Gluconeogenesis (GNG) is a process of glucose synthesis from non-carbohydrate precursors, which is enhanced in diabetic kidneys, contributing to elevations of blood glucose. Podocytes mainly utilize glucose to maintain glomerular filtration barrier integrity. However, their gluconeogenic potential has not yet been elucidated and the magnitude of this process has not been assessed under high glucose (HG) conditions. The present study demonstrated that podocytes express a set of key gluconeogenic enzymes at the mRNA and protein levels, including cytosolic and mitochondrial isoforms of phosphoenolpyruvate carboxykinase (PCK). High glucose-exposed podocytes exhibited an increase in PCK activity, which was linked to a decrease in intracellular levels of oxaloacetate (a PCK substrate), an increase in intracellular glucose levels and an increase in glucose efflux to the extracellular environment. Moreover, PCK inactivation in hyperglycemic podocytes increased the intracellular content of lactate, a preferred substrate for GNG, and decreased glycogen levels, suggesting the direction of glucose to glycogen synthesis under HG conditions. Additionally, the PCK inhibitor abolished the negative effect of HG on the activity of hexokinase and pyruvate kinase in podocytes, suggesting that inhibiting PCK may enhance the glycolytic pathway in hyperglycemia. Overall, an increase in PCK activity in response to HG concentrations may enhance GNG in podocytes, contributing to cell glucose overload. The present results broaden our understanding of metabolic changes that underlie HG-induced podocyte dysfunction and spotlight potential therapeutic targets for the treatment of diabetic kidney disease.

Podocyte injury is a hallmark of diabetic nephropathy (DN) and significantly contributes to disease progression. Accumulating evidence suggests that impaired autophagy exacerbates podocyte dysfunction under hyperglycaemic conditions. However, the underlying regulatory mechanisms remain incompletely understood. This study aimed to investigate the role and mechanism of microRNA-10a-5p (miR-10a-5p) in regulating podocyte injury and autophagy in DN. The function of miR-10a-5p was explored in high glucose (HG)-induced murine podocyte (MPC5) cells and streptozotocin (STZ)-induced diabetic mice. Gain- and loss-of-function experiments were conducted using miR-10a-5p mimics or inhibitors. E2F transcription factor 7 (E2f7) was predicted as a downstream target of miR-10a-5p via bioinformatics, and the binding between miR-10a-5p and E2f7 was assessed using dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Podocyte injury, cell apoptosis, inflammation and autophagy were assessed using flow cytometry, Western blotting, enzyme-linked immunosorbent assay (ELISA), transmission electron microscopy (TEM) and green fluorescent protein-monomeric red fluorescent protein-LC3 (GFP-mRFP-LC3) staining. Rescue experiments were conducted by silencing or overexpressing E2f7 to confirm its role in miR-10a-5p-mediated effects. miR-10a-5p was significantly upregulated in HG-stimulated podocytes and kidney tissues of diabetic mice. Inhibition of miR-10a-5p alleviated HG-induced podocyte injury, as evidenced by enhanced cell viability, reduced apoptosis and inflammatory cytokine production, and restored autophagic activity. E2f7 was identified as a direct target of miR-10a-5p. Notably, silencing E2f7 abrogated the protective effects of miR-10a-5p inhibition on podocyte survival and autophagy, and E2f7 overexpression restored the detrimental effects of miR-10a-5p overexpression on podocytes. Invivo, administration of AntagomiR-10a-5p in STZ-induced diabetic mice ameliorated renal injury and restored autophagic flux, and increased E2f7 expression in mouse renal tissues. This study identifies the miR-10a-5p/E2f7 axis as a critical regulator of podocyte injury and autophagy in DN. Therapeutic inhibition of miR-10a-5p may represent a promising strategy for preserving podocyte function and attenuating DN injury.

Epigenetic mechanism of LncRNA KCNQ1OT1 in high-glucose-induced podocyte injury via m6A methylation modification.

