miR-192-5p overexpression exacerbated I/R-induced renal damage. Then, the downstream target of miR-192-5p was analyzed by combining the differentially expressed mRNAs and the predicted genes and confirmed using a dual-luciferase reporter assay. It was found that miR-192-5p was found to regulate fat mass and obesity-associated (FTO) protein expression by directly targeting the 3' untranslated region of FTO mRNA. Moreover, in vivo and in vitro studies unveiled that FTO overexpression alleviated renal I/R injury and promoted HK-2 cell viability via stimulating autophagy flux. In conclusion, miR-192-5p aggravated I/R-induced renal injury by blocking autophagy flux via down-regulating FTO.

Mineralocorticoid Receptor Antagonism Attenuates Multiple Organ Failure after Renal Ischemia and Reperfusion in Mice.

Renal ischemia reperfusion (IR) injury is a major cause of acute kidney injury (AKI) that is often complicated by multiple organ failure of the liver and intestine. The mineralocorticoid receptor (MR) is activated in patients with renal failure associated with glomerular and tubular damage. We thus investigated whether canrenoic acid (CA), a mineralocorticoid receptor (MR) antagonist, protects against AKI-induced hepatic and intestinal injury, suggesting the underlying mechanisms. Mice were divided into five groups: sham mice, mice subjected to renal IR, and mice pretreated with canrenoic acid (CA; 1 or 10 mg/kg) 30 min prior to renal IR. At 24 h after renal IR, the levels of plasma creatinine, alanine aminotransferase and aldosterone were measured, and structural changes and inflammatory responses of the kidney, liver, and intestine were analyzed. We found that CA treatment reduced plasma creatinine levels, tubular cell death and oxidative stress induced by renal IR. CA treatment also decreased renal neutrophil infiltration and inflammatory cytokine expression and inhibited the release of high-mobility group box 1 induced by renal IR. Consistently, CA treatment reduced renal IR-induced plasma alanine transaminase, hepatocellular injury and neutrophil infiltration, and inflammatory cytokine expression. CA treatment also decreased small intestinal cell death, neutrophil infiltration and inflammatory cytokine expression induced by renal IR. Taken together, we conclude that MR antagonism by CA treatment protects against multiple organ failure in the liver and intestine after renal IR.

The Sigma-1 Receptor Is a Novel Target for Improving Cold Preservation in Rodent Kidney Transplants.

Kidney transplantation is the preferred treatment for patients with end-stage kidney disease. Maintaining organ viability between donation and transplantation, as well as minimizing ischemic injury, are critically important for long-term graft function and survival. Moreover, the increasing shortage of transplantable organs is a considerable problem; thus, optimizing the condition of grafts is a pivotal task. Here, rodent models of kidney transplantation and cold storage were used to demonstrate that supplementation of a preservation solution with Sigma-1 receptor (S1R) agonist fluvoxamine (FLU) reduces cold and warm ischemic injury. Post-transplant kidney function was improved, histological injury was mitigated, and mRNA expression of two tubular injury markers-kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin-was robustly reduced. In addition, renal inflammation was diminished, as shown by reduced leukocyte infiltration and pro-inflammatory cytokine expression. In the cold ischemia model, FLU ameliorated structural injury profoundly after 2 h as well as 24 h. The reduced number of TUNEL-positive and Caspase 3-positive cells suggests the anti-apoptotic effect of FLU. None of these beneficial effects of FLU were observed in S1R-/- mice. Of note, organ damage in FLU-treated kidneys after 24 h of cold storage was similar to just 2 h without FLU. These results indicate that S1R agonists can prolong storage time and have great potential in improving organ preservation and in alleviating the problem of organ shortages.

GADD45A and GADD45B as Novel Biomarkers Associated with Chromatin Regulators in Renal Ischemia-Reperfusion Injury.

