WWP2 deletion aggravates acute kidney injury by targeting CDC20/autophagy axis.

INTRODUCTION: Acute kidney injury (AKI) is associated with high morbidity and mortality rates. The molecular mechanisms underlying AKI are currently being extensively investigated. WWP2 is an E3 ligase that regulates cell proliferation and differentiation. Whether WWP2 plays a regulatory role in AKI remains to be elucidated. OBJECTIVES: We aimed to investigate the implication of WWP2 in AKI and its underlying mechanism in the present study. METHODS: We utilized renal tissues from patients with AKI and established AKI models in global or tubule-specific knockout (cKO) mice strains to study WWP2's implication in AKI. We also systemically analyzed ubiquitylation omics and proteomics to decipher the underlying mechanism. RESULTS: In the present study, we found that WWP2 expression significantly increased in the tubules of kidneys with AKI. Global or tubule-specific knockout of WWP2 significantly aggravated renal dysfunction and tubular injury in AKI kidneys, whereas WWP2 overexpression significantly protected tubular epithelial cells against cisplatin. WWP2 deficiency profoundly affected autophagy in AKI kidneys. Further analysis with ubiquitylation omics, quantitative proteomics and experimental validation suggested that WWP2 mediated poly-ubiquitylation of CDC20, a negative regulator of autophagy. CDC20 was significantly decreased in AKI kidneys, and selective inhibiting CDC20 with apcin profoundly alleviated renal dysfunction and tubular injury in the cisplatin model with or without WWP2 cKO, indicating that CDC20 may serve as a downstream target of WWP2 in AKI. Inhibiting autophagy with 3-methyladenine blocked apcin's protection against cisplatin-induced renal tubular cell injury. Activating autophagy by rapamycin significantly protected against cisplatin-induced AKI in WWP2 cKO mice, whereas inhibiting autophagy by 3-methyladenine further aggravated apoptosis in cisplatin-exposed WWP2 KO cells. CONCLUSION: Taken together, our data indicated that the WWP2/CDC20/autophagy may be an essential intrinsic protective mechanism against AKI. Further activating WWP2 or inhibiting CDC20 may be novel therapeutic strategies for AKI.

NSC228155 alleviates septic cardiomyopathy via protecting mitochondria and inhibiting inflammation.

Septic cardiomyopathy is a lethal symptom of sepsis. Discovery of effective therapy that prevents cardiac injury in sepsis is critical in the clinical management of sepsis. NSC228155 is a novel compound with therapeutic potential on acute kidney injury by preventing apoptosis and protecting mitochondria. Whether NSC228155 protects against septic cardiomyopathy is unclear. In the present study, adult C57BL/6J mice were i.p injected with 5 mg/kg/day NSC228155 for 2 days before 10 mg/kg lipopolysaccharide (LPS) injection. Cardiac functional testing and sampling for serum and tissue were performed 12 and 24 h post LPS injection, respectively. NSC228155 significantly improved cardiac function examined by echocardiography, decreased the serum lactate dehydrogenase (LDH) and creatine kinase-MB, and pathologically alleviated cardiac injury in LPS mice. Accordingly, NSC228155 attenuated cardiomyocytes' mitochondrial damage as shown by decreased damaged mitochondrial ratio and activated signals for mitochondrial biogenesis, dynamics and mitophagy in LPS mice model. Metabolomics analysis demonstrated that NSC228155 corrected the metabolic disturbance involved in oxidative stress and energy metabolism, and decreased tissue injury metabolites in LPS-stimulated cardiac tissue. In the LPS-stimulated cardiac cell culture derived from human induced pluripotent stem cells, NSC228155 effectively restored the beating frequency, decreased LDH release, and protected mitochondria. NSC228155 also inhibited inflammation shown by decreased pro-inflammatory mediators in both serum and cardiac tissue in LPS model. Taken together, NSC228155 significantly improved cardiac function by directly preventing against cardiac cell injury and inhibiting inflammation in LPS model, hence may be a potential novel therapy against septic cardiomyopathy.

Maintaining homeostasis of mitochondria and endoplasmic reticulum with NSC228155 alleviates cisplatin-induced acute kidney injury.