The analysis of diabetic nephropathy (DN)-related gene dataset demonstrated that C-X-C motif chemokine ligand 3 (CXCL3) is highly expressed in DN. Exploring the impact of CXCL3 in the course of DN is the core goal of this study. The cell model used in this study was CIHP-1 cells induced by high glucose (HG). qRT-PCR and western blot analysis were carried out to determine the expression difference of CXCL3. After down-regulating the CXCL3 level, we analyzed HG-induced CIHP-1 cell viability by MTT assay, proliferation by EdU staining, apoptosis by flow cytometry, and changes in related protein expression by western blot. In order to analyze the possible regulatory relationship between endothelial cellspecific molecule 1 (ESM-1) and CXCL3 in DN, we constructed an over-expressed ESM-1 plasmid and carried out a rescue experiment. CXCL3 and ESM-1 were highly expressed in HG-induced podocytes (p<0.05). Silenced CXCL3 (siCXCL3) increased the viability and proliferation of CIHP- 1 cells induced by HG, reduced the proportion of apoptosis, and produced corresponding protein changes (p<0.01). After the overexpression of ESM-1, the effects of siCXCL3 were partially offset (p<0.05). In this study, ESM-1 increased HG-induced podocyte damage by promoting CXCL3 expression.

The extremely complex, multivariate, and systemic pathophysiology of diabetic nephropathy is brought on by prolonged exposure to hyperglycemia, making the quest for the best therapeutic approach crucial and urgent. Accumulating evidence suggests that pyroptosis and inflammation contribute to the development of diabetic nephropathy. Zinc fingers and homeoboxes 2 (ZHX2) were recently discovered to be a new regulator of inflammatory response. However, the role and potential molecular mechanisms of ZHX2 in diabetic nephropathy remain unclear. Exosomes derived from gingival mesenchymal stem cells (GMSCs-Exo) were successfully isolated and characterized. GMSCs-Exo reversed high glucose-induced podocyte pyroptosis and inflammation. ZHX2 was highly expressed in GMSCs-Exo. Furthermore, ZHX2 derived from GMSCs-Exo reversed podocyte pyroptosis and inflammation induced by HG. Additionally, ZHX2 was enriched in the FABP4 promoter region and transcriptionally inhibited the mRNA and protein levels of FABP4. GMSCs-Exo-derived ZHX2 abolished HG-induced podocyte pyroptosis and inflammation by inhibiting FABP4 and blocking the advanced glycation endproducts/the receptor of advanced glycation endproducts/NOD-like receptor family pyrin domain-containing 3 (AGEs/RAGE/NLRP3) pathway. Similarly, GMSCs-Exo-derived ZHX2 alleviated renal injury, pyroptosis, and inflammation in diabetic nephropathy mice. In conclusion, our findings demonstrated that ZHX2 protects against diabetic nephropathy by binding to the FABP4 promoter and reducing the expression of FABP4. We also showed that GMSCs-Exo-derived ZHX2 reversed HG-induced podocyte pyroptosis and inflammation by inhibiting the AGEs/RAGE/NLRP3 pathway. ZHX2 derived from GMSCs-Exo also alleviated tubular injury, pyroptosis, and inflammation in diabetic nephropathy mice. The therapeutic potential of targeting ZHX2 to treat diabetic nephropathy is clarified by these findings.

