Reduction of anaerobic glycolysis contributes to angiotensin II-induced podocyte injury with foot process effacement

Activation of the renin-angiotensin system is associated with podocyte injury and has been well demonstrated as a pivotal factor in the progression of chronic kidney disease. Podocyte energy metabolism is crucial for maintaining their physiological functions. However, whether renin-angiotensin system activation promotes chronic kidney disease progression by disturbing the energy metabolism of podocytes has not been elucidated. Angiotensin II, the main active molecule of the renin-angiotensin system, plays a crucial role in chronic kidney disease initiation and progression, but its impact on podocyte metabolism remains unclear. Here, we demonstrate a rapid decrease in the expression of pyruvate kinase M2, a key glycolytic enzyme, and reduced glycolytic flux in podocytes exposed to angiotensin II in vivo and in vitro. Podocyte-specific deletion of pyruvate kinase M2 in mice aggravated angiotensin II-induced glomerular and podocyte injury with foot process effacement and proteinuria. The inhibition of glycolysis was accompanied by adenosine triphosphate deficiency, cytoskeletal remodeling and podocyte apoptosis. Mechanistically, we found that angiotensin II-induced glycolysis impairment contributed to an insufficient energy supply to the foot process, leading to podocyte injury. Additionally, pyruvate kinase M2 expression was found to be reduced in podocytes from kidney biopsies of patients with hypertensive nephropathy and diabetic kidney disease. Thus, our findings suggest that glycolysis activation is a potential therapeutic strategy for podocyte injury. Activation of the renin-angiotensin system is associated with podocyte injury and has been well demonstrated as a pivotal factor in the progression of chronic kidney disease. Podocyte energy metabolism is crucial for maintaining their physiological functions. However, whether renin-angiotensin system activation promotes chronic kidney disease progression by disturbing the energy metabolism of podocytes has not been elucidated. Angiotensin II, the main active molecule of the renin-angiotensin system, plays a crucial role in chronic kidney disease initiation and progression, but its impact on podocyte metabolism remains unclear. Here, we demonstrate a rapid decrease in the expression of pyruvate kinase M2, a key glycolytic enzyme, and reduced glycolytic flux in podocytes exposed to angiotensin II in vivo and in vitro. Podocyte-specific deletion of pyruvate kinase M2 in mice aggravated angiotensin II-induced glomerular and podocyte injury with foot process effacement and proteinuria. The inhibition of glycolysis was accompanied by adenosine triphosphate deficiency, cytoskeletal remodeling and podocyte apoptosis. Mechanistically, we found that angiotensin II-induced glycolysis impairment contributed to an insufficient energy supply to the foot process, leading to podocyte injury. Additionally, pyruvate kinase M2 expression was found to be reduced in podocytes from kidney biopsies of patients with hypertensive nephropathy and diabetic kidney disease. Thus, our findings suggest that glycolysis activation is a potential therapeutic strategy for podocyte injury. Translational StatementAngiotensin II, the main active molecule of the renin-angiotensin system, plays a crucial role in chronic kidney disease initiation and progression, but its impact on podocyte metabolism remains unclear. Herein, we demonstrated that the glycolysis-related enzyme pyruvate kinase M2 and glycolytic flux are significantly downregulated in podocytes in angiotensin II–induced kidney injury. We further showed that compromised glycolysis contributed to an insufficient energy supply to the foot process, leading to cytoskeletal remodeling and podocyte apoptosis. Our results provide new insight into the role of glycolysis in the treatment of podocyte injury. Angiotensin II, the main active molecule of the renin-angiotensin system, plays a crucial role in chronic kidney disease initiation and progression, but its impact on podocyte metabolism remains unclear. Herein, we demonstrated that the glycolysis-related enzyme pyruvate kinase M2 and glycolytic flux are significantly downregulated