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. 2018 Jun 15;19(1):140.
doi: 10.1186/s12882-018-0940-3.

IP3R-Grp75-VDAC1-MCU calcium regulation axis antagonists protect podocytes from apoptosis and decrease proteinuria in an Adriamycin nephropathy rat model

Han Xu  1 , Na Guan  2 , Ya-Li Ren  3 , Qi-Jiao Wei  1 , Ying-Hong Tao  4 , Guo-Sheng Yang  4 , Xiao-Ya Liu  1 , Ding-Fang Bu  5 , Ying Zhang  5 , Sai-Nan Zhu  6
Affiliations

IP3R-Grp75-VDAC1-MCU calcium regulation axis antagonists protect podocytes from apoptosis and decrease proteinuria in an Adriamycin nephropathy rat model

Han Xu et al. BMC Nephrol. .

Abstract

Background: The mechanism of podocyte apoptosis is not fully understood. In addition, the role of the inositol 1,4,5-triphosphate receptor (IP3R)/glucose-regulated protein 75 (Grp75)/voltage-dependent anion channel 1 (VDAC1)/mitochondrial calcium uniporter (MCU) calcium regulation axis, which is located at sites of endoplasmic reticulum (ER) mitochondria coupling, in the mechanism of podocyte apoptosis is unclear. This study aimed to understand the roles of this axis in podocyte apoptosis and explore potential targets for podocyte protection.

Methods: The expression of IP3R, Grp75, VDAC1, and MCU and mitochondrial Ca2+ were analyzed during Adriamycin- or angiotensin II-induced apoptosis in cultured mouse podocytes. The interaction between IP3R, Grp75, and VDAC1 was investigated using co-immunoprecipitation experiments. The effects of IP3R, Grp75, and MCU agonists and antagonists on mitochondrial Ca2+ and apoptosis were investigated in cultured podocytes. The podocyte-protective effects of an MCU inhibitor were further investigated in rats with Adriamycin-induced nephropathy.

Results: Increased expression of IP3R, Grp75, VDAC1 and MCU, enhanced interaction among the IP3R-Grp75-VDAC1 complex, mitochondrial Ca2+ overload, and increased active caspase-3 levels were confirmed during Adriamycin- or angiotensin II-induced mouse podocyte apoptosis. Agonists of this axis facilitated mitochondrial Ca2+ overload and podocyte apoptosis, whereas specific antagonists against IP3R, Grp75, or MCU prevented mitochondrial Ca2+ overload and podocyte apoptosis. A specific MCU inhibitor prevented Adriamycin-induced proteinuria and podocyte foot process effacement in rats.

Conclusions: This study identified a novel pathway in which the IP3R-Grp75-VDAC1-MCU calcium regulation axis mediated podocyte apoptosis by facilitating mitochondrial Ca2+ overload. Antagonists that inhibit Ca2+ transfer from ER to mitochondria protected mouse podocytes from apoptosis. An MCU inhibitor protected podocytes and decreased proteinuria in rats with Adriamycin-induced nephropathy. Therefore, antagonists to this pathway have promise as novel podocyte-protective drugs.

Keywords: Apoptosis; Calcium; Endoplasmic reticulum mitochondria coupling; Mitochondria; Podocyte.

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Conflict of interest statement

Ethics approval

The rats used in this study were bought from Beijing Vital River Laboratory Animal Technology Co., Ltd. All protocols were approved by the Institutional Animal Care and Use Committee of Peking University First Hospital (Number: 11400700229305).