Acute kidney injury (AKI) is a common complication of hospitalization with high mortality. Approximately 30% of patients receiving cisplatin, the first-line chemotherapy treatment, develop AKI. NSC228155 is a novel compound with potential anti-cancer and anti-bacterial effects. Its therapeutic efficacy in other diseases is unclear. In the present study, we investigated the effect of NSC228155 on cisplatin-induced AKI. The mice were consecutively treated with 2.5 mg/kg of NSC228155 for five days and injected with cisplatin (22 mg/kg) via intraperitoneal injection on day three. NSC228155 strikingly improved the renal function by decreasing the serum creatinine by 52.6% in the cisplatin-induced AKI mice model. Pathologically, NSC228155 profoundly alleviated the tubular damage in Periodic Acid-Schiff staining, and significantly reduced the expression of tubular injury markers and apoptosis in the cisplatin-injured mice kidneys. NSC228155 effectively restored the mitochondrial homeostasis by decreasing damaged mitochondria, activating signals for mitochondrial dynamics and recycling, and corrected mitochondrial dysfunction in ATP production and oxidative stress in the cisplatin model. Transcriptomics and metabolomics analysis on the mice renal cortex suggested that NSC228155 profoundly corrected energy metabolism, especially citrate cycle-related pathways, oxidative stress, and endoplasmic reticulum (ER) stress in the cisplatin-induced AKI kidneys. NSC228155 effectively inhibited ER stress induced by cisplatin or tunicamycin in mice kidneys and HK-2 cells. Co-treatment of NSC228155 with 4-phenylbutyrate or MnTBAP showed a similar therapeutic effect in AKI as the inhibitors or NSC228155 alone did, and corrected the mitochondrial dysfunction and ER stress, respectively, indicating the crosstalk between ER and mitochondria played essential roles in the therapeutic effect of NSC228155 in AKI. Together, these results consistently demonstrated that NSC228155 alleviated cisplatin-induced AKI by restoring the homeostasis in mitochondria and ER, suggesting a therapeutic potential and perhaps a novel strategy for drug discovery.

Effects of autophagy on macrophage adhesion and migration in diabetic nephropathy.

Objective: Macrophage infiltration in kidney is a major pathological feature of diabetic nephropathy (DN), which has been demonstrated associate with macrophages autophagy homeostasis. However, the relationships between autophagy and the infiltration response related of macrophages adhesion and migration are unknown. This study aims to investigate the impact of macrophages adhesion and migration by modulating autophagy. Methods: In vivo, rats were randomly distributed into control (NC) and DN groups. The pathological changes in renal tissue were assessed, and expression of CD68, LC3, P62 were analyzed. In vitro, RAW264.7 cells were divided into NC and high glucose (HG) groups. The capacity of macrophages adhesion migration and the expression of autophagy markers were observed with and without autophagy modulators (rapamycin, 3-methyladenine, chloroquine, and bafilomycin A1 for RAPA, 3-MA, CQ, BAFA). The macrophages autophagosome and the process of degradation and fusion of autophagosome-lysosome were observed by electron microscopy. Results: In vivo, renal injury is aggravated in diabetic rat compared with NC group. The autophagy level is inhibited in renal tissues of DN group with the increasing expression of CD68 and P62, while expression level of LC3 decreased (p < .05). In vitro, HG and 3-MA reduce the numbers of autophagosome of macrophages to inhibit autophagy level with decrease expression of LC3 and Beclin-1, but increase expression of P62, which promote the adhesion and migration capacity of macrophages (p < .05). Moreover, CQ and BAFA suppress autophagy level by inhibiting the process of autophagosome-lysosome degradation and fusion of macrophages, as well as the expression of LC3 and Beclin-1. We notice an increase expression of P62 by CQ and BAFA stimulation (p < .05). CQ and BAFA further facilitate the adhesion and migration capacity of macrophages. However, RAPA increases the numbers of macrophages autophagosome that inhibited by HG, resulting in a recovery of autophagy level with increase expression of LC3 and Beclin-1, whereas a reduction expression of P62, which lead to inhibition of adhesion and migration of macrophages induced by HG (p < .05) Conclusions: High glucose efficiently reduced the level of macrophage autophagy, following macrophages adhesion and migration enhanced when autophagy is suppressed. Activation of autophagosome improve the level of autophagy, but leading to a reduction of the macrophages adhesion and migration. While, inhibiting the process of degradation and fusion of autophagosome-lysosome suppress the level of autophagy and promote the macrophages adhesion and migration. These results indicate that high glucose may play an important role in macrophages adhesion and migration through modulating autophagy activities in diabetic nephropathy.