in podocytes in angiotensin II–induced kidney injury. We further showed that compromised glycolysis contributed to an insufficient energy supply to the foot process, leading to cytoskeletal remodeling and podocyte apoptosis. Our results provide new insight into the role of glycolysis in the treatment of podocyte injury. Proteinuria is a common clinical feature of chronic kidney disease.1Hahr A.J. Molitch M.E. Management of diabetes mellitus in patients with CKD: core curriculum 2022.Am J Kidney Dis. 2022; 79: 728-736Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar Urinary albumin leakage usually indicates glomerular injury and dysfunction.2Haraldsson B. A new era of podocyte-targeted therapy for proteinuric kidney disease.N Engl J Med. 2013; 369: 2453-2454Crossref PubMed Scopus (19) Google Scholar Among glomerular cells, podocyte injury and loss are closely related to glomerular filtration barrier damage.3Sun H. Li H. Yan J. et al.Loss of CLDN5 in podocytes deregulates WIF1 to activate WNT signaling and contributes to kidney disease.Nat Commun. 2022; 13: 1600Crossref PubMed Scopus (5) Google Scholar, 4Zhou D. Wang Y. Gui Y. et al.Non-canonical Wnt/calcium signaling is protective against podocyte injury and glomerulosclerosis.Kidney Int. 2022; 102: 96-107Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar, 5Nagata M. Podocyte injury and its consequences.Kidney Int. 2016; 89: 1221-1230Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar Activation of the renin-angiotensin system (RAS) is a major risk factor for the progression of hypertensive nephropathy (HN) and diabetic kidney disease (DKD).6Fu E.L. Evans M. Clase C.M. et al.Stopping renin-angiotensin system inhibitors in patients with advanced CKD and risk of adverse outcomes: a nationwide study.J Am Soc Nephrol. 2021; 32: 424-435Crossref PubMed Scopus (39) Google Scholar In addition to increasing glomerular pressure, angiotensin II (Ang Ⅱ), a major RAS effector, can directly lead to podocyte injury, but the exact mechanism by which this occurs remains unclear.7Yang Q. Hu J. Yang Y. et al.Sirt6 deficiency aggravates angiotensin II-induced cholesterol accumulation and injury in podocytes.Theranostics. 2020; 10: 7465-7479Crossref PubMed Scopus (22) Google Scholar,8Ma Y. Yang Q. Zhong Z. et al.Role of c-Abl and nephrin in podocyte cytoskeletal remodeling induced by angiotensin II.Cell Death Dis. 2018; 9: 185Crossref PubMed Scopus (19) Google Scholar The energy metabolism is the core of cell biology, and all organisms are inseparable from their energy supply.9Clark A.J. Parikh S.M. Targeting energy pathways in kidney disease: the roles of sirtuins, AMPK, and PGC1α.Kidney Int. 2021; 99: 828-840Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar As a high-energy demanding organ, the kidney is more likely to sustain injury during aberrant energy metabolism.10Zhu Z. Hu J. Chen Z. et al.Transition of acute kidney injury to chronic kidney disease: role of metabolic reprogramming.Metabolism. 2022; 131155194Abstract Full Text Full Text PDF Scopus (5) Google Scholar The energy is mainly generated by mitochondria-dependent oxidative phosphorylation and mitochondria-independent glycolysis. The above processes are exquisitely orchestrated to allow cells to survive under stress conditions.11Li Z. Lu S. Li X. The role of metabolic reprogramming in tubular epithelial cells during the progression of acute kidney injury.Cell Mol Life Sci. 2021; 78: 5731-5741Crossref PubMed Scopus (19) Google Scholar Previous studies mainly focused on the causes of podocyte injury from the perspective of substance metabolism and signaling pathway activation; however, whether RAS activation interferes with energy metabolism in podocytes remains unclear. A recent study showed that hypertension promoted renal metabolic insults with oxidative stress, and energy-equivalent metabolite depletion occurred mainly in the glomeruli but not the tubules.12Rinschen M.M. Palygin O. Guijas C. et al.Metabolic rewiring of the hypertensive kidney.Sci Signal. 2019; 12eaax9760Crossref PubMed Scopus (26) Google Scholar Our previous studies found that RAS activation decreased adenosine triphosphate (ATP) production in podocytes.13Zhu Z. Liang W. Chen Z. et al.Mitoquinone protects podocytes from angiotensin II-induced mitochondrial dysfunction and injury via the Keap1-Nrf2 signaling pathway.Oxid Med Cell Longev. 