Dihydroxyacetone phosphate accumulation leads to podocyte pyroptosis in diabetic kidney disease.

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.

LRH-1 activation alleviates diabetes-induced podocyte injury by promoting GLS2-mediated glutaminolysis.

Alteration of metabolic phenotype in podocytes directly contributes to the development of albuminuria and renal injury in conditions of diabetic kidney disease (DKD). This study aimed to identify and evaluate liver receptor homologue-1 (LRH-1) as a possible therapeutic target that alleviates glutamine (Gln) metabolism disorders and mitigates podocyte injury in DKD. Metabolomic and transcriptomic analyses were performed to characterize amino acid metabolism changes in the glomeruli of diabetic mice. Next, Western blotting, immunohistochemistry assays, and immunofluorescence staining were used to detect the expression of different genes in vitro and in vivo. Furthermore, Gln and glutamate (Glu) content as well as ATP generation were examined. A decrease in LRH-1 and glutaminase 2 (GLS2) expression was detected in diabetic podocytes. Conversely, the administration of LRH-1 agonist (DLPC) upregulated the expression of GLS2 and promoted glutaminolysis, with an improvement in mitochondrial dysfunction and less apoptosis in podocytes compared to those in vehicle-treated db/db mice. Our study indicates the essential role of LRH-1 in governing the Gln metabolism of podocytes, targeting LRH-1 could restore podocytes from diabetes-induced disturbed glutaminolysis in mitochondria.

Compromised glycolysis contributes to foot process fusion of podocytes in diabetic kidney disease: Role of ornithine catabolism.

INTRODUCTION: Compromised glycolysis in podocytes contributes to the initiation of diabetic kidney disease (DKD). Podocyte injury is characterized by cytoskeletal remodeling and foot process fusion. Compromised glycolysis in diabetes likely leads to switch of energy supply in podocyte. However, the underlying mechanism by which disturbed energy supply in podocytes affects the cytoskeletal structure of podocytes remains unclear. METHODS: Metabolomic and transcriptomic analyses were performed on the glomeruli of db/db mice to examine the catabolism of glucose, fatty, and amino acids. Ornithine catabolism was targeted in db/db and podocyte-specific pyruvate kinase M2 knockout (PKM2-podoKO) mice. In vitro, expression of ornithine decarboxylase (ODC1) was modulated to investigate the effect of ornithine catabolism on mammalian target of rapamycin (mTOR) signaling and cytoskeletal remodeling in cultured podocytes. RESULTS: Multi-omic analyses of the glomeruli revealed that ornithine metabolism was enhanced in db/db mice compared with that in db/m mice under compromised glycolytic conditions. Additionally, ornithine catabolism was exaggerated in podocytes of diabetic PKM2-podoKO mice compared with that in diabetic PKM2flox/flox mice. In vivo, difluoromethylornithine (DFMO, inhibitor of ODC1) administration reduced urinary albumin excretion and alleviated podocyte foot process fusion in db/db mice. In vitro, 2-deoxy-d-glucose (2-DG) exposure induced mTOR signaling activation and cytoskeletal remodeling in podocytes, which was alleviated by ODC1-knockdown. Mechanistically, a small GTPase Ras homolog enriched in the brain (Rheb), a sensor of mTOR signaling, was activated by exposure to putrescine, a metabolic product of ornithine catabolism. CONCLUSION: These findings demonstrate that compromised glycolysis in podocytes under diabetic conditions enhances ornithine catabolism. The metabolites of ornithine catabolism contribute to mTOR signaling activation via Rheb and cytoskeletal remodeling in podocytes in DKD.

PFKP Activation Ameliorates Foot Process Fusion in Podocytes in Diabetic Kidney Disease.

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 cytoskeletal remodeling and alterations of glycolysis in podocytes in DKD have not been clarified. Methods: mRNA sequencing of glomeruli obtained from db/db and db/m mice with albuminuria was performed to analyze the expression profiling of genes in glucose metabolism. Expressions of phosphofructokinase platelet type (PFKP) in the glomeruli of DKD patients were detected. Clotrimazole (CTZ) was used to explore the renal effects of PFKP inhibition in diabetic mice. Using Pfkp siRNA or recombinant plasmid to manipulate PFKP expression, the effects of PFKP on high glucose (HG) induced podocyte damage were assessed in vitro. The levels of fructose-1,6-bisphosphate (FBP) were measured. Targeted metabolomics was performed to observe the alterations of the metabolites in glucose metabolism after HG stimulation. Furthermore, aldolase type b (Aldob) siRNA or recombinant plasmid were applied to evaluate the influence of FBP level alteration on podocytes. FBP was directly added to podocyte culture media. Db/db mice were treated with FBP to investigate its effects on their kidney. Results: mRNA sequencing showed that glycolysis enzyme genes were altered, characterized by upregulation of upstream genes (Hk1, and Pfkp) and down-regulation of downstream genes of glycolysis (Pkm, and Ldha). Moreover, the expression of PFKP was increased in glomeruli of DKD patients. The CTZ group presented more severe renal damage. In vitro, the Pfkp siRNA group and ALDOB overexpression group showed much more induced cytoskeletal remodeling in podocytes, while overexpression of PFKP and suppression of ALDOB in vitro rescued podocytes from cytoskeletal remodeling through regulation of FBP levels and inhibition of the RhoA/ROCK1 pathway. Furthermore, targeted metabolomics showed FBP level was significantly increased in HG group compared with the control group. Exogenous FBP addition reduced podocyte cytoskeletal remodeling and renal damage of db/db mice. Conclusions: These findings provide evidence that PFKP may be a potential target for podocyte injury in DN and provide a rationale for applying podocyte glycolysis enhancing agents in patients with DKD.