BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a common glomerulopathy with an unclear mechanism. The demand for FSGS clinical diagnostic biomarkers has not yet been met. Circular RNA (circRNA) is a novel non-coding RNA with multiple functions, but its diagnostic value for FSGS remains unexplored. This study aimed to identify circRNAs that could aid in early clinical diagnosis and to investigate their mechanisms in podocyte injury.
METHODS: The signature of plasma circRNAs for FSGS was identified by circRNA microarray. The existence of circRNAs was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR), RNase R assay, and DNA sequencing. Plasma levels of circRNAs were evaluated by qRT-PCR. The diagnostic value was appraised by the receiver operating characteristic curve. The circRNA-miRNA-mRNA network was built with Cytoscape 7.3.2. Statistically significant differences were calculated by the Mann-Whitney U test.
RESULTS: A total of 493 circRNAs (165 upregulated, 328 downregulated) were differentially expressed in the plasma of FSGS patients (n = 3) and normal controls (n = 3). Eight candidate circRNAs were demonstrated to be circular and stable transcripts. Among them, hsa_circ_0001230 and hsa_circ_0023879 were significantly upregulated in FSGS patients (n = 29) compared to normal controls (n = 51). The areas under the curve value of hsa_circ_0001230 and hsa_circ_0023879 were 0.668 and 0.753, respectively, while that of the two-circRNA panel was 0.763. The RNA pull-down analysis revealed that hsa_circ_0001230 and hsa_circ_0023879 could sponge hsa-miR-106a. Additionally, hsa_circ_0001230 and hsa_circ_0023879 positively regulated hsa-miR-106a target genes phosphatase and tensin homolog (PTEN) and Bcl-2-like protein 11 (BCL2L11) in podocytes.
CONCLUSIONS: hsa_circ_0001230 and hsa_circ_0023879 are novel blood biomarkers for FSGS. They may regulate podocyte apoptosis by competitively binding to hsa-miR-106a.

Podocyte apoptosis exerts a crucial role in the pathogenesis of DN. Recently, long noncoding RNAs (lncRNAs) have been gradually identified to be functional in a variety of different mechanisms associated with podocyte apoptosis. This study aimed to investigate whether lncRNA Glis2 could regulate podocyte apoptosis in DN and uncover the underlying mechanism. The apoptosis rate was detected by flow cytometry. Mitochondrial membrane potential (ΔΨM) was measured using JC-1 staining. Mitochondrial morphology was detected by MitoTracker Deep Red staining. Then, the histopathological and ultrastructure changes of renal tissues in diabetic mice were observed using periodic acid-Schiff (PAS) staining and transmission electron microscopy. We found that lncRNA Glis2 was significantly downregulated in high-glucose cultured podocytes and renal tissues of db/db mice. LncRNA Glis2 overexpression was found to alleviate podocyte mitochondrial dysfunction and apoptosis. The direct interaction between lncRNA Glis2 and miR-328-5p was confirmed by dual luciferase reporter assay. Furthermore, lncRNA Glis2 overexpression alleviated podocyte apoptosis in diabetic mice. Taken together, this study demonstrated that lncRNA Glis2, acting as a competing endogenous RNA (ceRNA) of miRNA-328-5p, regulated Sirt1-mediated mitochondrial dysfunction and podocyte apoptosis in DN.

BACKGROUND: Podocyte apoptosis plays a vital role in proteinuria pathogenesis in diabetic nephropathy (DN). The regulatory relationship between long noncoding RNAs (lncRNAs) and podocyte apoptosis has recently become another research hot spot in the DN field.
OBJECTIVE: To investigate whether lncRNA protein-disulfide isomerase-associated 3 (Pdia3) could regulate podocyte apoptosis through miR-139-3p and revealed the underlying mechanism.
METHODS: Using normal glucose or high glucose (HG)-cultured podocytes, the cellular functions and exact mechanisms underlying the regulatory effects of lncRNA Pdia3 on podocyte apoptosis and endoplasmic reticulum stress (ERS) were explored. LncRNA Pdia3 and miR-139-3p expression were measured through quantitative real-time polymerase chain reaction. Relative cell viability was detected through the cell counting kit-8 colorimetric assay. The podocyte apoptosis rate in each group was measured through flow cytometry. The interaction between lncRNA Pdia3 and miR-139-3p was examined through the dual luciferase reporter assay. Finally, western blotting was performed to detect the effect of lncRNA Pdia3 on podocyte apoptosis and ERS via miR-139-3p.
RESULTS: The expression of lncRNA Pdia3 was significantly downregulated in HG-cultured podocytes. Next, lncRNA Pdia3 was involved in HG-induced podocyte apoptosis. Furthermore, the dual luciferase reporter assay confirmed the direct interaction between lncRNA Pdia3 and miR-139-3p. LncRNA Pdia3 overexpression attenuated podocyte apoptosis and ERS through miR-139-3p in HG-cultured podocytes.
CONCLUSIONS: Taken together, this study demonstrated that lncRNA Pdia3 overexpression could attenuate HG-induced podocyte apoptosis and ERS by acting as a competing endogenous RNA of miR-139-3p, which might provide a potential therapeutic target for DN.

