Renal Tubular Cell Apoptosis Research Articles

404.7: Study on the Regulatory Effect of iPS-Derived Exosomes on the Local Immune Microenvironment in DCD Kidney Transplanted Rats

Introduction: Ischemia reperfusion injury (IRI) of donation After Cardiac death (DCD) transplanted kidney is the key factor of acute renal injury, acute and chronic rejection of renal transplantation and transplanted kidney disease.The latest researches show that mesenchymal stem cells (MSCs) have the function of protecting damaged tissues and organs and strong immune regulation. Induced pluripotent stem (iPS) cells have higher proliferative capacity and exosome secretion potential than MSCs. In addition to the functions of stem cells, exosomes have the unique ability to cross the blood-brain barrier and deliver remotely, which means that exosomes have better advantages in clinical disease treatment compared with the therapeutic potential of stem cells. Therefore, the main purpose of this study is to clarify the role of IPS derived exosomes in protecting the IRI of DCD transplanted kidney and regulating its local immune microenvironment. Methods: The iPS cells were passaged to the third passage and massively expanded. After 48 hours of culture, 100 mL of cell culture supernatant was collected and exosomes were extracted from the supernatant by the kit method, and the morphology and surface membrane marker proteins of the exosomes were identified. The SD rats were established with DCD renal transplantation model and 10, 50, and 100 μg of exosomes were infused into that SD rats respectively, and a PBS control group was set at the same time. On the 3rd, 5th, 7th, 14th and 28th days after transplantation, the content of cytokines in the serum of the recipient rats, the proportion of lymphocyte subsets CD4+CD25+Foxp3+Tregs in the spleen and the relative expression of Foxp3 mRNA were detected. The proliferation of renal tubular cells was detected by PCNA immunofluorescence. The pathological changes of renal tissue were determined by HE staining, and the apoptosis of renal tubular epithelial cells was detected by Tunel staining. The infiltration of inflammatory cells was detected by immunohistochemistry. The expression changes of regulatory T cells in renal tissue and macrophage phenotypic characteristics were analyzed by flow cytometry. Results: The experimental amount of exosomes was successfully obtained, and the morphology was observed by transmission electron microscope. The exosomes were round or oval membranous vesicles with uniform size, and the edges were clearly visible. The TSG101, CD63, and CD9 proteins were highly expressed on the exosome membrane surface by western-blot. A DCD rat kidney transplantation model was successfully established. The expression levels of IL-12, IL-2, IFN-γ, and TNF-α in the serum of each group were increased to varying degrees from the 3rd day after operation. On the 3rd and 7th days, the levels of IL-12, IL-2, IFN-γ, and TNF-α in the 100 μg exosome infusion group were significantly lower than those in the 10 μg, 50 μg and PBS groups (P<0.05), and the expression level of IL-10 in the 100 μg exosome infusion group was significantly higher than 10μg, 50μg and PBS groups (P<0.05). On the 28th day, the expression level of IL-10 in the 100 μg exosome infusion group was significantly higher than other groups (P<0.01). Compared with other groups, the CD4+CD25+Tregs and CD4+CD25+Foxp3+Tregs accounted for the highest proportion of CD4+T cells in the spleen of the 100μg infusion group (P<0.05). On the 28th day, the expression level of Foxp3 mRNA in the 100 μg infusion group was significantly higher than that in the 50 μg group (P<0.05). The stainings of PCNA, HE and Tunel showed that SD rats infused with 100μg exosomes could significantly reduce the pathological damage, inflammatory response and apoptosis of renal tubular epithelial cells in the transplanted kidney, promote the proliferation of renal tubular epithelial cells, and have a protective effect on IRI kidney. On the 28th day, the expression of CD4+CD25+Foxp3+Tregs cells in the transplanted kidney tissue was significantly increased, which further promoted the differentiation of macrophages into the immunomodulatory M2 subset. Conclusions: The infusion of iPS-derived high-dose exosomes is involved in the protection of the DCD kidney transplant during IRI process, and alleviates the acute and chronic injury of the DCD kidney transplant, and promotes the formation of the local immunosuppressive microenvironment in the DCD kidney transplantation. The National Natural Science Foundation of China (No.81900686).

Astragaloside IV Protects Sepsis-induced Acute Kidney Injury by Attenuating Mitochondrial Dysfunction and Apoptosis in Renal Tubular Epithelial Cells.

