FIGURE 6 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
| Deletion of PFKP aggravated HG-induced cytoskeletal remodeling in podocytes in vitro. (A) Western blots of PFKP expression after transfection with various siPfkps, with GAPDH set as the loading control. (B) PFK enzymatic activity and FBP level in each group. (C) Cytoskeletal structures of podocytes labeled with phalloidin and the cytoskeletal rearrangement ratio in each group (original magnification ×600). (D) Western blotting analyses of the expression of ROCK1, PFKP, and RhoA in podocytes of each group. GAPDH was set as the loading control. (E) Motility of podocytes measured by a wound-healing assay using an inverted microscope. For all figures, n=3 independent experiments/group. *P < 0.05, ns, No significance. Scale bars: 10 µm. HG, high glucose; ROCK1, Rhoassociated, coiled-coil containing protein kinase 1; PFKP, phosphofructokinase 1 platelet type; RhoA, RhoA-GTPase.
Source publication
Background
Glycolysis dysfunction is an important pathogenesis of podocyte injury in diabetic kidney disease (DKD). Foot process fusion of podocytes and increased albuminuria are markers of early DKD. Moreover, cytoskeletal remodeling has been found to be involved in the foot process fusion of podocytes. However, the connections between cytoskeleta...
Similar publications
Aims: To evaluate the effect of Topical antifungal Clotrimazole in Radiotherapy induced mucositis, burning sensation, xerostomia and loss of taste sensation. Study Design: Randomised Controlled Trial. Methodology: 64 patients (52 males and 12 females) undergoing Co 60 teletherapy for cervicofacial malignancies. Patients who received a total 60 Gray...
Citations
... When we reduced the generation of oxidized cardiolipin by knocking down ALCAT1 or using SS-31, the structure of podocyte mitochondria was more stable, and the indicators related to mitophagy and apoptosis were significantly improved. Glomerular disease is associated with mitochondrial fragmentation in podocytes [46,51]. Our studies have demonstrated that mitochondrial dynamic homeostasis in podocytes plays a crucial role in the progression of DKD and hypertensive nephropathy [52]. ...
Background
Cardiolipin (CL) plays a critical role in maintaining mitochondrial membrane integrity and overall mitochondrial homeostasis. Recent studies have suggested that mitochondrial damage resulting from abnormal cardiolipin remodelling is associated with the pathogenesis of diabetic kidney disease (DKD). Acyl-coenzyme A:lyso-cardiolipin acyltransferase-1 (ALCAT1) was confirmed to be involved in the progression of Parkinson’s disease, diet-induced obesity and other ageing-related diseases by regulating pathological cardiolipin remodelling. Thus, the purpose of this investigation was to determine the role of ALCAT1-mediated CL remodelling in DKD and to explore the potential underlying mechanism.
Methods
In vivo study, the mitochondrial structure was examined by transmission electron microscopy (TEM). The colocalization of ALCAT1 and synaptopodin was evaluated by double immunolabelling. Western blotting (WB) was performed to assess ALCAT1 expression in glomeruli. Lipidomics analysis was conducted to evaluate the composition of reconstructed cardiolipins. In vitro study, the lipidomics, TEM and WB analyses were similar to those in vivo. Mitochondrial function was evaluated by measuring the mitochondrial membrane potential (MMP) and the production of ATP and ROS.
Results
Here, we showed that increased oxidized cardiolipin (ox-CL) and significant mitochondrial damage were accompanied by increased ALCAT1 expression in the glomeruli of patients with DKD. Similar results were found in db/db mouse kidneys and in cultured podocytes stimulated with high glucose (HG). ALCAT1 deficiency effectively prevented HG-induced ox-CL production and mitochondrial damage in podocytes. In contrast, ALCAT1 upregulation enhanced ox-CL levels and podocyte mitochondrial dysfunction. Moreover, treatment with the cardiolipin antioxidant SS-31 markedly inhibited mitochondrial dysfunction and cell injury, and SS-31 treatment partly reversed the damage mediated by ALCAT1 overexpression. We further found that ALCAT1 could mediate the key regulators of mitochondrial dynamics and mitophagy through the AMPK pathway.
Conclusions
Collectively, our studies demonstrated that ALCAT1-mediated cardiolipin remodelling played a crucial role in DKD, which might provide new insights for DKD treatment.