BACKGROUND: Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease and currently there are no specific and effective drugs for its treatment. Podocyte injury is a detrimental feature and the major cause of albuminuria in DN. We previously reported Tangshen Formula (TSF), a Chinese herbal medicine, has shown therapeutic effects on DN. However, the underlying mechanisms remain obscure.
OBJECTIVE: This study aimed to explore the protective effect of TSF on podocyte apoptosis in DN and elucidate the potential mechanism.
METHODS: The effects of TSF were assessed in a murine model using male KKAy diabetic mice, as well as in advanced glycation end products-stimulated primary mice podocytes. Transcription factor EB (TFEB) knockdown primary podocytes were employed for mechanistic studies. In vivo and in vitro studies were performed and results assessed using transmission electron microscopy, immunofluorescence staining, and western blotting.
RESULTS: TSF treatment alleviated podocyte apoptosis and structural impairment, decreased albuminuria, and mitigated renal dysfunction in KKAy mice. Notably, TSF extracted twice showed a more significant reduction in proteinuria than TSF extracted three times. Accumulation of autophagic biomarkers p62 and LC3, and aberrant autophagic flux in podocytes of DN mice were significantly altered by TSF therapy. Consistent with the in vivo results, TSF prevented the apoptosis of primary podocytes exposed to AGEs and activated autophagy. However, the anti-apoptosis capacity of TSF was countered by the autophagy-lysosome inhibitor chloroquine. We found that TSF increased the nuclear translocation of TFEB in diabetic podocytes, and thus upregulated transcription of its several autophagic target genes. Pharmacological activation of TFEB by TSF accelerated the conversion of autophagosome to autolysosome and lysosomal biogenesis, further augmented autophagic flux. Conversely, TFEB knockdown negated the favorable effects of TSF on autophagy in AGEs-stimulated primary podocytes.
CONCLUSIONS: These findings indicate TSF appears to attenuate podocyte apoptosis and promote autophagy in DN via the TFEB-mediated autophagy-lysosome system. Thus, TSF may be a therapeutic candidate for DN.

Diabetic kidney disease (DKD) is a leading cause of end-stage renal disease without early diagnostic and specific therapeutic approaches. Podocyte apoptosis and loss play important roles in the pathological process of DKD. This study aimed to explore whether urinary exosomes from type 2 diabetes patients with DKD could induce podocyte apoptosis and the underlying pathological mechanisms. The exosomes were isolated from the urine samples of patients with DKD (DKD-Exo). Later, they were taken up and internalized by MPC5 cells. MPC5 cells were co-cultured with DKD-Exo (45 μg/ml) for 24 h in the presence or absence of microRNA-145-5p (miR-145-5p) inhibitor, fasudil and pcDNA-Srgap2 transfection. MiR-145-5p and Srgap2 expression was evaluated using real-time quantitative PCR. The protein levels of Srgap2, Bcl-2, Bax, and cleaved caspase-3, as well as ROCK activity were determined using Western blotting. Cell apoptosis was measured using flow cytometry and the TUNEL assay. miR-145-5p expression in MPC5 cells exposed to DKD-Exo was markedly upregulated. miR-145-5p negatively regulated Srgap2 levels. Exposure of MPC5 cells to DKD-Exo reduced Srgap2 expression and activated ROCK, which was partly reversed by the presence of the miR-145-5p inhibitor or Srgap2 overexpression. The apoptosis of MPC5 cells exposed to DKD-Exo increased significantly, which was counteracted by the addition of the miR-145-5p inhibitor and fasudil. The results showed that urinary exosomal miR-145-5p from patients with DKD induced podocyte apoptosis by inhibiting Srgap2 and activating the RhoA/ROCK pathway, suggesting that urinary exosomal miR-145-5p is involved in the pathological process of DKD and could become a noninvasive diagnostic biomarker for DKD.