Chromatin regulators (CRs) are essential upstream regulatory factors of epigenetic modification. The role of CRs in the pathogenesis of renal ischemia-reperfusion injury (IRI) remains unclear. We analyzed a bioinformatic analysis on the differentially expressed chromatin regulator genes in renal IRI patients using data from public domains. The hub CRs identified were used to develop a risk prediction model for renal IRI, and their expressions were also validated using Western blot, qRT-PCR, and immunohistochemistry in a murine renal IRI model. We also examined the relationships between hub CRs and infiltrating immune cells in renal IRI and used network analysis to explore drugs that target hub CRs and their relevant downstream microRNAs. The results of machine learning methods showed that five genes (DUSP1, GADD45A, GADD45B, GADD45G, HSPA1A) were upregulated in renal IRI, with key roles in the cell cycle, p38 MAPK signaling pathway, p53 signaling pathway, FoxO signaling pathway, and NF-κB signaling pathway. Two genes from the network, GADD45A and GADD45B (growth arrest and DNA damage-inducible protein 45 alpha and beta), were chosen for the renal IRI risk prediction model. They all showed good performance in the testing and validation cohorts. Mice with renal IRI showed significantly upregulated GADD45A and GADD45B expression within kidneys compared to sham-operated mice. GADD45A and GADD45B showed correlations with plasmacytoid dendritic cells (pDCs) in infiltrating immune cell analysis and enrichment in the MAPK pathway based on the weighted gene co-expression network analysis (WGCNA) method. Candidate drugs that target GADD45A and GADD45B include beta-escin, sertraline, primaquine, pimozide, and azacyclonol. The dysregulation of GADD45A and GADD45B is related to renal IRI and the infiltration of pDCs, and drugs that target GADD45A and GADD45B may have therapeutic potential for renal IRI.

The effects of tarantula cubensis extract on renal ischemia reperfusion injury in rats.

BACKGROUND: Renal ischemia-reperfusion (IR) related acute kidney injury (AKI) is an important health problem and has not yet been fully treated. Tarantula cubensis extract (TCE) is a homeopathic drug that has antiinflammatory and antioxidant effects. This study aimed to investigate the effects of TCE on renal ischemia-reperfusion injury in rats. METHODS: This study was carried out on 48 Spraque-Dawley male rats, which were divided into six groups. The first, second, and third groups were control, sham, and IR groups, respectively. Group four received IR and 0.2 mL of 96% ethanol. Group five and six received ischemia and reperfusion and TCE 0.01 and 0.1 mg per rat (which correspond to approximately 0.04 mg/kg, and 0.4 mg/kg), respectively. Tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1ß), total antioxidant status (TAS), and total oxidant status (TOS) levels in renal tissue were measured by enzyme-linked immunosorbent assay (ELISA). Oxidative stress index (OSI) was obtained by proportioning TAS and TOS. Superoxide dismutase (SOD), myeloperoxidase (MPO) activities, and malondialdehyde (MDA) levels were determined by manual spectrophotometric methods. The histopathological changes were evaluated via hematoxylineosin and immunohistochemical staining. RESULTS: In IR group, renal tissue TNF-α and IL-1ß levels were significantly higher than control group (p < 0.0001 for both), and low(p < 0.0001 for both) and high dose (p < 0.0001 for both) TCE administration decreased these markers. Low and high doses of TCE decreased OSI values compared with IR group (p = 0.04 and p = 0.001 respectively). Although TCE decreased MDA levels, it was not statistically significant. MPO levels significantly decreased. In addition, TCE has been found to prevent hemorrhage, cast formation, and dilatation caused by IR in renal tissues stained with hematoxylin-eosin. And also, the most intense nuclear factor kappa B (NFκB) and caspase-3 immunopositivity found in IR group was decreased in both of the TCE groups. DISCUSSION: Although TCE showed a protective effect by inhibiting inflammation against IR damage in renal tissues, there was no clear effect on oxidative stress. Larger and more detailed studies are needed to clarify the issue.

THE POTENTIAL RENOPROTECTIVE EFFECT OF TILIANIN IN RENAL ISCHEMIA REPERFUSION INJURY IN MALE RAT MODEL.

OBJECTIVE: The aim: To determine whether Tilianin (TIL) may have Nephroprotective effects on bilateral renal IRI in rats by analyzing kidney function biomarkers U and Cr, inflammatory cytokines like TNF α and IL-1ß, antioxidant marker total anti-oxidant Capacity (TAC), anti-apoptotic markers caspase-3, and histopathological scores. PATIENTS AND METHODS: Materials and methods: 20 rats divided into even 4 groups as: Sham group: Rats underwent median laparotomies without having their ischemia induced. Control group: Rats had bilateral renal ischemia for 30 minutes, followed by 2 hours of reperfusion. Vehicle group: 30 minutes prior to the onset of ischemia, rats were given a pretreatment of corn oil and DMSO. Tilianin treated group: Rats administered Tilianin 5 mg/kg for 30 min prior to ischemia induction, then IRI. RESULTS: Results: The study found that the serum levels of TNF, IL-1, caspase-3, urea and creatinine, as well as TNF and creatinine in the Tilianin group were significantly lower than those of the control and vehicle groups. On the other hand, it revealed that TAC levels are remarkably higher in the Tilianin group than they are in the control and vehicle groups. CONCLUSION: Conclusions: This study concluded that Tilianin have a Nephroprotective effect via multiple impacts as anti-inflammatory, anti-apoptotic, and anti-oxidant agents.