... In the glomerulus, podocytes exhibit a strong preference for anaerobic glycolysis and are almost independent of mitochondrial OXPHOS to generate energy [41]. Indeed, it has been reported that the expression of PFKP is increased in the glomeruli of patients with DKD, and PFKP activation ameliorates podocyte cytoskeletal remodeling in podocytes through regulation of F-1,6-BP levels and inhibition of the Ras homology A/Rho-associated kinase 1 (RhoA/ROCK1) pathway in vitro [42]. Exogenous F-1,6-BP addition reduced foot process fusion and renal damage in db/db mice. ...
... Exogenous F-1,6-BP addition reduced foot process fusion and renal damage in db/db mice. These findings provide evidence that PFKP may be a potential target for podocyte injury in DKD [42]. In contrast to podocytes, PTECs rely almost entirely on mitochondrial FAO to generate energy [41]. ...
Metabolic reprogramming to glycolysis is closely associated with the development of chronic kidney disease (CKD). Although it has been reported that phosphofructokinase 1 (PFK) is a rate-limiting enzyme in glycolysis, the role of the platelet isoform of PFK (PFKP) in kidney fibrosis initiation and progression is as yet poorly understood. Here, we investigated whether PFKP could mediate the progression of kidney interstitial fibrosis by regulating glycolysis in proximal tubular epithelial cells (PTECs). We induced PFKP overexpression or knockdown in renal tubules via an adeno-associated virus (AAV) vector in the kidneys of mice following unilateral ureteral occlusion. Our results show that the dilated tubules, the area of interstitial fibrosis, and renal glycolysis were promoted by proximal tubule-specific overexpression of PFKP, and repressed by knockdown of PFKP. Furthermore, knockdown of PFKP expression restrained, while PFKP overexpression promoted TGF-β1-induced glycolysis in the human PTECs line. Mechanistically, Chip-qPCR revealed that TGF-β1 recruited the small mothers against decapentaplegic (SMAD) family member 3-SP1 complex to the PFKP promoter to enhance its expression. Treatment of mice with isorhamnetin notably ameliorated PTEC-elevated glycolysis and kidney fibrosis. Hence, our results suggest that PFKP mediates the progression of kidney interstitial fibrosis by regulating glycolysis in PTECs.
... Diabetic kidney disease (DKD), also known as diabetic nephropathy, is one of the serious microvascular complications of diabetes and one of the key factors leading to end-stage renal disease (ESRD). 1 Due to the global prevalence of DKD, it has brought huge economic burden and serious health damage to human beings. The lesions of DKD mainly involve widening of mesangial matrix, glomerulosclerosis, loss of podocytes, tubular atrophy, apoptosis, renal inflammatory infiltration, and tubulointerstitial fibrosis (TIF). ...
Background:
The primary pathophysiology of diabetic kidney disease (DKD) is tubulointerstitial fibrosis (TIF), and an essential contributing element is excessive extracellular matrix deposition. Irisin is a polypeptide formed by splitting fibronectin type III domain containing 5 (FNDC5), which participates in a number of physiological and pathological processes.
Methods:
The purpose of this article is to examine irisin's function in DKD and analyze both its in vitro and in vivo effects. The Gene Expression Omnibus (GEO) database was used to download GSE30122, GSE104954, and GSE99325. Analysis of renal tubule samples from nondiabetic and diabetic mice identified 94 differentially expressed genes (DEGs). The transforming growth factor beta receptor 2 (TGFBR2), irisin, and TGF-β1 were utilized as DEGs to examine the impact of irisin on TIF in diabetic kidney tissue, according to the datasets retrieved from the GEO database and Nephroseq database. Additionally, the therapeutic impact of irisin was also examined using Western blot, RT-qPCR, immunofluorescence, immunohistochemistry, and kits for detecting mouse biochemical indices.
Results:
In vitro, the findings demonstrated that irisin not only down-regulated the expression of Smad4 and β-catenin but also reduced the expression of proteins linked to fibrosis, the epithelial-mesenchymal transition (EMT), and mitochondrial dysfunction in HK-2 cells maintained in high glucose (HG) environment. In vivo, overexpressed FNDC5 plasmid was injected into diabetic mice to enhance its expression. Our studies found that overexpressed FNDC5 plasmid not only reversed the biochemical parameters and renal morphological characteristics of diabetic mice but also alleviated EMT and TIF by inhibiting Smad4/β-catenin signaling pathway.
Conclusion:
The above experimental results revealed that irisin could reduce TIF in diabetic mice via regulating the Smad4/β-catenin pathway.
