Circular RNA mediates cardiomyocyte death via miRNA-dependent upregulation of MTP18 expression

Background/Aims: The aim of our study is to investigate the molecular mechanism by which mammalian STE20-like kinase 1 (Mst1) participates in renal I/R injury through modifying mitophagy and the AMPK-YAP signalling pathway. Methods: WT mice and Mst1-knockout mice were subjected to renal ischaemia-reperfusion (I/R) in vivo. In vitro, the hypoxia-reoxygenation model was used with renal tubular epithelial cells to mimic renal I/R injury. Mitochondrial function was monitored via western blotting and immunofluorescence. Pathway blocker and siRNA knockout technology were used to establish the role of the AMPK-YAP signalling pathway in Mst1-mediated mitochondrial apoptosis in the setting of renal I/R injury. Results: Our data demonstrated that Mst1 expression was upregulated in response to renal I/R injury in vivo, and a higher Mst1 content was positively associated with renal dysfunction and more tubular epithelial cell apoptosis. However, genetic ablation of Mst1 improved renal function, alleviated reperfusion-mediated tubular epithelial cell apoptosis, and attenuated the vulnerability of kidney to I/R injury. In vitro, Mst1 upregulation induced mitochondrial damage including mitochondrial potential reduction, ROS overloading, cyt-c liberation and caspase-9 apoptotic pathway activation. At the molecular levels, I/R-mediated mitochondrial damage via repressing mitophagy and Mst1 suppressed mitophagy via inactivating AMPK signalling pathway and dowregulating OPA1 expression. Re-activation of AMPK-YAP-OPA1 signalling pathway provided a survival advantage for the tubular epithelial cell in the context of renal I/R injury by repressing mitochondrial fission. Conclusion: Overall, our results demonstrate that the pathogenesis of renal I/R injury is closely associated with an increase in Mst1 expression and the inactive AMPK-YAP-OPA1 signalling pathway. Based on this, strategies to repress Mst1 expression and activate mitophagy could serve as therapeutic targets to treat kidney ischaemia-reperfusion injury.

Section: Methodsmentioning

confidence: 99%

Mammalian STE20-Like Kinase 1 Deletion Alleviates Renal Ischaemia-Reperfusion Injury via Modulating Mitophagy and the AMPK-YAP Signalling Pathway

Feng¹,

Li²,

Zhang³

et al. 2018

“…Samples were resolved by 10% SDS-PAGE. Electroblotting apparatus was used to transfer the proteins to PVDF membrane by operating the apparatus at 85V for 60 min using transfer buffer [37]. After transfer, the membrane was blocked by 5% non fat dried milk in Tris buffered saline and then incubated with the respective antibody for overnight at 4⁰C.…”

Section: Western Blot Analysismentioning

confidence: 99%

NR4A1 Promotes Diabetic Nephropathy by Activating Mff-Mediated Mitochondrial Fission and Suppressing Parkin-Mediated Mitophagy

Sheng

Dai

et al. 2018

Background/Aims: Disrupted mitochondrial dynamics, including excessive mitochondrial fission and mitophagy arrest, has been identified as a pathogenic factor in diabetic nephropathy (DN), although the upstream regulatory signal for mitochondrial fission activation and mitophagy arrest in the setting of DN remains unknown. Methods: Wild-type (WT) mice and NR4A1 knockout (NR4A1-KO) mice were used to establish a DN model. Mitochondrial fission and mitophagy were evaluated by western blotting and immunofluorescence. Mitochondrial function was assessed by JC-1 staining, the mPTP opening assay, immunofluorescence and western blotting. Renal histopathology and morphometric analyses were conducted via H&E, Masson and PASM staining. Kidney function was evaluated via ELISA, western blotting and qPCR. Results: In the present study, we found that nuclear receptor subfamily 4 group A member 1 (NR4A1) was actually activated by a chronic hyperglycemic stimulus. Higher NR4A1 expression was associated with glucose metabolism disorder, renal dysfunction, kidney hypertrophy, renal fibrosis, and glomerular apoptosis. At the molecular level, increased NR4A1 expression activated p53, and the latter selectively stimulated mitochondrial fission and inhibited mitophagy by modulating Mff and Parkin transcription. Excessive Mff-related mitochondrial fission caused mitochondrial oxidative stress, promoted mPTP opening, exacerbated proapoptotic protein leakage into the cytoplasm, and finally initiated mitochondria-dependent cellular apoptosis in the setting of diabetes. In addition, defective Parkin-mediated mitophagy repressed cellular ATP production and failed to correct the uncontrolled mitochondrial fission. However, NR4A1 knockdown interrupted the Mff-related mitochondrial fission and recused Parkin-mediated mitophagy, reducing the hyperglycemia-mediated mitochondrial damage and thus improving renal function. Conclusion: Overall, we have shown that NR4A1 functions as a novel malefactor in diabetic renal damage and operates by synchronously enhancing Mff-related mitochondrial fission and repressing Parkin-mediated mitophagy. Thus, finding strategies to regulate the balance of the NR4A1-p53 signaling pathway and mitochondrial homeostasis may be a therapeutic option for treating diabetic nephropathy in clinical practice.