Acute kidney injury (AKI) is closely linked to the pathogenesis of sepsis. Oxidative stress can affect the development of AKI by increasing damage to renal tubular epithelial cells. Astragaloside IV (AS-IV) is a natural saponin widly verified beneficial for ameliorating sepsis-induced kidney injury. However, the underlying mechanisms of AS-IV on relieving oxidative stress in renal tubular epithelial cells are yet to be established. We aimed to investigate whether AS-IV could attenuate mitochondrialdysfunction and apoptosis in renal tubular epithelial cells and reveal its underlying mechanisms. For the in vivo study, mice were divided into four groups (n=6): sham+saline, CLP+saline, CLP+ASIV- low dosage (5 mg/kg), CLP+AS-IV-high dosage (10 mg/kg), After 6 h or 24 h of treatment, the renal injuries were assessed based on related parameters of blood, protein and histopathological examination. Immunohistochemistry and ELISA were used to examine renal function. The molecular mechanism of AS-IV inhibited apoptosis and mitochondrial damage were monitored by flow cytometry and western blot analysis in HK-2 cells. We found that AS-IV ameliorates renal vacuolization, brush border loss, mitochondrial ultrastructure changes in sepsis-induced AKI, and the apoptosis and oxidative damage were greatly mitigated by AS-IV (10 mg/kg)-treated group. Abnormal changes in mitochondrial morphology and mitochondrial membrane potential were alleviated, and the expression of mitochondrial complex protein I (NDUFB8) and mitochondrial complex protein II (SDHB8) increased with (10 mg/kg)-treated group. Tubular epithelial cell apoptosis in AS-IV (20 μM)-treated cells was reduced by the Bax and cleaved caspase3 pathway. These studies demonstrated that AS-IV protects against sepsis-induced kidney tubular injury by alleviating oxidative stress, mitochondrial dysfunction possibly associated with the restored cleaved caspase3 pathway.

A biocompatible nanoparticle-based approach to inhibiting renal ischemia reperfusion injury in mice by blocking thrombospondin-1 activity

Thrombospondin-1 (TSP-1) is a key mediator of renal ischemia-reperfusion injury (IRI), a major cause of kidney dysfunction under various disease conditions and a risk factor of renal allograft rejection. In this study, we developed a nanotechnology-based therapy targeting TSP-1 to prevent renal IRI. A biocompatible nanoparticle (NP) capable of specific binding to TSP-1 was prepared by conjugating NPs with TSP-1-binding (LSKL) peptides. LSKL/NPs not only effectively adsorbed recombinant TSP-1 proteins in vitro, but also efficiently neutralized TSP-1 in mice undergoing renal IRI. IRI-induced elevation of TSP-1 in the kidney was significantly inhibited by post-IR treatment with LSKL/NPs, but not free LSKL or NPs. Furthermore, TSP-1 proteins adsorbed on LSKL/NPs were functionally inactive and unable to induce apoptosis in renal tubular epithelial cells. Importantly, LSKL/NPs induced strong protection against renal IRI, as shown by markedly diminished serum creatinine levels and improved histological lesions of the kidney. Thus, LSKL/NPs provide a useful means of depleting and inactivating TSP-1 and a potential therapy for renal IRI.

MiR-6918-5p prevents renal tubular cell apoptosis by targeting MBD2 in ischemia/reperfusion-induced AKI

AimsAlthough previous studies reported that miRNAs are involved in the progression of acute kidney injury (AKI), their exact function and mechanism in ischemic AKI remains largely unknown. This study aims to define the role of miR-6918-5p in ischemia-reperfusion AKI. Materials and methodsThe renal arteries of C57BL/6J mice were clamped to establish a model of ischemia-reperfusion renal injury. BUMPT cells were added with Antimycin A and calcium ionophore to establish a model of ATP depletion in vitro. Cell apoptosis was detected by CCK8, flow cytometry and western blot, while HE staining and TUNEL staining were used to assess the degree of kidney damage. Key findingsWe suppressed mmu_miR-6918-5p by ischemic injury in vitro and in vivo. We found that ischemia-reperfusion (I/R)-induced renal tubular cell apoptosis and the expression of cleaved caspase3 were enhanced by the inhibitor of mmu_miR-6918-5p; this effect was attenuated by an mmu_miR-6918-5p mimic. Mechanistically, mmu_miR-6918-5p binds to the 3′ UTR region of MBD2 and represses its expression. The mmu_miR-6918-5p mimic alleviated the ischemic AKI by targeting MBD2. Conversely, the inhibitor of mmu_miR-6918-5p enhanced the ischemic AKI; this was diminished by MBD2-KO. SignificanceMmu_miR-6918-5p protected against the development of ischemic AKI by targeting MBD2.

A novel contrast-induced acute kidney injury mouse model based on low-osmolar contrast medium

The contrast-induced acute kidney injury (CI-AKI) has been becoming the third common cause of hospital-acquired acute kidney injury. An ideal animal model is essential for understanding the pathophysiology of CI-AKI. Previous CI-AKI studies were mostly performed on rats with high-osmolar contrast medium (HOCM), which is unsuitable for transgenic researches. This study provides a novel, efficient and reproducible CI-AKI model which was developed in mouse by administrating a low-osmolar contrast medium (LOCM). First of all, we applied the frequently used pretreatments (uninephrectomy and water deprivation), which combined with HOCM on rats could induce CI-AKI, on mice with LOCM. Secondly, we attempted to find a novel pretreatment suitable for mouse and LOCM by combining two classic pretreatments(uninephrectomy, water deprivation and furosemide administration). Finally, we evaluate the kidney damage of the novel model. We found that this mouse model possessed a significant reduction in renal function, severe renal tissue damage, and increased renal tubular cells apoptosis, indicating that LOCM is a feasible inducer for CI-AKI mice model. Taken together, we found that uninephrectomy (UPHT) combined with 24 h water deprivation and furosemide administration 20 min before LOCM (iohexol, 10 ml/kg) application is a feasible pretreatment to establish a novel CI-AKI mouse model.

