Backgroud/Aims: The aim of this study was to investigate the potential renoprotective effect of rapamycin on the autophagy of podocytes treated with the supernatant of mesangial cells cultured with aggregated IgA1 (aIgA1) from immunoglobulin A nephropathy (IgAN) patients. Methods: Monomeric IgA1 (mIgA1) was isolated from the serum of IgAN patients or healthy volunteers, and then transformed to aIgA1 by heating. Subsequently, the aIgA1-mesangial cell supernatant was prepared by collecting the medium of mouse mesangial cells (MSC1097) cultured with aIgA1 (100 mg/L) from different IgAN patients or healthy volunteers for 48 h. Subsequently mouse podocytes (MPC5) were exposed to the supernatant of the aIgA1-mesangial cells for 24 h, using 100 mg/L aIgA1 from healthy volunteers as the control group or 100 mg/L aIgA1 from IgAN patients as the IgANs group, in RPMI 1640 medium. The MPC5 cells in the IgANs+Rap group were cultured with rapamycin (10 nmol/L) and the supernatant of MSC-1097 cells cultured with aIgA1 from IgAN patients in RPMI 1640 medium. Autophagy was assessed by western blot analysis (LC3, p62), electron microscopy, and immunofluorescence staining (LC3, p62, and CD63). The apoptosis of podocytes was evaluated by flow cytometry, and the expression of apoptosis-associated proteins cleaved-caspase-3 and caspase-3 were determined by western blot analysis. Results: Deficient autophagy, which was evident by decreased LC3-II and CD63 levels, caused accumulation of p62, and fewer autophagosomes were observed in the MPC5 cells cultured with the IgAN supernatant, along with stronger expression of cleaved caspase-3 and a higher apoptosis rate. Inhibition of autophagy was alleviated in the IgANs+Rap group. The LC3-II/LC3-I ratio increased by almost 30%, the accumulated p62 amount was reduced by 50%, and the number of autophagosomes per podocyte increased to about 7 times that of the IgAN groups. These results were confirmed by immunofluorescence staining. In addition, the apoptosis rate of MPC5 cells decreased from 19.88% in the IgAN group to 16.78% in the IgANs+Rap group, which was accompanied by a weaker expression level of cleaved caspase-3. Conclusions: Rapamycin can reduce the apoptosis of podocytes by inducing autophagy in IgAN.

Therefore, the results of the present study indicate that autophagy participates in podocyte injury and the progression of IgAN and IMN.

Objective: To identify the significance of autophagy in lupus nephritis (LN). Methods: The number of autophagosomes in podocytes was counted and the expression of multiple molecular markers associated with autophagy was evaluated in LN specimens: in renal biopsy specimens from 90 patients with LN and 15 healthy controls, autophagosomes in podocytes were counted using transmission electron microscopy and the expression levels of four autophage related proteins including Beclin-1, microtubule-associated protein light chain 3 (LC3), autophagy-related gene 7 (Atg7), and UNC-51-like kinase 1 (ULK1) were measured using immunohistochemistry. Results: The number of autophagosomes in patients with LN types III, IV, and combined V–IV type were significantly higher than in controls ( p < 0.0001; p < 0.0001; p = 0.009, respectively). However, the autophagosomes numbers in patients with II and V types LN were significantly lower than controls (both p < 0.0001). Various levels of marker expression were identified, and they correlated significantly with LN pathology classifications. Moreover, the percentage of marker expression in LN types III, IV and V-IV were significantly higher than controls ( p < 0.05), while that in types II and V were lower than controls, although the difference for LC3 and ULK1 was not statistically significant. Conclusions: Autophagy activity and expression pattern of autophagy-related markers in podocytes were significantly positively correlated with LN of types III, IV, and V–IV, but negatively correlated with II and V types. Therefore autophagy could be a useful predictor of LN pathology type, and be informative and helpful in the development of treatment strategies in clinical settings.

Triptolide is often used to treat patients with immunoglobulin A nephropathy (IgAN), especially in Asia. However, its detailed mechanism remains unclear. In vitro experiments were conducted with podocytes exposed to aggregated IgA (aIgA)-MSC1097-conditioned media. A total of four groups were compared in this study: A control group (CON); a healthy supernatant group (HEAs); an IgAN supernatant group (IgANs); and a triptolide group (TRI). First, aggregated IgA1 (aIgA1) was generated by heating monomeric IgA1 (mIgA1) from IgAN patients or healthy subjects. Next, the conditioned supernatant of MSC-1097 cells cultured with aIgA1 (100 mg/l) from IgAN patients (IgANs) or healthy subjects (HEAs) or without aIgA1 (CON) were harvested and used to incubate MPC5 cells. MPC5 cells in the TRI group were cultured with triptolide (10 ng/ml) and conditioned media from MSC-1097 cells cultured with aIgA1 from IgAN patients. After 24 h of treatment, MPC5 cells were collected to measure autophagy-related protein levels, including microtubule-associated protein light chain 3 (LC3), p62, cluster of differentiation (CD)63, phosphorylated-protein kinase B (Akt), Akt, p-mammalian target of rapamycin (mTOR), and mTOR, via western blotting, immunofluorescence or both, and to determine apoptosis by flow cytometry. All the results showed no difference between the CON and the HEAs. Compared to the CON and the HEAs, MPC5 cells in the IgANs group showed reduced autophagy, which was presented as decreased levels of LC3-II and CD63, as well as accumulation of p62, and an increased podocyte apoptosis rate. This was partly rescued by the addition of triptolide. Moreover, the p-mTOR/mTOR ratio increased in the IgANs group and decreased in the TRI group. Therefore, these results suggest that triptolide protects podocyte autophagy in IgAN patients.