2021; 20211394486Crossref Scopus (16) Google Scholar These findings suggested that RAS activation may lead to podocyte damage by inducing abnormal energy metabolism. As a key driver of self-renewal and cell proliferation, glycolysis plays an important role in angiogenesis and tumor invasion.14Cantelmo A.R. Conradi L.C. Brajic A. et al.Inhibition of the glycolytic activator PFKFB3 in endothelium induces tumor vessel normalization, impairs metastasis, and improves chemotherapy.Cancer Cell. 2016; 30: 968-985Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar In recent years, the role of glycolysis in podocyte injury has attracted considerable attention.15Qi W. Keenan H.A. Li Q. et al.Pyruvate kinase M2 activation may protect against the progression of diabetic glomerular pathology and mitochondrial dysfunction.Nat Med. 2017; 23: 753-762Crossref PubMed Scopus (266) Google Scholar, 16Yuan Q. Miao J. Yang Q. et al.Role of pyruvate kinase M2-mediated metabolic reprogramming during podocyte differentiation.Cell Death Dis. 2020; 11: 355Crossref PubMed Scopus (21) Google Scholar, 17Fu J. Shinjo T. Li Q. et al.Regeneration of glomerular metabolism and function by podocyte pyruvate kinase M2 in diabetic nephropathy.JCI Insight. 2022; 7e155260Crossref Scopus (6) Google Scholar, 18Gordin D. Shah H. Shinjo T. et al.Characterization of glycolytic enzymes and pyruvate kinase M2 in type 1 and 2 diabetic nephropathy.Diabetes Care. 2019; 42: 1263-1273Crossref PubMed Scopus (53) Google Scholar Crucial findings were obtained from the Medalist study, which revealed that higher glycolysis flux protects against hyperglycemia-induced podocyte injury.15Qi W. Keenan H.A. Li Q. et al.Pyruvate kinase M2 activation may protect against the progression of diabetic glomerular pathology and mitochondrial dysfunction.Nat Med. 2017; 23: 753-762Crossref PubMed Scopus (266) Google Scholar In addition, Yang et al. reported that glycolytic flux significantly increased during podocyte differentiation.16Yuan Q. Miao J. Yang Q. et al.Role of pyruvate kinase M2-mediated metabolic reprogramming during podocyte differentiation.Cell Death Dis. 2020; 11: 355Crossref PubMed Scopus (21) Google Scholar The glycolysis provides intermediate metabolites for the biosynthesis of amino acids, lipids, and nucleic acids.19Lunt S.Y. Vander H.M. Aerobic glycolysis: meeting the metabolic requirements of cell proliferation.Annu Rev Cell Dev Biol. 2011; 27: 441-464Crossref PubMed Scopus (1902) Google Scholar In mature podocytes, higher glycolytic flux may be indispensable for the synthesis of slit diaphragm proteins.16Yuan Q. Miao J. Yang Q. et al.Role of pyruvate kinase M2-mediated metabolic reprogramming during podocyte differentiation.Cell Death Dis. 2020; 11: 355Crossref PubMed Scopus (21) Google Scholar However, whether glycolysis disorders are involved in Ang II–induced podocyte injury remains unknown. Herein, we investigated whether Ang II could induce glycolysis dysmetabolism in podocytes and explored how these changes affect podocyte fate. For details, please see Supplementary Complete Materials and Methods. All the animal studies were approved by the Committee on the Ethics of Animal Experiments of Renmin Hospital of Wuhan University. Podocyte-specific pyruvate kinase M2 (PKM2) deletion mice (PKM2fl/fl/Cre+) were generated by cross-breeding PKM2-floxed mice (Jackson Laboratory; strain 024048) with B6. Cg-Tg(NPHS2-cre)295Lbh/J mice (Jackson Laboratory; strain 008205). Littermates with homozygous floxed mice without Cre expression (PKM2fl/fl/Cre–) were used as controls. Eight-week-old PKM2fl/fl/Cre+ and PKM2fl/fl/Cre– mice were embedded by an osmotic mini-pump (Alzet model 2004), and randomly assigned to receive a normal saline or Ang II (700 ng/kg per minute; Sigma-Aldrich) infusion for 4 weeks.7Yang Q. Hu J. Yang Y. et al.Sirt6 deficiency aggravates angiotensin II-induced cholesterol accumulation and injury in podocytes.Theranostics. 