β-Hydroxybutyrate against Cisplatin-Induced acute kidney injury via inhibiting NLRP3 inflammasome and oxidative stress

Cisplatin, as a commonly used anticancer drug, can easily lead to acute kidney injury (AKI), and has received more and more attention in clinical practice. β-hydroxybutyric acid (BHB) is a metabolite in the body and acts as an inhibitor of oxidative stress and NLRP3 inflammasome, reducing inflammatory responses and apoptosis. However, the role of BHB in cisplatin-induced AKI is currently not fully elucidated. In this study, C57BL/6 male mice were randomly divided into normal control group, cisplatin-induced AKI group and AKI with BHB treatment group. Compared to the control, cisplatin-treated mice exhibited high level of serum creatinine, blood urea nitrogen and severe tubular injury, which accompanied with significantly increased expression level of NLRP3, IL-1β, IL-18, BAX, cleaved-caspase 3, as well as aggravated oxidative stress and renal tubular cell apoptosis. However, these changes were significantly improved in that of BHB treatment. In vitro, our study showed that the expression of cleaved-caspase3, IL-1β and IL-18 were significantly increased in human proximal tubular epithelial cell line (HK-2) treated with cisplatin compared with the control group, while decreased in cells treated with BHB. Furthermore, a significantly increased expression of cGAS and STING in HK-2 cells treated with cisplatin were found, whereas notably decreased in cells treated with BHB. This data indicates that BHB protects against cisplatin-induced AKI and renal tubular damage mediated by NLRP3 inflammasome and cGAS-STING pathway.

Oxalate-induced apoptosis through ERS-ROS–NF-κB signalling pathway in renal tubular epithelial cell

BackgroundKidney stones are composed of approximately 70–80% calcium oxalate. However, the exact mechanism of formation of calcium oxalate kidney stones remains unclear. In this study, we investigated the roles of endoplasmic reticulum stress (ERS), reactive oxygen species (ROS), and the NF-κB signalling pathway in the pathogenesis of oxalate-induced renal tubular epithelial cell injury and its possible molecular mechanisms.MethodsWe established a model to evaluate the formation of kidney stones by intraperitoneal injection of glyoxylic acid solution into mice and assessed cell morphology, apoptosis, and the expression levels of ERS, ROS, and NF-κB signalling pathway-related proteins in mouse renal tissues. Next, we treated HK-2 cells with potassium oxalate to construct a renal tubular epithelial cell injury model. We detected the changes in autophagy, apoptosis, and mitochondrial membrane potential and investigated the ultrastructure of the cells by transmission electron microscopy. Western blotting revealed the expression levels of apoptosis and autophagy proteins; mitochondrial structural and functional proteins; and ERS, ROS, and NF-κB (p65) proteins. Lastly, we studied the downregulation of NF-κB activity in HK-2 cells by lentivirus interference and confirmed the interaction between the NF-κB signalling and ERS/ROS pathways.ResultsWe observed swelling of renal tissues, increased apoptosis of renal tubular epithelial cells, and activation of the ERS, ROS, and NF-κB signalling pathways in the oxalate group. We found that oxalate induced autophagy, apoptosis, and mitochondrial damage in HK-2 cells and activated the ERS/ROS/NF-κB pathways. Interestingly, when the NF-κB signalling pathway was inhibited, the ERS/ROS pathway was also inhibited.ConclusionOxalate induces HK-2 cell injury through the interaction between the NF-κB signalling and ERS/ROS pathways.

Effect of intravenous glutamine on the protein expression changes, caspase-12 and apoptosis in renal tubulus russ male, rattusnovergicusstrain wistar, exposed by cisplastin

Cisplatin efficacy is dependent on dose. The cisplatin-induced apoptosis pathway can go through the ER stress pathway which will activate Bax and Bak (proapoptosis) causing calcium homeostasis disorders to occur. Glutamine protective effect on caspase-12 expression on the prevention of apoptotic events in renal proximal tubular epithelial cells is unknown. This further study analyzes the effects of intravenous glutamine on caspase- 12 expression in the apoptosis of renal proximal tubular cells of male white rats exposed to cisplatin. The control group (P0) consisted of 10 individuals, group P1 on day 7 was given a single dose of 20mg / kg of intraperitoneal cisplatin injection, group P2 on day 1-7 was given intravenous glutamine injection of 100mg / kg on day 7 also given a single intraperitoneal cisplatin injection 20mg / kg. The number of cells expressing caspase-12 and the number of apoptotic cells was observed after 72 hours. Caspase-12 expression and apoptosis data were collected for processing and analysis. There is a significant effect (p &lt;0.0001) in giving intravenous glutamine on caspase-12 expression in renal proximal tubular cell apoptosis. Glutamine can inhibit the expression of caspase-12, and the incidence of proximal tubular cell apoptosis were exposed by cisplatin.