UNASSIGNED: Podocyte apoptosis is a common mechanism driving progression in Alport syndrome (AS). This study aimed to investigate the mechanism of podocyte apoptosis caused by COL4A3 mutations.
UNASSIGNED: We recruited patients with autosomal dominant AS (ADAS). Patients with minimal change disease (MCD) were recruited as controls. Microarray analysis was carried out on isolated glomeruli from the patients and validated. Then, corresponding mutant human podocytes (p.C1616Y) and 129 mice (p.C1615Y, the murine homolog to the human p.C1616Y) were constructed. The highest differentially expressed genes (DEGs) from microarray analysis were validated in transgenic mice and podocytes before and after administration of MMP-2 inhibitor (SB-3CT) and NOX4 inhibitor (GKT137831). We further validated NOX4/MMP-2/apoptosis pathway by real-time polymerase chain reaction (PCR), immunohistochemistry, and western blot in renal tissues from the ADAS patients.
UNASSIGNED: Using microarray analysis, we observed that DEGs, including NOX4/H2O2, MMP-2, and podocyte apoptosis-related genes were significantly upregulated. These genes were validated by real-time PCR, histologic analysis, and western blot in corresponding mutant human podocyte (p.C1616Y) and/or mice models (p.C1615Y). Moreover, we found podocyte apoptosis was abrogated and MMP-2 expression was down-regulated both in vivo and in vitro by NOX4 inhibition, urinary albumin-to-creatinine ratio, 24-hour proteinuria; and renal pathologic lesion was attenuated by NOX4 inhibition in vivo. Furthermore, podocyte apoptosis was attenuated whereas NOX4 expression remained the same by inhibition of MMP-2 both in vivo and in vitro.
UNASSIGNED: These results indicated that NOX4 might induce podocyte apoptosis through the regulation of MMP-2 in patients with COL4A3 mutations. Our findings provided new insights into the mechanism of ADAS.

Glucagon like peptide-1 receptor agonists (GLP-1RAs) belong to the class of incretin drugs. Incretin is a hormone secreted into blood by intestinal cells after food stimulation that induces insulin secretion. Liraglutide is a long-acting GLP-1RA that can reduce blood pressure, blood lipids, and inflammation. Previous studies showed that liraglutide can promote white fat browning and improve renal outcomes in patients with type 2 diabetes mellitus. However, no studies have linked white fat browning to kidney damage. The objective of this study was to investigate the effects of liraglutide-induced white fat browning on podocyte apoptosis in diabetic nephropathy. We also aimed to determine whether podocytes express glucagon like peptide-1 receptor (GLP-1R) and if liraglutide directly affects podocytes via GLP-1R. We assessed fat and renal function in db/db and wild-type mice and the effects of adipocyte conditioned medium on cultured podocytes. Liraglutide (400 mg/kg/d) was subcutaneously injected for 8 weeks. Liraglutide promoted white fat browning in vivo. During adipogenic differentiation of 3T3-L1 cells in vitro, liraglutide also upregulated expression of peroxisome proliferator-activated receptor γ coactivator-1 alpha (PGC1α) and uncoupling protein 1 (UCP1), which can induce white fat browning in vitro. Furthermore, we found that supernatant from 3T3-L1 cells stimulated by liraglutide reduced podocyte apoptosis. The inhibitory effect of liraglutide on apoptosis was eliminated by exogenous TNF-α. Finally, podocytes express GLP-1R. In vivo and in vitro studies showed that the apoptosis of podocytes in diabetic nephropathy may be related to the effect of liraglutide on promoting white lipid browning. Similarly, liraglutide may directly affect podocytes via GLP-1R.