2020; 10: 7465-7479Crossref PubMed Scopus (22) Google Scholar The mean serum Ang II concentration in Ang II (700 ng/kg per minute) infusion mice was 95.37 ± 9.82 pg/ml on day 28 (Supplementary Table S1). For pharmacologic treatment, the Ang II infusion mice received gavage administration of TEPP-46, a PKM2 activator (30 mg/kg biweekly for 4 weeks; MCE).15Qi W. Keenan H.A. Li Q. et al.Pyruvate kinase M2 activation may protect against the progression of diabetic glomerular pathology and mitochondrial dysfunction.Nat Med. 2017; 23: 753-762Crossref PubMed Scopus (266) Google Scholar In all of the experiments, only male mice were used. Twenty-four-hour urine samples were collected weekly for biochemical analysis from individual mice via metabolic cages (Tecniplast). Primary podocytes were isolated as previously described.20Liang W. Yamahara K. Hernando-Erhard C. et al.A reciprocal regulation of spermidine and autophagy in podocytes maintains the filtration barrier.Kidney Int. 2020; 98: 1434-1448Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar Briefly, glomeruli were collected from mT/mG, Nphs-cre+ mice, which were obtained by crossing Gt (ROSA)26Sortm4(ACTB-tdTomato, -EGFP) Luo/J (Jackson Laboratory; strain 007676) with B6. Cg-Tg(NPHS2-cre)295Lbh/J (Jackson Laboratory; strain 008205) mice by sieving and cultured in collagen-coated dishes. Flow cytometry was performed to isolate the green fluorescent protein–positive population of primary podocytes, and the cells were cultured at 37 °C in 5% CO2 condition. Data were analyzed using GraphPad Prism 7 software. Values are presented as mean ± SD. Statistical comparisons of groups were performed using 1-way analysis of variance, and multiple comparisons of groups were performed using the least-significant difference test. P < 0.05 was considered of statistical significance. We first established an Ang II–infused mouse model based on our previous study7Yang Q. Hu J. Yang Y. et al.Sirt6 deficiency aggravates angiotensin II-induced cholesterol accumulation and injury in podocytes.Theranostics. 2020; 10: 7465-7479Crossref PubMed Scopus (22) Google Scholar (Figure 1a). Ang II–infused mice displayed significant kidney injury that manifested as glomerular sclerosis, mesangial matrix expansion (Figure 1b), and an increased urinary albumin excretion rate (Figure 1c). To further explore the role of Ang II in podocyte glucose metabolism, we performed quantitative reverse transcription–polymerase chain reaction to assay the glycolysis and tricarboxylic acid cycle–related enzymes in the glomeruli of mice with or without Ang II infusion. Surprisingly, of these enzymes, PKM, a key enzyme that catalyzes the final step of glycolysis metabolism, was significantly downregulated in the glomeruli from Ang II–infused mice (Figure 1d and e). However, there were no significant alterations in the expressions of other isoforms of pyruvate kinase, pyruvate kinase L/R (PKLR), and tricarboxylic acid cycle–related enzymes. The expressions of PKM1, PKM2, and PKLR were further analyzed by immunoblotting. Similarly, we only observed reduced PKM2 expression in glomeruli from Ang II–infused mice, whereas those of PKM1 and PKLR were not significantly altered (Figure 1f). The downregulated expression of PKM2 in glomeruli from Ang II–infused mice was also confirmed by histochemical staining analysis (Figure 1g). Moreover, double-immunofluorescence staining for PKM2 and synaptopodin, a specific marker for podocytes, showed decreased PKM2 expression in podocytes from Ang II–infused mice (Figure 1h). Pyruvate kinase (PK) activity was also measured in glomeruli. In line with lower PKM2 expression, PK activity declined in glomeruli from Ang II–infused mice (Figure 1i). Given that PKM2 is a key rate-limiting enzyme in glycolysis, these results indicate that Ang II may disturb glycolysis in podocytes. To further evaluate whether Ang II could induce glycolysis dysmetabolism in podocytes, we cultured primary podocytes from mT/mG, Cre+ mice (Supplementary Figure S1A). The expression of PKM2 in primary podocytes was significantly reduced after exposure to Ang II (Supplementary Figure S1B). In addition, Ang II decreased PKM2 expression and PK activity in human podocytes (Figure 1j–1). Moreover, treatment with the angiotensin receptor blocker candesartan rescued the decrease in PKM2, suggesting that the Ang Ⅱ–induced reduction in PKM2 in podocytes was mediated through Ang II type 1 receptor (Supplementary Figure S2A). To gain further