Diabetic nephropathy (DN), a destructive complication of diabetes mellitus (DM), is one of the leading causes of end-stage renal disease (ESRD). This study aimed to investigate the role of long non-coding RNA (lncRNA) MIAT in high-glucose (HG)-induced podocyte injury associated with DN.
Three human kidney podocyte (HKP) cultures were treated with HG to mimic DN. Expression of lncRNA MIAT, podocyte-specific and injury-related proteins, and apoptosis were assessed before and after MIAT knockdown using MIAT shRNAs.
MIAT expression was upregulated in HKPs in response to glucose stress. HG treatment resulted in a significant increase in the apoptotic rate, Bax level, and levels of injury-related proteins desmin, fibroblast-specific protein 1 (FSP-1), and smooth muscle α-actin (α-SMA), and a significant reduction in Bcl-2 levels and the levels of podocyte-specific proteins synaptopodin and podocin. Transfection of HKPs with shRNAs significantly reduced MIAT levels (p < 0.05) and attenuated apoptosis in HG-medium. Correspondingly, the levels of synaptopodin and podocin were upregulated, and desmin, FSP-1, and α-SMA were reduced (p < 0.05). Western blot analysis also showed that anti-apoptotic active caspase-3 and Bax and proapoptotic Bcl-2 were elevated and decreased, respectively, after MIAT knockdown, suggesting that apoptosis pathways are deactivated after MIAT downregulation.
High glucose upregulates MIAT level in HKPs and induces cellular injury. Knockdown of MIAT alleviates the injury likely via deactivating apoptosis pathways.
Diabetic kidney disease (DKD) can lead to accumulation of glucose upstream metabolites due to dysfunctional glycolysis. But the effects of accumulated glycolysis metabolites on podocytes in DKD remain unknown. The present study examined the effect of dihydroxyacetone phosphate (DHAP) on high glucose induced podocyte pyroptosis. By metabolomics, levels of DHAP, GAP, glucose‐6‐phosphate and fructose 1, 6‐bisphosphate were significantly increased in glomeruli of db/db mice. Furthermore, the expression of LDHA and PKM2 were decreased. mRNA sequencing showed upregulation of pyroptosis‐related genes ( Nlrp3 , Casp1 , etc.). Targeted metabolomics demonstrated higher level of DHAP in HG‐treated podocytes. In vitro, ALDOB expression in HG‐treated podocytes was significantly increased. siALDOB‐transfected podocytes showed less DHAP level, mTORC1 activation, reactive oxygen species (ROS) production, and pyroptosis, while overexpression of ALDOB had opposite effects. Furthermore, GAP had no effect on mTORC1 activation, and mTORC1 inhibitor rapamycin alleviated ROS production and pyroptosis in HG‐stimulated podocytes. Our findings demonstrate that DHAP represents a critical metabolic product for pyroptosis in HG‐stimulated podocytes through regulation of mTORC1 pathway. In addition, the results provide evidence that podocyte injury in DKD may be treated by reducing DHAP.
Clinicians have long been interested in understanding the molecular basis of diabetic kidney disease (DKD)and its potential treatment targets. Its pathophysiology involves protein phosphorylation, one of the most recognizable post-transcriptional modifications, that can take part in many cellular functions and control different metabolic processes. In order to recognize the molecular and protein changes of DKD kidney, this study applied Tandem liquid chromatography-mass spectrometry (LC-MS/MS) and Next-Generation Sequencing, along with Tandem Mass Tags (TMT) labeling techniques to evaluate the mRNA, protein and modified phosphorylation sites between DKD mice and model ones. Based on Gene Ontology (GO) and KEGG pathway analyses of transcriptome and proteome, The molecular changes of DKD include accumulation of extracellular matrix, abnormally activated inflammatory microenvironment, oxidative stress and lipid metabolism disorders, leading to glomerulosclerosis and tubulointerstitial fibrosis. Oxidative stress has been emphasized as an important factor in DKD and progression to ESKD, which is directly related to podocyte injury, albuminuria and renal tubulointerstitial fibrosis. A histological study of phosphorylation further revealed that kinases were crucial. Three groups of studies have found that RAS signaling pathway, RAP1 signaling pathway, AMPK signaling pathway, PPAR signaling pathway and HIF-1 signaling pathway were crucial for the pathogenesis of DKD. Through this approach, it was discovered that targeting specific molecules, proteins, kinases and critical pathways could be a promising approach for treating DKD.