Oxidative stress and Cx43-mediated apoptosis are involved in PFOS-induced nephrotoxicity

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant that can cause nephrotoxicity. However, the underlying mechanisms are not fully understood and require further investigation. In the present study, we established a PFOS-exposed Sprague-Dawley (SD) rat kidney injury model by intraperitoneal injection of PFOS (1 mg/kg and 10 mg/kg body weight) every alternate day for 15 days and cytotoxicity models of normal rat kidney epithelial (NRK52E) and human renal proximal tubular (HK2) cells exposed to PFOS (20 μM and 60 μM) for 24 h to reveal the mechanisms underlying PFOS-induced nephrotoxicity. Data showed that PFOS increased the kidney index and induced nephrotoxicity in rats. Furthermore, PFOS significantly increased malondialdehyde (MDA) levels, decreased GSH peroxidase (GSH-PX) activity in kidney tissues, and increased intracellular reactive oxygen species (ROS) levels in NRK52E and HK2 cells. Following PFOS treatment, mitochondrial damage in the renal tubular epithelial cells of rats was observed and the mitochondrial membrane potential (ΔΨm) was decreased in NRK52E cells. PFOS upregulated apoptosis of tubular epithelial cells and expression of Connexin 43 (Cx43) in vitro and in vivo. The Cx43 inhibitor gap26 attenuated the apoptosis of tubular epithelial cells. In conclusion, our findings reveal that PFOS may trigger renal tubular cell apoptosis through oxidative stress and upregulation of Cx43, resulting in PFOS-induced nephrotoxicity.

Cyproheptadine, a SET7/9 inhibitor, reduces hyperglycaemia-induced ER stress alleviating inflammation and fibrosis in renal tubular epithelial cells

Context Persistent hyperglycaemia increases SET7/9 expression and endoplasmic reticulum (ER) stress which causes inflammation, apoptosis, and fibrosis in renal tubular epithelial cells leading to diabetic kidney disease (DKD). Objective Current study explores the renoprotective potential of a novel SET7/9 inhibitor, Cyproheptadine, and the underlying molecular mechanisms in hyperglycaemia-induced renal tubular epithelial cell injury. Methods Change in expression of SET7/9, histone H3 lysine (K4) monomethylation (H3K4Me1), inflammatory, fibrotic, and ER stress proteins were evaluated in-vivo and in-vitro. NRK-52E cells were used to study the preventive effect of Cyproheptadine against hyperglycaemia-induced ER stress and subsequent inflammation and fibrosis. Results SET7/9 and H3K4Me1 expression significantly increased with ER stress, inflammation, apoptosis, and fibrosis, in-vivo and in-vitro under hyperglycaemia. However, the cells treated with Cyproheptadine showed significant suppression of H3K4Me1 and reduction in ER stress, inflammation, apoptosis, and fibrosis. Conclusion Cyproheptadine prevented hyperglycaemia-induced renal fibrosis and inflammation by reducing H3K4Me1 expression and ER stress.

ROCK2 knockdown alleviates LPS-induced inflammatory injury and apoptosis of renal tubular epithelial cells via the NF-κB/NLRP3 signaling pathway.

Rho-associated protein kinase 2 (ROCK2) is an important regulator of the inflammatory response and has been reported to serve a role in sepsis. The present study aimed to investigate whether ROCK2 served a role in sepsis-associated acute kidney injury (S-AKI). HK-2 cells were stimulated with lipopolysaccharide (LPS) to simulate S-AKI in vitro. Subsequently, the change in ROCK2 expression levels were determined. ROCK2 in LPS-induced HK-2 cells was knocked down using short hairpin RNA-ROCK2, in the absence or presence of phorbol 12-myristate 13-acetate (PMA), an activator of NF-κB. Cell viability, cytotoxicity, inflammation and apoptosis were assessed using MTT, lactate dehydrogenase (LDH) release, reverse transcription-quantitative PCR, ELISA, TUNEL and western blotting assays. The protein expression levels of proteins involved in the NF-κB/NLR family pyrin domain containing 3 (NLRP3) signaling pathway were also assessed using western blotting. The results demonstrated that ROCK2 was upregulated in HK-2 cells upon LPS treatment. LPS also reduced cell viability, promoted LDH activity and increased TNF-α, IL-6 and IL-1β mRNA expression levels and concentrations. Apoptosis was also induced by LPS as indicated by an increase in the proportion of TUNEL-positive cells, decreased Bcl-2 protein expression levels and increased cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase protein expression levels. However, ROCK2 knockdown in LPS-induced HK-2 cells reversed cell viability damage and inhibited LDH activity, the generation of pro-inflammatory cytokines and apoptosis caused by LPS. Furthermore, ROCK2 knockdown inhibited the LPS-induced expression of phosphorylated-NF-κB p65, NLRP3, apoptosis-associated speck-like protein containing a CARD and caspase-1 p20. PMA treatment reversed all the aforementioned effects of ROCK2 knockdown on LPS-treated HK-2 cells. Therefore, ROCK2 knockdown may alleviate LPS-induced HK-2 cell injury via the inactivation of the NF-κB/NLRP3 signaling pathway.