BackgroundThe core pathological feature of Diabetic kidney disease is glomerular podocyte injury. A hyperglycemic milieu induces podocyte injury through the synergistic actions of multiple pathways, including oxidative stress, inflammation, and apoptosis. Protocatechuic Aldehyde (PCA), a naturally occurring phenolic acid compound, exhibits significant antioxidant activity. However, the protective effects and underlying mechanisms of PCA on podocyte function under high-glucose conditions remain incompletely elucidated.ObjectiveTo investigate the effects and mechanism of PCA on high glucose-induced podocyte inflammation, oxidative stress, and apoptotic injury.MethodsA podocyte injury model was established by treating mouse podocytes (MPC5) with high-glucose medium. Podocytes were concurrently treated with varying concentrations of Protocatechuic Aldehyde. To explore the mechanism, cells in different treatment groups were exposed to the GSK3β inhibitor TDZD-8 and the endoplasmic reticulum stress inducer Tunicamycin (TM). The levels of inflammatory cytokines and oxidative stress markers were measured using relevant assay kits. The expression of proteins associated with inflammation, oxidative stress, apoptosis, the GSK3β/Nrf2 signaling pathway, and endoplasmic reticulum stress was detected by Western blot. Apoptosis rate of podocytes was assessed using flow cytometry.ResultsHigh glucose significantly reduced MPC5 cell viability and increased lactate dehydrogenase release; these effects were significantly reversed by PCA treatment. PCA significantly reduced the secretion of inflammatory cytokines (TNF-α, IL-1β, IL-6), restored the activities of SOD and GSH-Px, decreased MDA content, and downregulated the expression of Cox-2, iNOS, Nox2, and Nox4 proteins, thereby suppressing HG-induced podocyte inflammation and oxidative stress. Furthermore, PCA upregulated Bcl-2 expression while downregulating Bax and cleaved-caspase 3 expression, effectively inhibiting HG-induced podocyte apoptosis. Mechanistically, PCA upregulated the expression of p-GSK3β and Nrf2 proteins, activating the GSK3β/Nrf2 signaling pathway. This activation was associated with downregulation of ER stress markers (CHOP, GRP78, p-PERK), indicating suppression of podocyte ER stress. Notably, the protective effects of PCA were abrogated by co-treatment with the GSK3β inhibitor TDZD-8 or the ER stress inducer TM.ConclusionPCA attenuates high glucose-induced podocyte injury, characterized by inflammation, oxidative stress, and apoptosis, suggesting that this protection involves inhibition of ER stress via activation of the GSK3β/Nrf2 signaling pathway.

As a predominant contributor to end-stage renal disease, diabetic nephropathy (DN) progression is critically influenced by podocyte impairment, which serves as a pivotal determinant in the initiation of proteinuria and subsequent renal functional decline. MicroRNAs (miRNAs) act as key post-transcriptional regulators that modulate target gene expression through mRNA degradation and/or translational repression. Previous studies have demonstrated that the upregulation of human runt-associated transcription factor 3 (RUNX3) suppresses the epithelial-to-mesenchymal transition (EMT) in renal tubular epithelial cells in DN. But, the role of RUNX3 in podocyte-specific EMT and its regulation by miRNAs has not been comprehensively explored. This study aimed to investigate whether the microRNA mmu-miR-664-5p contributes to high glucose (HG)-induced podocyte injury by targeting RUNX3 and to determine if Shenxiao decoction (SXD) exerts its protective effect through this pathway. We found that HG significantly upregulates mmu-miR-664-5p, which directly binds to the 3' UTR of RUNX3 mRNA. Inhibition of mmu-miR-664-5p in HG-induced mouse podocyte clone-5 (MCP5) enhanced cell viability, reduced apoptosis, migration, and invasion, and reversed EMT marker expression. We further discovered that SXD-containing serum (SCS), counteracts HG-induced podocyte EMT by downregulating mmu-miR-664-5p and restoring RUNX3. Conversely, adding exogenous miR-664-5p mimetic to SCS-treated HG media reversed these protective effects. In conclusion, mmu-miR-664-5p promoted the EMT effect of MPC5 podocytes in HG medium by targeting RUNX3, and SXD mitigates this process through its inhibition of miR-664-5p, collectively highlighting the mmu-miR-664-5p/RUNX3 axis as a prospective target for therapeutic intervention in DN.

ABSTRACT Podocyte injury significantly contributes to glomerular filtration dysfunction and albuminuria in diabetic nephropathy (DN). Circular RNAs, particularly circFAT1 (hsa_circ_0001461), have emerged as influential regulators in pathological processes. This research focused on exploring the function of hsa_circ_0001461 in high glucose (HG)-induced podocyte damage and the associated underlying mechanism. Here, we demonstrate that circFAT1 is significantly upregulated in HPCs under HG conditions. Inhibition of circFAT1 led to decreased podocyte migration and a restoration of differentiation markers, along with a reduction in mesenchymal markers. Mechanistically, circFAT1 was found to inhibit miR-30e-5p, resulting in enhanced SOX4 expression, which promoted epithelial-mesenchymal transition and migration in podocytes. Moreover, we identified EIF4A3 as a crucial regulator of circFAT1 biogenesis under hyperglycaemic conditions. Importantly, elevated levels of circFAT1 were also detected in DN patients, correlating with increased albuminuria and serum creatinine. In conclusion, this study elucidates the critical role of circFAT1 in HG-induced podocyte injury through the miR-30e-5p/SOX4 signalling pathway. The findings suggest that targeting circFAT1 May offer a potential strategy for DN intervention.