insights into the impact of Ang II on podocyte glycolysis metabolism, the extracellular acidification rate, a method for quantifying glycolytic flux, was monitored in podocytes. First, the nonglycolytic acidification rate did not change between the Ang II–treated and control cells. However, the acidification rate in Ang II–treated cells declined significantly after the addition of glucose and oligomycin compared with the controls, suggesting that Ang II exposure suppressed glycolytic flux in podocytes (Figure 1m). In addition to the change in glycolytic flux in podocytes, ATP generation significantly decreased in podocytes after Ang II stimulation (Figure 1n). Taken together, these results demonstrate that Ang II causes dysmetabolism of glycolysis in podocytes. To further corroborate the above results, we evaluated PKM2 expression in human samples from subjects with kidney biopsy–proven HN and DKD (Figure 2a). Immunohistochemical staining showed generalized downregulation of PKM2 in glomeruli from subjects with HN and DKD compared with that from healthy area of kidney tissue from cases undergoing nephrectomy (Figure 2b). Moreover, PKM2 expression in podocytes was decreased in these cases, especially in the kidney tissues from patients with HN, as evidenced by the costaining of PKM2 and synaptopodin (Figure 2c). In addition, the reduced expression of PKM2 in podocytes was associated with proteinuria level in subjects with HN. The higher the proteinuria level in patients with HN, the lower the PKM2 expression (Supplementary Figure S3A). To explore the role of glycolysis in Ang II–induced podocyte injury, we utilized the Cre-Loxp system to generate podocyte-specific PKM2 knockout mice, which were identified by tail genotyping (Supplementary Figure S4A and B). Western blot analysis and immunohistochemical staining revealed that glomerular PKM2 levels were significantly reduced in PKM2fl/fl/Cre+ mice (Supplementary Figure S4C and D). The deletion of PKM2 in podocytes was further confirmed by immunofluorescence double staining, and the efficacy of PKM2 knockout was ≈85% (Supplementary Figure S4E and F). PKM2fl/fl/Cre+ mice were born as normal, and no significant differences in urinary albumin excretion, kidney weight, kidney/body weight ratio, or kidney histology were present between the PKM2fl/fl/Cre+ and PKM2fl/fl/Cre– mice at 2 months after birth (Supplementary Figure S5), consistent with the characterization of PKM2fl/fl/Cre+ mice described previously.15Qi W. Keenan H.A. Li Q. et al.Pyruvate kinase M2 activation may protect against the progression of diabetic glomerular pathology and mitochondrial dysfunction.Nat Med. 2017; 23: 753-762Crossref PubMed Scopus (266) Google Scholar Next, we induced kidney injury in PKM2fl/fl/Cre+ and PKM2fl/fl/Cre– mice by Ang II infusion (Figure 3a). As shown in Figure 3b and c, Ang II–infused PKM2fl/fl/Cre+ mice exhibited a significant increase in albuminuria and showed notable glomerular injury, which manifested as mesangial expansion and glomerulosclerosis, compared with Ang II–infused PKM2fl/fl/Cre- mice. Moreover, the glomerular ultrastructure was evaluated by transmission electron microscopy, and more severe thickening of the basement membrane and effacement of podocyte foot processes were observed in Ang II–infused PKM2fl/fl/Cre+ mice than those in Ang II–infused PKM2fl/fl/Cre– mice (Figure 3d). Meanwhile, PKM2 deletion in podocytes aggravated Ang II–induced podocyte apoptosis and loss, as determined by double immunostaining for Wilms tumor-1 and terminal deoxynucleotidyl transferase dUTP nick end labeling (Figure 3e). Moreover, a significant loss of synaptopodin indicated podocyte foot process effacement in Ang II–infused PKM2fl/fl/Cre+ mice (Figure 3e). These results demonstrate that PKM2 deficiency aggravated Ang II–induced podocyte injury and foot process effacement. To evaluate the protective effects of PKM2 upregulation on Ang II–induced kidney injury, we overexpressed PKM2 using an adeno-associated virus system with intrarenal injection. Transfection efficiency was confirmed by Western blotting, which showed that glomerular PKM2 was markedly increased in mice that received adeno-associated virus–cytomegalovirus–PKM2 (Supplementary Figure S6A). In addition, PKM2 