ZnT8 Exerts Anti-apoptosis of Kidney Tubular Epithelial Cell in Diabetic Kidney Disease Through TNFAIP3-NF-κB Signal Pathways.

Apoptosis of kidney tubular epithelial cells contributes to diabetic kidney disease (DKD) pathophysiology, but the mechanisms are not fully understood. Zinc transporter protein member 8 (ZnT8, SLC30A8) is a susceptive gene in diabetes. Here, we aim to investigate whether ZnT8 has effects on pathophysiology of DKD. The animal groups include control, ZnT8KO mice, STZ-induced, and ZnT8-KO-STZ. STZ-induced DKD was performed in male C57BL/6J mice and in ZnT8-KO mice. High glucose (HG)-induced apoptosis in a normal rat kidney tubular epithelial cell line (NRK-52E cells) was performed in vitro. Transfection of hZnT8-EGFP or TNFAIP3 siRNA was done in NRK-52E cells. Flow cytometry with Annexin V-FITC/PI double staining and TUNEL analysis was performed for the detection of apoptosis. Gene expression at mRNA and protein levels was examined with real-time RT-PCR and Western blot. Urine albumin to creatinine ratio, proinflammatory cytokines, and apoptosis were enhanced in kidneys of STZ and ZnT8-KO-STZ mice compared to control or ZnT8-KO mice. ZnT8 overexpression after hZnT8-EGFP transfection decreased HG-stimulated inflammatory activity and apoptosis in NRK-52E cells. Furthermore, treatment with ZnSO4 blunted HG-induced apoptosis and NF-κB activation. ZnSO4 increased the abundance of zinc-finger protein TNF-α-induced protein 3 (TNFAIP3). Also, ZnT8 over-expression after hZnT8-EGFP transfection significantly ameliorates HG-induced NF-κB-dependent transcriptional activity and apoptotic protein expressions in NRK-52E cells, but the inhibitory effect of ZnT8 was significantly abolished with TNFAIP3 siRNA. Our study provides evidence that ZnT8 has protective effects against apoptosis of renal tubular epithelial cells through induction of TNFAIP3 and subsequent suppression of the NF-κB pathway.

Class IIa histone deacetylase inhibition ameliorates acute kidney injury by suppressing renal tubular cell apoptosis and enhancing autophagy and proliferation.

Expression and function of histone deacetylases (HDACs) vary with cell types and pathological conditions. Our recent studies showed that pharmacological targeting class IIa HDACs attenuated renal fibrosis, but the effect of class IIa HDAC inhibition on acute kidney injury (AKI) remains unknown. In this study, we found that four class IIa HDACs (4, 5, 7, 9) were highly expressed in the kidney of folic acid (FA) and ischemia/reperfusion (I/R)-induced AKI in mice. Administration of TMP269, a potent and selective class IIa HDAC inhibitor, improved renal function and reduced tubular cell injury and apoptosis, with concomitant suppression of HDAC4 and elevation of acetyl-histone H3. Mechanistical studies showed that TMP269 treatment inhibited FA and I/R-induced caspase-3 cleavage, Bax expression and p53 phosphorylation. Conversely, TMP269 administration preserved expression of E-cadherin, BMP7, Klotho and Bcl-2 in injured kidneys. Moreover, TMP269 was effective in promoting cellular autophagy as indicated by increased expression of Atg7, beclin-1, and LC3II, and promoted renal tubular cell proliferation as shown by increased number of proliferating cell nuclear antigen-positive cells and expression of cyclin E. Finally, blocking class IIa HDACs inhibited FA-and I/R-induced phosphorylation of extracellular signal-regulated kinases 1 and 2, and p38, two signaling pathways associated with the pathogenesis of AKI. Collectively, these results suggest that pharmacological inhibition of class IIa HDACs protects against AKI through ameliorating apoptosis, enhancing autophagy and promoting proliferation of renal tubular cells by targeting multiple signaling pathways.