“…After the cells were washed with cold PBS three times, the cells were collected using 0.25% pancreatin. After resuspension in cold PBS, the cells were analyzed using a flow cytometer (BD FACSVerse; BD Biosciences, San Jose, CA, USA) [34].…”

Section: Mitochondrial Function Analysismentioning

confidence: 99%

Hyperglycaemia Stress-Induced Renal Injury is Caused by Extensive Mitochondrial Fragmentation, Attenuated MKP1 Signalling, and Activated JNK-CaMKII-Fis1 Biological Axis

Zhang¹,

Feng²,

Wang³

et al. 2018

Background/Aims: Hyperglycaemia stress-induced renal injury is closely associated with mitochondrial dysfunction through poorly understood mechanisms. The aim of our study is to explore the upstream trigger and the downstream effector driving diabetic nephropathy via modulating mitochondrial homeostasis. Methods: A diabetic nephropathy model was generated in wild-type (WT) mice and MAP Kinase phosphatase 1 transgenic (MKP1-TG) mice using STZ injection. Cell experiments were conducted via high-glucose treatment in the human renal mesangial cell line (HRMC). MKP1 overexpression assay was carried out via adenovirus transfection. Renal function was evaluated via ELISA, western blotting, histopathological staining, and immunofluorescence. Mitochondrial function was determined via mitochondrial potential analysis, ROS detection, ATP measurement, mitochondrial permeability transition pore (mPTP) opening evaluation, and immunofluorescence for mitochondrial pro-apoptotic factors. Loss- and gain-of-function assays for mitochondrial fragmentation were performed using a pharmacological agonist and blocker. Western blotting and the pathway blocker were used to establish the signalling pathway in response to MKP1 overexpression in the presence of hyperglycaemia stress. Results: MKP1 was downregulated in the presence of chronic high-glucose stress in vivo and in vitro. However, MKP1 overexpression improved the metabolic parameters, enhanced glucose control, sustained renal function, attenuated kidney oxidative stress, inhibited the renal inflammation response, alleviated HRMC apoptosis, and repressed tubulointerstitial fibrosis. Molecular investigation found that MKP1 overexpression enhanced the resistance of HRMC to the hyperglycaemic injury by abolishing mitochondrial fragmentation. Hyperglycaemia-triggered mitochondrial fragmentation promoted mitochondrial dysfunction, as evidenced by decreased mitochondrial potential, elevated mitochondrial ROS production, increased pro-apoptotic factor leakage, augmented mPTP opening and activated caspase-9 apoptotic pathway. Interestingly, MKP1 overexpression strongly abrogated mitochondrial fragmentation and sustained mitochondrial homeostasis via inhibiting the JNK-CaMKII-Fis1 pathway. After re-activation of the JNK-CaMKII-Fis1 pathway, the beneficial effects of MKP1 overexpression on mitochondrial protection disappeared. Conclusion: Taken together, our data identified the protective role played by MKP1 in regulating diabetic renal injury via repressing mitochondrial fragmentation and inactivating the JNK-CaMKII-Fis1 pathway, which may pave the road to new therapeutic modalities for the treatment of diabetic nephropathy.