Defective antioxidant system as well as mitochondrial dysfunction contributes to the pathogenesis and progression of diabetic kidney disease (DKD). Nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated signaling is the central defensive mechanism against oxidative stress and therefore pharmacological activation of Nrf2 is a promising therapeutic strategy. In this study, using molecular docking we found that Astragaloside IV (AS-IV), an active ingredient from traditional formula of Huangqi decoction (HQD), exerted a higher potential to promote Nrf2 escape from Keap1-Nrf2 interaction via competitively bind to amino acid sites in Keap1. When podocyte exposed to high glucose (HG) stimulation, mitochondrial morphological alterations and podocyte apoptosis were presented and accompanied by Nrf2 and mitochondrial transcription factor A (TFAM) downregulation. Mechanistically, HG promoted a decrease in mitochondria-specific electron transport chain (ETC) complexes, ATP synthesis and mtDNA content as well as increased ROS production. Conversely, all these mitochondrial defects were dramatically alleviated by AS-IV, but suppression of Nrf2 with inhibitor or siRNA and TFAM siRNA simultaneously alleviated the AS-IV efficacy. Moreover, experimental diabetic mice exhibited significant renal injury as well as mitochondrial disorder, corresponding with the decreased expression of Nrf2 and TFAM. On the contrary, AS-IV reversed the abnormality and the Nrf2 and TFAM expression were also restored. Taken together, the present findings demonstrate the improvement of AS-IV on mitochondrial function, thereby resistance to oxidative stress-induced diabetic kidney injury and podocyte apoptosis, and the process is closely associated with activation of Nrf2-ARE/TFAM signaling.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a unique component of the ubiquitin-proteasome system (UPS), which has multiple activities in maintaining intracellular ubiquitin levels. We previously reported the aberrant low expression of UCHL1 in podocytes of non-immune complex-mediated glomerulonephritis, and recent studies indicate that anti-UCHL1 antibody was responsible for the refractory minimal change disease (MCD), but the specific effect of UCHL1 to the podocytopathy has not been determined. Therefore, we generated podocyte-specific UCHL1 gene knockout (UCHL1cre/cre) rats model. Podocyte-specific UCHL1 knockout rats exhibited severe kidney damage, including segmental/global glomerulosclerosis, kidney function damage and severe proteinuria, compared with littermate control. Subsequently, by carrying out mass spectrometry analysis of isolated glomeruli of rats, abnormal protein accumulation of ECM-receptor Interaction was found in UCHL1cre/cre rats. Mechanistic studies in vivo and in vitro revealed that aberrant protein accumulation after UCHL1 deficiency induced endoplasmic reticulum (ER) stress, unfolded protein reaction (UPR) to reduce the protein level of podocyte skeleton proteins, and CHOP mediated apoptosis as well, which related to the dysfunction of the ubiquitin-proteasome system with decreased free monomeric ubiquitin level, thereby affecting protein ubiquitination and degradation. In addition, inhibition of ER stress by 4-PBA could attenuate the degree of ER stress and podocyte dysfunction. Our study indicates that UCHL1 is a potential target for preventing podocytes injury in some non-immune complex-mediated glomerulopathy.

OBJECTIVE: To investigate the effect of emodin on high glucose (HG)-induced podocyte apoptosis and whether the potential anti-apoptotic mechanism of emodin is related to induction of adenosine-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)-mediated autophagy in podocytes (MPC5 cells) in vitro.
METHODS: MPC5 cells were treated with different concentrations of HG (2.5, 5, 10, 20, 40, 80 and 160 mmol/L), emodin (2, 4, 8 µ mol/L), or HG (40 mmol/L) and emodin (4 µ mol/L) with or without rapamycin (Rap, 100 nmol/L) and compound C (10 µ mol/L). The viability and apoptosis of MPC5 cells were detected using cell counting kit-8 (CCK-8) assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, autophagy marker light chain 3 (LC3) I/II, and AMPK/mTOR signaling pathway-related proteins were determined by Western blot. The changes of morphology and RFP-LC3 fluorescence were observed under microscopy.
RESULTS: HG at 20, 40, 80 and 160 mmol/L dose-dependently induced cell apoptosis in MPC5 cells, whereas emodin (4 µ mol/L) significantly ameliorated HG-induced cell apoptosis and caspase-3 cleavage (P<0.01). Emodin (4 µ mol/L) significantly increased LC3-II protein expression levels and induced RFP-LC3-containing punctate structures in MPC5 cells (P<0.01). Furthermore, the protective effects of emodin were mimicked by rapamycin (100 nmol/L). Moreover, emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C (10 µ mol/L) reversed emodin-induced autophagy activation.
CONCLUSIONS: Emodin ameliorated HG-induced apoptosis of MPC5 cells in vitro that involved induction of autophagy through the AMPK/mTOR signaling pathway, which might provide a potential therapeutic option for diabetic nephropathy.