Active vitamin D regulates macrophage M1/M2 phenotypes via the STAT-1-TREM-1 pathway in diabetic nephropathy.

AIM: Imbalance of M1/M2 macrophages phenotype activation is a key point in diabetic nephropathy (DN). This study aimed to investigate whether active vitamin D (VD) suppresses macrophage transition to the M1 phenotype via inhibiting the high glucose-induced STAT-1 phosphorylation to reduce TREM-1 expression. METHODS: In vivo, pathological changes in kidney tissue were detected and the expression of CD68 TREM-1, STAT-1, M1 makers, and M2 makers were acquired in renal tissue of patients with DN and 18w DN rats. In vitro, RAW 264.7 cells were incubated in the presence of high glucose with or without VD. Silencing and overexpression of TREM-1 and silencing and activate of STAT-1 were explored to elucidate the underlying mechanism. The expression of TREM-1 and STAT-1 and the changes of macrophage phenotype were examined separately by western blot and immunofluorescence staining. RESULTS: (a) Expression of TREM-1, p-STAT-1, and M1 markers (iNOS and TNF-α) were increased and positively correlated in kidneys from patients with DN. (b) In DN rats, the enlargement of glomerular surface area, expansion of glomerular mesangial matrix, the expression of CD68, TREM-1, p-STAT-1, and M1 marker (iNOS) were significantly increased in comparison with the normal control group, whereas above changes were markedly decreased in the diabetic group treated with the VD group. (c) In vitro, VD significantly decreased high glucose-induced CD68, TREM-1, p-STAT-1, and M1 marker (iNOS) expression. However, above-mentioned effects of VD are abolished when TREM-1 is overexpressed or STAT-1 is activated. Reductions in STAT-1 expression decreased the TREM-1 expression. CONCLUSION: VD can inhibit macrophage transition to the M1 phenotype through the STAT-1/TREM-1 pathway.

Vitamin D suppresses macrophage infiltration by down-regulation of TREM-1 in diabetic nephropathy rats.

This study intends to investigate the effect of active vitamin D (VD) on the expression of triggering receptor expressed on myeloid cells-1 (TREM-1) in the renal tissues of diabetic nephropathy (DN) rats and to explore the impact of TREM-1 on macrophage adhesion and migration. We find that the expressions of TREM-1 and CD68 protein are higher in DN rats compared with rats in the normal control group and that these changes are decreased in the DN + VD group. In vitro, the capacity for macrophage adhesion and migration and the expression of TREM-1 are increased under high-glucose conditions, but VD inhibits this progress. TREM-1 siRNA decreases high-glucose-induced macrophage adhesion and migration, whereas over-expression of TREM-1 inhibits its action. However, VD cannot suppress high glucose-induced TREM-1 expression and macrophage adhesion and migration when TREM-1 is over-expressed. These results demonstrate that VD can suppress macrophage adhesion and migration by reducing the expression of TREM-1.

Mitophagy regulates macrophage phenotype in diabetic nephropathy rats.

Imbalance of M1/M2 macrophages phenotype activation is a key point in diabetic nephropathy (DN). Macrophages mainly exhibit M1 phenotype, which contributes to the inflammation and fibrosis in DN. Studies indicate that autophage plays an important role in M1/M2 activation. However, the effect of mitophage on M1/M2 macrophage phenotype transformation in DN is unknown. This study investigates the role of mitophage on macrophage polarization in DN. In vivo experiments show that macrophages are exhibited to M1 phenotype and display a lower level of mitophagy in the kidney of streptozocin (STZ)-induced diabetic rats. Additionally, inducible nitric oxide synthase (iNOS) expression is positive correlated with the P62 expression, while negative correlated with LC3. Electronic microscope analysis shows mitochondria swelling, crista decrease and lysosome reduction in DN rats compared with NC rats. In vitro, RAW264.7 macrophages switch to M1 phenotype under high glucose conditions. Mitophagy is downregulated in such high glucose induced M1 macrophages. Furthermore, macrophages tend to switch to the M1 phenotype, expressing higher iNOS and TNF-α when impair mitophagy by 3-MA. Rapamycin, an activator of mitophagy, signifcantly blocks high-glucose induced M1 makers (iNOS and TNF-α) expression, meanwhile enhances M2 makers (MR and Arg-1) expression. These results demonstrate that mitophage participates in the regulation of M1/M2 macrophage phenotype in diabetic nephropathy.