Recent evidence highlights the critical role of 5-methylcytidine (m5C) as an epigenetic modification in the pathogenesis of various diseases. However, its regulatory mechanisms in diabetic nephropathy (DN) remain poorly understood. In this study, we observed a marked increase in m5C levels in the kidneys of type 2 diabetic (db/db) mice and in high glucose (HG)-stimulated podocytes, which was linked to reduced expression of the m5C demethylase ten-eleven translocation 2 (TET2). Moreover, renal biopsy samples from patients with DN exhibited decreased TET2 expression, correlating with impaired renal function. Gain-of-function assays revealed that TET2 overexpression in HG-induced podocytes enhanced mitophagy and ameliorated podocyte injury both invitro and invivo. Therapeutically, systemic delivery of AAV-TET2 in db/db mice reduced albuminuria, improved renal histopathology, and restored mitophagy. Mechanistically, TET2 regulated mitophagy by modulating the m5C methylation of Breast Carcinoma Amplified Sequence 3 (Bcas3). Furthermore, Bcas3 overexpression promoted mitophagy and attenuated podocyte damage under HG conditions. In conclusion, TET2-mediated m5C modification contributes to podocyte injury in DN, and targeting m5C via TET2 presents a promising therapeutic strategy for DN.

Human urine-derived stem cells from different donor sources ameliorate diabetic nephropathy in mice by activating autophagy and restoring mitochondrial function of podocyte.

Diabetic kidney disease (DKD) progression is strongly associated with podocyte mitochondrial dysfunction. The clinically effective Chinese herbal Baoshentongluo formula (BSTL) has demonstrated significant proteinuria reduction in DKD patients. HPLC-ESI-MS analysis identified characteristic bioactive components in BSTL including astragalosides, rehmanniosides, and tanshinones. However, the molecular mechanisms through which BSTL maintains podocyte homeostasis remain incompletely understood. Mouse podocyte clone-5 (MPC-5) cells and db/db mice were used. Db/db mice were randomized into db/db and db/db + BSTL (16.5 g/kg/d, intragastric administration for 12 weeks). A group of m/m mice served as the control. Renal function, urinary albumin-to-creatinine ratio (UACR), histopathological analysis, apoptotic, and mitophagy-related protein levels were evaluated. MPC-5 cells were exposed to high glucose (HG, 30 mM) and BSTL drug-containing serum (8%) for 24 h grouping as control, HG, HG + BSTL, and HG + siPINK1. Podocyte apoptosis, mitophagy levels, and expression of PTEN-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase (Parkin) were assessed. In db/db diabetic mice, oral administration of BSTL significantly lowered urinary albumin-to-creatinine ratio (P<0.05), improved glomerular filtration rate, and ameliorated renal histopathological changes, decreased LC3-II/LC3-I ratio, and downregulated expression of mitophagy-related proteins PINK1, Parkin, ATG5 and Beclin-1. Treatment with 8% BSTL-containing serum significantly attenuated HG-induced podocyte apoptosis (P<0.01) and suppressed excessive mitophagy, as evidenced by reduced TOM20/LC3 co-localization (P<0.01). Notably, BSTL treatment markedly reduced protein levels of both PINK1 and Parkin (P<0.01), key regulators of mitophagy initiation. Genetic silencing of PINK1 in podocytes phenocopied BSTL's protective effects, confirming the pathway specificity. Our integrated in vitro and in vivo findings establish that BSTL protects against DKD progression by selectively inhibiting PINK1/Parkin-dependent mitophagy in podocytes to inhibit podocyte injury, which provides both mechanistic insights and therapeutic potential for clinical DKD management.

Targeting the NF-κB p65-MMP28 axis: Wogonoside as a novel therapeutic agent for attenuating podocyte injury in diabetic nephropathy.