Folic acid-targeted pluronic F127 micelles improve oxidative stress and inhibit fibrosis for increasing AKI efficacy

The oxidative stress and activation of the fibrosis pathway are essential pathological mechanisms of acute kidney injury (AKI). In this article, we designed a drug delivery system that could effectively improve oxidative stress and relieve fibrosis by the combination of precise targeting, solubilization, and reducing the toxicity of nano-transport system to strengthen the efficacy of AKI. Folic acid (FA) was used as the targeting molecule, and curcumin (Cur) and resveratrol (Res), which are Chinese medicine monomers with anti-inflammatory and antioxidant effects, were used as model drugs. Here, the targeting nanosystem (Cur/Res@FA-F127/TPGS) co-loaded with Cur and Res was successfully synthesized. Finally, the comprehensive therapeutic effect of the nanosystem was evaluated through the targeted and pharmacodynamic researches on the AKI models induced by cisplatin (CDDP) in vitro and in vivo. The studies in vitro proved that the nanosystem could not only specifically target HK-2 cells and promote the effective accumulation of Cur and Res in the kidney, but also effectively improve oxidative stress by eliminating reactive oxygen species (ROS), stabilizing mitochondrial membrane potential (MMP), and reducing the expression of apoptosis-related proteins. The studies in vivo showed that the nanosystem could effectively play the role of anti-oxidation, anti-inflammatory and alleviate fibrosis to reduce the apoptosis and necrosis of renal tubular cells. The nanosystem could coordinately repair damaged HK-2 cells by improving oxidative stress, inhibiting inflammation and tissue fibrosis, which provided a new idea for the treatment of AKI.

Secretory leukocyte peptidase inhibitor inhibits the inflammatory response and apoptosis of lipopolysaccharide-induced human proximal renal tubular cells

To screen out the potential key genes of sepsis-associated acute kidney injury (AKI), and provide theoretical and experimental evidence for the treatment of sepsis-associated AKI. (1) Bioinformatics analysis: two gene expression datasets (GSE30718 and GSE53773) were downloaded for bioinformatics analysis from the Gene Expression Omnibus (GEO). These two datasets recorded mRNA microarray data from kidney biopsies before and after kidney transplantation, and a subset of patients developed AKI after kidney transplantation. Differential analysis was conducted, and the genes with the same differential expression and a higher area under the receiver operator characteristic curve (AUC) in both databases were used as the target gene for subsequent cell experiments. (2) Cell validation experiment: human proximal renal tubular cells HK2 were cultured in vitro, and lipopolysaccharide (LPS) was used for establishing LPS-HK2 cell model (LPS 10 mg/L for 6 hours, LPS model group), and the blank control group was set. Then, small interfering RNA (siRNA) technology was used to knock down the target gene obtained by bioinformatics analysis in LPS-HK2 cells (gene knockdown group), and a gene negative control group was set. The real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-qPCR) technique was used to detect the expression of the target gene in HK2 cells. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors in the cell supernatants. Western blotting was used to detect the expressions of key apoptosis proteins. (1) Results of bioinformatics analysis: 325 genes in the two datasets showed the same expression trend, of which 144 were significantly down-regulated and 181 were significantly up-regulated, while the expression difference of secretory leukocyte protease inhibitor (SLPI) in the two datasets was both statistically significant. Further receiver operator characteristic curve (ROC curve) analysis confirmed that the SLPI expression in GSE30718 and GSE53773 datasets had a high diagnostic efficiency for AKI, with AUC of 0.83 and 0.92, respectively. Therefore, SLPI was selected as the target gene for subsequent cell validation experiment. (2) Cell validation experiment: the RT-qPCR analysis showed that the expression of SLPI in LPS-HK2 cells of the LPS model group was significantly higher than that of the blank control group (2-ΔΔCT: 1.80±0.14 vs. 1.00±0.11, P < 0.01), and the change trend was the same with the results of bioinformatics analysis. Furthermore, knockdown SLPI gene analysis showed that the levels of inflammatory factors in LPS-HK2 cells supernatants in the gene knockdown group were significantly higher than those in the negative control group [Interleukin-6 (IL-6, ng/L): 509.58±27.08 vs. 253.87±75.83, IL-1β (ng/L): 490.99±49.52 vs. 239.67±26.97, tumor necrosis factor-α (TNF-α, ng/L): 755.22±48.66 vs. 502.06±10.92, all P < 0.01]. The above results indicated that SLPI could inhibit the inflammatory response of HK2 cells induced by LPS. The expressions of key apoptosis proteins Bax and caspase-3 in LPS-HK2 cells in the gene knockdown group were significantly higher than those in the negative control group [Bax protein (Bax/GAPDH): 1.38±0.12 vs. 1.00±0.10, caspase-3 protein (caspase-3/GAPDH): 1.44±0.15 vs. 1.00±0.11, both P < 0.05], and Bcl-2 expression was significantly decreased (Bcl-2/GAPDH: 0.83±0.08 vs. 1.00±0.05, P < 0.05), the above results indicated that SLPI could inhibit the apoptosis of cells in the inflammatory response. SLPI can inhibit the inflammatory response and apoptosis of HK2 cells induced by LPS, which may be involved in the protective mechanism of renal tubular cells in the response to sepsis, and is a potential target for the treatment of sepsis-associated AKI.

A Chinese Medicine Compound Alleviates Cisplatin-Induced Acute Kidney Injury via Its Antiapoptosis and Anti-Inflammation Effects in Mice.