overexpression significantly alleviated albuminuria, glomerulosclerosis, and foot process effacement in Ang II–infused mice (Supplementary Figure S6B–D). PKM2 can form monomers, dimers, or tetramers, with the latter possessing the highest catalytic activity.15Qi W. Keenan H.A. Li Q. et al.Pyruvate kinase M2 activation may protect against the progression of diabetic glomerular pathology and mitochondrial dysfunction.Nat Med. 2017; 23: 753-762Crossref PubMed Scopus (266) Google Scholar As a PKM2 agonist, TEPP-46 can markedly increase PK activity by inducing PKM2 tetramerization.15Qi W. Keenan H.A. Li Q. et al.Pyruvate kinase M2 activation may protect against the progression of diabetic glomerular pathology and mitochondrial dysfunction.Nat Med. 2017; 23: 753-762Crossref PubMed Scopus (266) Google Scholar,21Apostolidi M. Vathiotis I.A. Muthusamy V. et al.Targeting pyruvate kinase M2 phosphorylation reverses aggressive cancer phenotypes.Cancer Res. 2021; 81: 4346-4359Crossref PubMed Scopus (17) Google Scholar We demonstrated that TEPP-46 significantly increased glomerular PK activity (Supplementary Figure S7A), and then utilized TEPP-46 to evaluate whether activating PKM2 could prevent glomerular and podocyte injury in Ang II–infused mice (Figure 4a). As shown in Figure 4b and c, after 4 weeks of TEPP-46 treatment, a significant increase in PK activity and ATP levels was observed in the glomeruli of Ang II–infused mice. Furthermore, Ang II–infused mice treated with TEPP-46 showed lower urinary albumin excretion and less glomerulosclerosis, foot process fusion, podocyte loss, and apoptosis than Ang II–infused mice (Figure 4d–g). In addition, TEPP-46 administration increased the expressions of podocyte-associated proteins, including synaptopodin and nephrin (Figure 4h and i), indicating that enhancing PKM2 activity by TEPP-46 partially alleviated Ang II–induced loss of podocyte and foot process effacement. We further assessed whether the inhibition of glycolysis could aggravate Ang II–induced podocyte injury in vitro. Knockdown of PKM2 by small, interfering RNA markedly decreased PKM2 expression and exacerbated the decreases in PK activity, lactate, and ATP content caused by Ang II (Figure 5a–d), indicating that PKM2 knockout further promoted Ang II–induced glycolysis impairment. Meanwhile, PKM2 knockdown exacerbated the Ang II–induced actin cytoskeleton derangement in podocytes (Figure 5e). Previous studies demonstrated that podocyte cytoskeletal reorganization affected cell motility, thereby promoting cell detachment and apoptosis.8Ma Y. Yang Q. Zhong Z. et al.Role of c-Abl and nephrin in podocyte cytoskeletal remodeling induced by angiotensin II.Cell Death Dis. 2018; 9: 185Crossref PubMed Scopus (19) Google Scholar Thus, we further assessed podocyte migration and apoptosis and found that PKM2 deficiency significantly promoted Ang II–induced podocyte migration and apoptosis (Figure 5f and g). Simultaneously, we utilized 2-deoxyglucose, a glucose analog that inhibits glucose-6-phosphate generation, to inhibit glycolysis. The administration of 2-deoxyglucose exacerbated Ang II–induced glycolytic impairment, as evidenced by a significant reduction in lactate and ATP generation (Figure 5h and i). The 2-deoxyglucose markedly provoked podocyte actin cytoskeleton derangement, migration, and apoptosis (Figure 5j–m). These results further confirm that glycolysis is indispensable for podocytes to maintain the distinct cytoskeleton architecture. We next evaluated whether enhanced glycolysis could ameliorate Ang II–induced podocyte injury. PKM2 overexpression by transfection of the recombinant plasmid (pcDNA3.1-PKM2) into podocytes significantly increased PKM2 expression and rescued the Ang II–induced decreases in PK activity, lactate, and ATP content in podocytes (Figure 6a–d), suggesting that PKM2 overexpression attenuated the Ang II–induced impairments in glycolytic flux. In addition, PKM2 overexpression markedly ameliorated Ang II–induced podocyte actin cytoskeleton derangement, migration, and apoptosis (Figure 6e–g). Furthermore, PKM2 activation with TEPP-46 ameliorated the Ang II–induced reduction in PK activity, lactate, and ATP levels in podocytes (Figure 6h–j). TEPP-46 also reversed Ang II–induced podocyte actin cytoskeleton