Recently, microRNAs (miRNAs) have been found to mediate the development of diabetic kidney disease (DKD) by regulating podocyte injury. The aim of this study was to investigate the influence of miR-504-3p on high glucose (HG)-treated mouse renal podocytes (MPC5) and its potential regulatory mechanisms. First, a DKD cell model was established. Next, RT-qPCR was performed to measure miR-504-3p and HNF1 Homeobox B (HNF1B) expression levels. Additionally, the proliferation and apoptosis of MPC5 cells were assessed using CCK-8 assay and Flow cytometry, respectively. The protein expression levels of cell fibrotic markers, podocyte injury marker, epithelial-mesenchymal transition (EMT) markers and HNF1B were measured by Western Blotting. ROS, MDA, SOD and GSH kits were used to assess oxidative stress levels. Furthermore, the interplay between miR-504-3p and HNF1B was confirmed by luciferase reporter experiments. The miR-504-3p expression was significantly upregulated in GEO database (GSE161884) and in HG-induced MPC5 cells. The results revealed that HG treatment decreased MPC5 cell proliferation, promoted cell apoptosis and fibrosis, and ultimately led to podocyte injury. However, miR-504-3p knockdown could reverse these phenotypes and reduce podocyte injury. Moreover, online database screening combined with dual luciferase reporter assay confirmed HNF1B as a specific target of miR-504-3p. Finally, overexpression of HNF1B mitigated the proliferation inhibition and apoptosis promotion induced by oxidative stress and inhibited EMT-mediated cell fibrosis, thereby counteracting the effects of miR-504-3p on podocyte injury under HG treatment. In summary, our data indicate that miR-504-3p regulates HG-induced podocyte injury by sponging HNF1B, providing a new direction for the treatment of DKD.

BackgroundDysfunction in podocyte mitophagy has been identified as a contributing factor to the onset and progression of diabetic nephropathy (DN), and BMAL1 plays an important role in the regulation of mitophagy. Thus, this study intended to examine the impact of BMAL1 on podocyte mitophagy in DN and elucidate its underlying mechanisms.Materials and methodsHigh D-glucose (HG)-treated MPC5 cells was used as a podocyte injury model for investigating the potential roles of BMAL1 in DN. Mitophagy was examined by detecting autophagosomes using transmission electron microscopy, and detecting the colocalization of LC3 and Tom20 using immunofluorescence staining. The interaction between BMAL1 and SIRT1 was conducted by immunoprecipitation (Co-IP) assay.ResultsIn HG-induced podocyte injury model, we found that BMAL1 and SIRT1 mRNA level was significantly decreased, and positively correlated with mitophagy dysfunction. BMAL1 overexpression could ameliorate HG-induced podocyte injury, evidenced by improved cell viability, decreased cell apoptosis and inflammatory cytokines expression (TNF-α, IL-1β, and IL-6). BMAL1 overexpression could promote podocyte mitophagy coupled with increased expression of mitophagy markers PINK1 and Parkin. In terms of mechanism, Co-IP suggested that BMAL1 could interact with SIRT1. SIRT1 inhibitor Ex-527 addition obviously inhibit the effect of BMAL1 overexpression on the mitophagy, demonstrating that BMAL1 may act on mitophagy by SIRT1//PGC-1α axis.ConclusionsOur in vitro experiments demonstrate that BMAL1/SIRT1/PGC-1α pathway may protect podocytes against HG-induced DN through promoting mitophagy.

Diabetic nephropathy (DN) is a major complication of diabetes, imposing substantial socioeconomic and public health challenges. N6-methyladenosine (m6A) modification, a prevalent epigenetic mechanism, influences cellular processes and disease progression. Wilms' tumor 1-associating protein (WTAP), an m6A methyltransferase subunit, was investigated for its role in DN. Bioinformatics identified differentially expressed genes in DN, and a high glucose (HG)-induced podocyte model was established to mimic DN in vitro. Techniques like Western blot, CCK-8, ELISA, flow cytometry, and TUNEL evaluated protein expression, cell viability, inflammation, oxidative stress, and apoptosis. SRAMP predicted m6A sites in DDX3Y mRNA, validated by MeRIP, while xenograft models confirmed in vivo effects. DDX3Y expression was elevated in DN and HG-induced podocytes, and sh-DDX3Y attenuated HG-induced injury. WTAP promoted DDX3Y mRNA stability via m6A methylation, exacerbating podocyte dysfunction. In diabetic mice, WTAP modulated DDX3Y to induce renal insufficiency and histopathological damage. Collectively, WTAP regulates DDX3Y via m6A methylation to promote HG-induced podocyte injury and DN progression.