Cisplatin, also known as cis-diamine dichloroplatinum (CDDP), is a widely used chemotherapeutic drug. However, its application is limited by the occurrence of serious nephrotoxicity. Currently, no effective therapy is available for combating CDDP-induced acute kidney injury (AKI). In the present study, we investigated the efficacy of Jianpi Yishen Tang (JPYST), a traditional Chinese medicine (TCM) compound commonly used to treat chronic kidney disease, against CDDP-induced AKI. In the CDDP + JPYST group, male mice were pretreated with JPYST (18.35 g/kg/day) for 5 consecutive days before receiving a single dose of CDDP (20 mg/kg), all mice were sacrificed 72 h after the CDDP injection. Results showed that JPYST suppressed CDDP-induced kidney dysfunction and tubular damage scores in the mice. Mechanistically, JPYST treatment attenuated CDDP-induced renal tubular cell apoptosis in AKI mice, as manifested by a marked decreased in TUNEL-positive cell counts, downregulation of the pro-apoptotic proteins Bax, Bad and caspase 3, and upregulation of the antiapoptotic protein Bcl-2 in kidney tissues. Meanwhile, JPYST decreased the expression of inflammatory cytokines TNF-α, IL-1β, and IL-6 in the serum and renal tissues of mice following CDDP administration. These factors are involved in suppressing the activation of phospho-NF-κB p65 in tubular epithelial cells. Taken together, these findings demonstrated that JPYST exerts renoprotective effects against CDDP-induced AKI through antiapoptosis and anti-inflammation effects, and these are associated with downregulation of NF-κB activation. Therefore, JPYST has potential for development of treatment therapies against CDDP-induced AKI.

Pharmacological and Genetic Inhibition of HDAC4 Alleviates Renal Injury and Fibrosis in Mice.

Histone deacetylase 4 (HDAC4) has been shown to be involved in cell proliferation, differentiation, and migration and is associated with a variety of cancers. However, the role of HDAC4 in renal fibrogenesis and its mechanisms are unclear. We assessed the role of HDAC4 and possible mechanisms of fibrosis in a murine model of kidney injury induced by unilateral ureteral obstruction (UUO) using tasquinimod, a highly selective HDAC4 inhibitor, and knockout mice with depletion of HDAC4 in renal tubular cells. UUO injury resulted in increased expression of HDAC4 and fibrotic proteins fibronectin and α-smooth muscle actin, while treatment with tasquinimod or knockout of HDAC4 significantly reduced their expression. Pharmacological and genetic inhibition of HDAC4 also decreased tubular epithelial cell arrest in the G2/M phase of the cell cycle, expression of transforming growth factor-β1 and phosphorylation of Smad3, signal transducer and activator of transcription 3, and extracellular signal-regulated kinase 1/2 in the injured kidney. Moreover, tasquinimod treatment or HDAC4 deletion inhibited UUO-induced renal tubular cell injury and apoptosis as indicated by reduced expression of neutrophil gelatinase–associated lipocalin, Bax, and inhibition of caspase-3. Finally, administration of tasquinimod or knockdown of HDAC4 prevented injury-related repression of Klotho, a renoprotective protein. Our results indicate that HDAC4 is critically involved in renal tubular injury and fibrosis and suggest that HDAC4 is a potential therapeutic target for treatment of chronic fibrotic kidney disease.

Inhibition of PLK3 Attenuates Tubular Epithelial Cell Apoptosis after Renal Ischemia-Reperfusion Injury by Blocking the ATM/P53-Mediated DNA Damage Response.

Objective Renal ischemia–reperfusion (I/R) injury is a major cause of acute kidney injury (AKI) in transplanted kidneys. This study was aimed at exploring the role of PLK3 (polo-like kinase 3) in renal I/R injury, focusing on its relationship with oxidative stress-induced DNA damage and renal tubular epithelial cell (TEC) apoptosis. Methods TRAP-seq data from the development dataset GSE52004 and the validation dataset GSE121191 were analyzed using GEO2R. PLK3 overexpression plasmids and targeted silencing siRNAs were used in a model of hypoxia/reoxygenation (H/R) injury, and rAAV-9-PLK3-KD were administered to C57BL/6J mice exposed to I/R injury. The ATM-specific inhibitor KU-60019 was used to block the DNA damage response (DDR). Western blotting was performed to measure DDR- and apoptosis-associated protein expression. Cell viability was measured by CCK-8 reagent, and apoptosis was examined by flow cytometry and TUNEL assay. Furthermore, the fluorescent probes H2DCFH-DA and DHE were used to measure ROS production in vitro. The MDA level and SOD activity were measured to assess oxidative stress in vivo. KIM-1 staining and Scr and BUN were used to evaluate kidney injury. Results The mRNA and protein levels of PLK3 were markedly increased in the H/R injury and I/R injury models. GO terms showed that PLK3 was mainly involved in oxidative stress and DNA damage after renal I/R injury. Overexpression of PLK3 decreased cell viability and increased apoptosis. In contrast, targeted silencing of PLK3 expression decreased the Bax/Bcl-2 ratio by decreasing P53 phosphorylation, thereby reducing TEC apoptosis. Furthermore, KU-60019 reduced PLK3 activation and DDR-induced apoptosis, while overexpression of PLK3 reversed the mitigating effect of KU-60019 on TEC apoptosis. Similarly, rAAV-9-PLK3 KD mice exhibited a lower rate of TEC apoptosis and milder renal damage after I/R injury. Conclusion We demonstrate for the first time that PLK3 is involved in oxidative stress-induced DNA damage and TEC apoptosis in renal I/R injury. Inhibition of PLK3 attenuates TEC apoptosis after I/R injury by blocking the ATM/P53-mediated DDR. Therefore, PLK3 may serve as a potential therapeutic target for ischemic AKI.

