Signal regulatory protein α protects podocytes through promotion of autophagic activity
Limin Li, Ying Liu, Shan Li, Rong Yang, Caihong Zeng, Weiwei Rong, Hongwei Liang, Mingchao Zhang, Xiaodong Zhu, Koby Kidder, Yuan Liu, Zhihong Liu, Ke Zen
Limin Li, Ying Liu, Shan Li, Rong Yang, Caihong Zeng, Weiwei Rong, Hongwei Liang, Mingchao Zhang, Xiaodong Zhu, Koby Kidder, Yuan Liu, Zhihong Liu, Ke Zen
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Research Article Nephrology

Signal regulatory protein α protects podocytes through promotion of autophagic activity

Abstract

High autophagic activity in podocytes, terminally differentiated cells that serve as main components of the kidney filtration barrier, is essential for podocyte survival under various challenges. How podocytes maintain such a high level of autophagy, however, remains unclear. Here we report that signal regulatory protein α (SIRPα) plays a key role in promoting podocyte autophagy. Unlike other glomerular cells, podocytes strongly expressed SIRPα, which was, however, downregulated in patients with focal segmental glomerulosclerosis and mice with experimental nephropathy. Podocyte SIRPα levels were inversely correlated with the severity of podocyte injury and proteinuria but positively with autophagy. Compared with WT littermates, Sirpa-deficient mice displayed greater age-related podocyte injury and proteinuria and developed more rapid and severe renal injury in various models of experimental nephropathy. Mechanistically, podocyte SIRPα strongly reduced Akt/GSK-3β/β-catenin signaling, leading to an increase in autophagic activity. Our findings thus demonstrate a critical protective role of SIRPα in podocyte survival via maintenance of autophagic activity.

Authors

Limin Li, Ying Liu, Shan Li, Rong Yang, Caihong Zeng, Weiwei Rong, Hongwei Liang, Mingchao Zhang, Xiaodong Zhu, Koby Kidder, Yuan Liu, Zhihong Liu, Ke Zen

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Figure 6

SIRPα critically controls podocyte stress adaptation through promotion of autophagy in vitro.

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SIRPα critically controls podocyte stress adaptation through promotion o...
(A) Level of autophagosomes in GFP-LC3–transgenic HPCs, transfected with SIRPα siRNA or SIRPα-overexpressing lentivirus. The cells were treated with or without HG, PAN for 48 hours. Autophagosomal degradation was inhibited with CQ for 3 hours. Numbers of punctate signals in GFP-LC3–transgenic HPCs were determined by counting approximately 60 cells from 3 independent experiments. (B) Comparison of LC3-II accumulation in PAN-induced HPCs transfected with or without SIRPα siRNA or SIRPα-overexpressing lentivirus in the presence of CQ. Immunoblots are representative of 3 independently performed experiments. Histograms are quantification of LC3-II level, and GAPDH was used as internal control. (C) Autophagic flux of HPCs transfected with or without SIRPα siRNA treated with HG, PAN, and/or Rapa for 48 hours in the presence of CQ was detected. (D) Comparison of p62 accumulation in PAN-induced HPCs transfected with SIRPα siRNA or SIRPα-overexpressing lentivirus in the absence of CQ. Immunoblots are representative of 3 independently performed experiments. Histograms show quantification of p62 level, and GAPDH was used as internal control. (E) Impaired or improved acidification of lysosomes was found by staining with LysoTracker Green in podocytes transfected with SIRPα siRNA or SIRPα-overexpressing lentivirus, respectively. (F) The level of phosphatidylserine-positive cells in HPCs transfected with or without SIRPα siRNA or SIRPα-overexpressing lentivirus treated with HG, PAN, 3-MA, and/or Rapa for 48 hours. Quantification of phosphatidylserine-positive cells was determined from 3 independent experiments. (G) Detection of kinase phosphorylation in HPCs and HPCs transfected with SIRPα-expressing plasmid using human phosphokinase arrays. Rectangles 1–3 highlight p–GSK-3α/β (S21/S9), p–Akt1/2/3 (S473), and β-catenin, respectively. The levels of p-GSK-3α/β (S21/S9), p–Akt1/2/3 (S473), and β-catenin were quantified using ImageJ software and are shown in the histograms. (H) The effect of SIRPα overexpression on autophagy through targeting p-Akt, Akt, p–GSK-3β, GSK-3β, and β-catenin. Immunoblots are representative of 3 independently performed experiments. Histograms are quantification of protein level from 3 independent experiments. Scale bar in E: 10 μm. Data in A–F and H represent mean ± SEM, and P value was analyzed by ANOVA with Tukey-Kramer test. *P < 0.05, **P < 0.01.