derangement, migration, and apoptosis (Figures 6k–n). Several recent studies revealed that anaerobic glycolysis plays a central role in maintaining normal podocyte function.15Qi W. Keenan H.A. Li Q. et al.Pyruvate kinase M2 activation may protect against the progression of diabetic glomerular pathology and mitochondrial dysfunction.Nat Med. 2017; 23: 753-762Crossref PubMed Scopus (266) Google Scholar, 16Yuan Q. Miao J. Yang Q. et al.Role of pyruvate kinase M2-mediated metabolic reprogramming during podocyte differentiation.Cell Death Dis. 2020; 11: 355Crossref PubMed Scopus (21) Google Scholar, 17Fu J. Shinjo T. Li Q. et al.Regeneration of glomerular metabolism and function by podocyte pyruvate kinase M2 in diabetic nephropathy.JCI Insight. 2022; 7e155260Crossref Scopus (6) Google Scholar,22Brinkkoetter P.T. Bork T. Salou S. et al.Anaerobic glycolysis maintains the glomerular filtration barrier independent of mitochondrial metabolism and dynamics.Cell Rep. 2019; 27: 1551-1566Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar Given the role of PKM2 in Ang Ⅱ–induced podocyte injury, we speculated that the energy generated by glycolysis is indispensable for podocytes. Because energy is mainly generated by mitochondria-dependent oxidative phosphorylation and mitochondria-independent glycolysis, we used transmission electron microscopy to visualize mitochondrial abundance and localization in podocytes from murine glomeruli. Compared with renal tubular cells, there were scanty mitochondria in podocytes (Figure 7a), which was also evidenced by the costaining of mitochondria-label translocase of outer mitochondrial membrane 20 (TOMM20) and nephrin (Figure 7b). In addition, podocyte mitochondria were distributed primarily in the cytoplasm but rarely distributed in the foot process (Figure 7a). We also evaluated the abundance and localization of mitochondria and glycolysis-associated enzymes in cultured podocytes using immunofluorescence double staining. Similarly, the results showed that podocyte mitochondria were mainly distributed in the perinuclear region, and rarely found at the cellular edge (Figure 7c). Nevertheless, key enzymes in glycolysis, including hexokinase 1, phosphofructokinase 1, PKM2, and lactate dehydrogenase, were ubiquitous (Figure 7c), suggesting that glycolysis was highly active in podocytes. To evaluate the distribution of PKM2 in podocytes in vivo, we performed immunoelectron microscope analysis and found abundant PKM2 immunoreactive signals in the primary and secondary foot processes (Supplementary Figure S8). Finally, we assessed the effects of inhibition of the mitochondrial respiratory chain and glycolysis on the podocyte cytoskeleton and apoptosis. PKM2 knockdown or 2-deoxyglucose administration to inhibit glycolysis subsequently led to cytoskeletal derangement and apoptosis; however, the inhibition of mitochondrial respiration by treatment with oligomycin did not cause these changes (Figure 7d and e). In summary, podocyte foot processes have fewer mitochondria and are primarily dependent on glycolysis for energy supply. The present study demonstrated that the key glycolytic enzyme PKM2 and glycolytic flux were significantly downregulated in podocytes following Ang II–induced kidney injury. Similarly, we observed reduced PKM2 expression in podocytes from patients with HN and DKD. Furthermore, podocyte-specific PKM2 knockout exacerbated Ang II–induced glomerulosclerosis and podocyte injury, whereas pharmacologic activation or transgenic PKM2 overexpression reversed these alterations. The compromised glycolysis may contribute to insufficient energy supply to foot processes, thus leading to Ang II–induced podocyte injury with foot process effacement. For decades, RAS activation has been known as an important factor leading to podocyte injury in chronic kidney disease.6Fu E.L. Evans M. Clase C.M. et al.Stopping renin-angiotensin system inhibitors in patients with advanced CKD and risk of adverse outcomes: a nationwide study.J Am Soc Nephrol. 2021; 32: 424-435Crossref PubMed Scopus (39) Google Scholar Our previous studies demonstrated that Ang II induced podocyte injury, including foot process effacement, cellular apoptosis, and proteinuria.7Yang Q. Hu J. Yang Y. et al.Sirt6 deficiency agg