LncRNA HOXB3OS improves high glucose-mediated podocyte damage and progression of diabetic kidney disease through enhancing SIRT1 mRNA stability.

BackgroundSodium-glucose cotransporter 2 (SGLT2) inhibitors have changed the therapeutic landscape for diabetic kidney disease (DKD) patients, but their underlying mechanisms are complicated and not fully understood. Mitochondria-associated endoplasmic reticulum membranes (MAMs), the dynamic contact sites between mitochondria and the endoplasmic reticulum (ER), serve as intracellular platforms important for regulating cellular fate and function. This study explored the roles and mechanisms of SGLT2 inhibitors in regulating MAMs formation in diabetic podocytes.MethodsWe assessed MAMs formation in podocytes from DKD patients’ renal biopsy samples and induced an increase in MAMs formation in cultured human podocytes by transfecting OMM-ER linker plasmid to investigate the effects of MAMs imbalance on podocyte injury. Empagliflozin-treated diabetic mice and podocyte-specific SGLT2 knockout diabetic mice (diabetic states were induced by streptozotocin and a high-fat diet), empagliflozin-treated podocytes, SGLT2-downregulated podocytes, and SGLT2-overexpressing podocytes were used to investigate the effects and mechanisms of SGLT2 inhibitors on MAMs formation in diabetic podocytes.ResultsMAMs were increased in podocytes and were associated with renal dysfunction in DKD patients. Increased MAMs aggravated HG-induced podocyte injury. The expression of SGLT2 was increased in diabetic podocytes. In addition, empagliflozin-treatment and podocyte-specific SGLT2 knockout attenuated MAMs formation and podocyte injury in diabetic mice. Empagliflozin treatment and SGLT2 knockdown decreased podocyte MAMs formation by activating the AMP-activated protein kinase (AMPK) pathway, while SGLT2 overexpression had the opposite effect.ConclusionsInhibition of SGLT2 attenuates MAMs imbalance in diabetic podocytes by activating the AMPK pathway. This study expands our knowledge of the roles of SGLT2 inhibitors in improving DKD podocyte injury and provides new insights into DKD treatment.

Podocyte injury plays a pivotal role in the pathogenesis of diabetic nephropathy (DN), leading to proteinuria formation. β-Sitosterol is a natural compound with anti-inflammatory, anti-diabetic, nephroprotective and antioxidant properties. The studyaimed to explore whether and how β-Sitosterol protected podocytes against high glucose (HG)-induced inflammatory andoxidative injury. DN cell models were established by stimulating podocytes or renal tubular epithelial cells (HK-2) cells with 25 mM glucose. Cell viability and apoptosis were evaluated using cell counting kit-8 assays and flow cytometry analyses. Westernblotting was used to quantify protein levels of genes related to podocyte injury, HK-2 cell damage, inflammation, and TLR4/NF-кB pathway. Contents of oxidative stress biomarkers were evaluated by corresponding commercial kits while proinflammatorycytokine levels were determined by enzyme-linked immunosorbent assay. Immunofluorescence staining was performed todetect intracellular levels of reactive oxygen species (ROS) and Nrf2 nuclear translocation. Experimental results revealed that HG treatment induced podocyte dysfunction by impairing cell viability while accelerating theapoptosis, and the changes were reversed by β-sitosterol treatment. Moreover, β-sitosterol repressed HG-evoked oxidative stressby reducing ROS and malondialdehyde (MDA) levels while increasing activities of antioxidant enzymes. The reduction ofproinflammatory cytokines mediated by β-sitosterol in HG-stimulated podocytes suggested the anti-inflammatory role of β-sitosterol. Additionally, the activation of the TLR4/NF-кB signaling induced by HG was inhibited by β-sitosterol in podocytes.Inactivation of the TLR4 using TAK-242 enhanced the protective effects of β-sitosterol against HG-mediated oxidative stressand inflammation. Similarly, β-sitosterol also protected HK-2 cells from HG-induced oxidative stress, inflammation, andapoptosis. In summary, β-sitosterol exerts anti-inflammatory, anti-oxidative, and anti-apoptotic activities in HG-induced podocytes or HK-2 cells by inhibiting TLR4/NF-кB signaling.