Inhibition of polycomb repressive complex 2 by targeting EED protects against cisplatin-induced acute kidney injury.

Polycomb repressive complex 2 (PRC2) is a multicomponent complex with methyltransferase activity that catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3). Interaction of the epigenetic reader protein EED with EZH2, a catalytic unit of PRC, allosterically stimulates PRC2 activity. In this study, we investigated the role and underlying mechanism of the PRC2 in acute kidney injury (AKI) by using EED226, a highly selective PRC2 inhibitor, to target EED. Administration of EED226 improved renal function, attenuated renal pathological changes, and reduced renal tubular cell apoptosis in a murine model of cisplatin‐induced AKI. In cultured renal epithelial cells, treatment with either EED226 or EED siRNA also ameliorated cisplatin‐induced apoptosis. Mechanistically, EED226 treatment inhibited cisplatin‐induced phosphorylation of p53 and FOXO3a, two transcriptional factors contributing to apoptosis, and preserved expression of Sirtuin 3 and PGC1α, two proteins associated with mitochondrial protection in vivo and in vitro. EED226 was also effective in enhancing renal tubular cell proliferation, suppressing expression of multiple inflammatory cytokines, and reducing infiltration of macrophages to the injured kidney. These data suggest that inhibition of the PRC2 activity by targeting EED can protect against cisplatin‐induced AKI by promoting the survival and proliferation of renal tubular cells and inhibiting inflammatory response.

Ferrostatin‑1 alleviates oxalate‑induced renal tubular epithelial cell injury, fibrosis and calcium oxalate stone formation by inhibiting ferroptosis.

The present study aimed to evaluate the role and mechanism of ferrostatin-1 (Fer-1) in oxalate (Ox)-induced renal tubular epithelial cell injury, fibrosis, and calcium oxalate (CaOx) stone formation. A CaOx model in mice kidneys was established via intraperitoneal injection of 80 mg/kg glyoxylic acid for 14 days. The mice were randomly divided into three groups (n=6), namely, the control (Con), the CaOx group, and the CaOx + Fer-1 group. Cultured human renal tubular epithelial cells (HK-2 cells) were randomly divided into three groups (n=3), namely, the control (Con), the Ox group, and the Ox + Fer-1 group. The levels of heme oxygenase 1 (HO-1), superoxide dismutase 2 (SOD2), glutathione peroxidase 4 (GPX4), and solute carrier family 7 member 11 (SLC7A11) were assessed by immunofluorescence and western blot analysis. Renal tubular injury and apoptosis were evaluated by H&E and TUNEL staining. Kidney interstitial fibrosis was evaluated by Masson and Sirius red staining, and the levels of E-cadherin, vimentin and α-SMA were detected by immunofluorescence or western blot analysis. Mitochondrial structure was observed using a transmission electron microscope. The levels of reactive oxygen species (ROS) were determined by flow cytometry and CaOx stone formation was evaluated by von Kossa staining. The results revealed that in comparison with the Con group, mitochondrial injury under glyoxylic acid treatment was observed by TEM. The expression of GPX4 and SLC7A11 in the CaOx and Ox groups was downregulated (P<0.05), whereas the expression of HO-1 and SOD2 was upregulated (P<0.05). Renal tissue damage, apoptosis of renal tubular epithelial cells, and interstitial fibrosis were increased in the CaOx and Ox groups (P<0.05). In comparison with the CaOx or Ox group, the expression of GPX4 and SLC7A11 in the CaOx + Fer-1 or Ox + Fer-1 group was upregulated (P<0.05), whereas that of HO-1 and SOD2 was downregulated (P<0.05). Renal tissue damage, apoptosis of renal tubular epithelial cells and interstitial fibrosis were decreased following Fer-1 treatment (P<0.05). The ROS level was also decreased following Fer-1 treatment. Moreover, CaOx stone formation was decreased in the CaOx + Fer-1 group (P<0.05). In conclusion, Fer-1 alleviated Ox-induced renal tubular epithelial cell injury, fibrosis, and CaOx stone formation by inhibiting ferroptosis.