Background Podocyte injury plays an important role in the occurrence and progression of diabetic kidney disease (DKD), which leads to albuminuria. Cytoskeletal remodeling is an early manifestation of podocyte injury in DKD. However, the underlying mechanism of cytoskeletal remodeling has not been clarified. Histone deacetylase sirtuin6 (Sirt6) has been found to play a key role in DKD progression, and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) pathway directly regulates the cytoskeletal structure of podocytes. Whereas, the relationship between Sirt6, the PI3K/AKT pathway and DKD progression remains unclear. Methods Renal injury of db/db mice was observed by PAS staining and transmission electron microscope. Expression of Sirt6 in the glomeruli of db/db mice was detected by immunofluorescence. UBCS039, a Sirt6 activator, was used to explore the renal effects of Sirt6 activation on diabetic mouse kidneys. We also downregulating Sirt6 expression in podocytes using the Sirt6 inhibitor, OSS_128167, and induced upregulation of Sirt6 using a recombinant plasmid, after which the effects of Sirt6 on high glucose (HG)-induced podocyte damage were assessed in vitro. Podocyte cytoskeletal structures were observed by phalloidin staining. The podocyte apoptotic rate was assessed by flow cytometry, and PI3K/AKT signaling activation was measured by Western blotting. Results Db/db mice exhibited renal damage including elevated urine albumin-to-creatinine ratio (ACR), increased mesangial matrix, fused podocyte foot processes, and thickened glomerular basement membrane. The expression of Sirt6 and PI3K/AKT pathway components was decreased in db/db mice. UBCS039 increased the expressions of Sirt6 and PI3K/AKT pathway components and ameliorated renal damage in db/db mice. We also observed consistent Sirt6 expression was in HG-induced podocytes in vitro. Activation of the PI3K/AKT pathway via a Sirt6 recombinant plasmid ameliorated podocyte cytoskeletal remodeling and apoptosis in HG-treated immortalized human podocytes in vitro, whereas Sirt6 inhibition by OSS_128167 accelerated HG-induced podocyte damage in vitro. Conclusions Sirt6 protects podocytes against HG-induced cytoskeletal remodeling and apoptosis through activation of the PI3K/AKT signaling pathway. These findings provide evidence supporting the potential efficacy of Sirt6 activation as a promising therapeutic strategy for addressing podocyte injury in DKD.

Poricoic acid A (PAA), a major triterpenoid component of Poria cocos with anti-tumor, anti-fibrotic, anti-inflammatory, and immune-regulating activities, has been shown to induce podocyte autophagy in diabetic kidney disease (DKD) by downregulating FUN14 domain containing 1 (FUNDC1). This study aimed to identify the role of adenosine monophosphate-activated protein kinase alpha (AMPKα) in PAA-mediated phosphorylation of FUNDC1 in podocyte injury occurring in the pathogenesis of DKD. A cellular model of renal podocyte injury was established by culturing MPC5 cells under high-glucose (HG) conditions. MPC5 cells were subjected to transfection with small interfering RNA (siRNA) targeting AMPKα or siRNA targeting FUNDC1, an AMPKα activator, or PAA. PAA treatment induced the phosphorylation of AMPKα in HG-cultured podocytes. AMPKα activation was implicated in the inhibitory effect of PAA on FUNDC phosphorylation in HG-cultured podocytes. Treatment targeting the AMPKα activator also significantly augmented proliferation, migration, mitochondrial membrane potential, and autophagy levels, while reducing apoptosis levels, inhibiting oxidative stress, and suppressing the release of proinflammatory factors in HG-cultured MPC5 cells. In contrast, insufficient expression of AMPKα reversed the effects of PAA on the proliferation, migration, and apoptosis of podocytes and further exacerbated the reduction of phosphorylated FUNDC1 expression in podocytes under HG conditions. AMPKα is involved in the regulation of FUNDC1 phosphorylation by PAA in HG-induced podocyte injury. Furthermore, the AMPKα/FUNDC1 pathway plays a crucial regulatory role in HG-induced podocyte injury. These findings support AMPKα, FUNDC1, and the AMPKα/FUNDC1 pathway as targets for PAA intervention.