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
IP3R-Grp75-VDAC1-MCU axis, mitochondrial Ca2+, and apoptosis in podocytes treated with ADR and Ang II. Ctl, control; ADR, Adriamycin; Ang II, angiotensin II; IP3R, inositol 1,4,5-triphosphate receptor; Grp75, glucose-regulated protein 75; VDAC1, voltage dependent anion channel 1; MCU, mitochondrial calcium uniporter; a, compared with the Ctl group; **, P < 0.01; *, P < 0.05. Compared with the Ctl podocytes, a) The cells in Q3 which were annexin high and PI low were counted as apoptotic cells. Significantly increased apoptosis rate was found in ADR- (n = 3) and Ang II-treated podocytes (n = 3), b) Significantly increased levels of active caspase-3 was found in podocytes treated with ADR (n = 4) and Ang II (n = 4), c) Mitochondrial Ca2+ levels were increased in podocytes treated with ADR (n = 12) and Ang II (n = 12), d) Significantly increased expression of IP3R, Grp75, VDAC1, and MCU were found in podocytes treated with ADR (n = 6) and Ang II (n = 6). e Co-IP were performed to analyze the IP3R-Grp75-VDAC1 interaction. Normal rabbit IgG without antigenicity was used as a negative control. Lysates from both Ctl and ADR- or Ang-II treated podocytes without immunoprecipitation were used as a positive control (input). The proteins pulled down by anti-Grp75 antibodies was analyzed by western blotting. Compared with the Ctl podocytes, there was a significant increase in the amount of IP3R, Grp75, and VDAC1 in pulled down samples from ADR- (n = 3) or Ang II-treated podocytes (n = 3)
Fig. 2
Fig. 2
IP3 and MCU agonists induced podocyte mitochondrial Ca2+ overload and apoptosis in vitro. Ctl, control; IP3: D-myo-inositol 1, 4, 5-triphosphate tripotassium salt; Spm: Spermine; a, compared with the Ctl group; b, compared with the IP3 group; **, P < 0.01; *, P < 0.05. a The cells in Q3 which were annexin high and PI low were counted as apoptotic cells. Compared with the Ctl podocytes, IP3 (n = 9) and Spm (n = 3) all induced significant increase of the podocyte apoptosis rate. b Compared with the Ctl podocytes, IP3 and Spm all induced significant increase of mitochondrial Ca2+ in mouse podocytes (n = 12). The increase of mitochondrial Ca2+ induced by IP3 was partially prevented by EDTA
Fig. 3
Fig. 3
IP3R antagonist prevented podocytes from ADR- and Ang II-induced apoptosis in vitro. Ctl, control; ADR, Adriamycin; XeC, Xestospongin C (IP3R inhibitor); a, compared with Ctl group; b, compared with the ADR or Ang II group; *, P < 0.05; **, P < 0.01. a The cells in Q3 which were annexin high and PI low were counted as apoptotic cells. Compared with ADR or Ang II -treated podocytes, XeC decreased the podocyte apoptosis rate significantly (n = 3). b Compared with ADR or Ang II -treated podocytes, XeC decreased active caspase-3 in podocytes significantly. c Compared with ADR or Ang II -treated podocytes, XeC decreased mitochondrial Ca2+ levels in podocytes significantly
Fig. 4
Fig. 4
MCU antagonist prevented podocytes from ADR- and Ang II-induced apoptosis in vitro. Ctl, control; ADR, Adriamycin; Ang II, angiotensin II; Ru360, MCU inhibitor; a, compared with Ctl group; b, compared with the ADR or Ang II group; *, P < 0.05; **, P < 0.01. a The cells in Q3 which were annexin high and PI low were counted as apoptotic cells. Compared with ADR or Ang II -treated podocytes, Ru360 decreased the podocyte apoptosis rate significantly (n = 3). b Compared with ADR or Ang II -treated podocytes, Ru360 decreased active caspase-3 in mouse podocytes significantly (n = 4). c Compared with ADR or Ang II -treated podocytes, Ru360 decreased mitochondrial Ca2+ levels in mouse podocytes significantly (n = 10)
Fig. 5
Fig. 5
Knocking down Grp75 protected podocytes from ADR- and Ang II-induced apoptosis in vitro. Ctl, control; Ang II, angiotensin II; Grp75, glucose-regulated protein 75; siCtl, non-targeted negative control siRNA; siGrp75, siRNA to knock down Grp75; a, compared with the Ctl group; b, compared with the ADR or Ang II group; *, P < 0.05; **, P < 0.01. a Knocking down Grp75 decreased Grp75 compared with non-targeted negative control siRNA group; b) The cells in Q3 which were annexin high and PI low were counted as apoptotic cells. Compared with ADR or Ang II -treated podocytes, knock down of Grp75 decreased the podocyte apoptosis rate significantly. c Compared with ADR or Ang II -treated podocytes, knock down of Grp75 decreased active caspase-3 significantly. d Compared with ADR or Ang II -treated podocytes, knock down of Grp75 decreased mitochondrial Ca2+ levels significantly
Fig. 6
Fig. 6
The protective effects of ruthenium red in an ADR nephropathy rat model. W, week; Ctl, normal saline group; RR, ruthenium red group; ADR, Adriamycin group; ADR + RR, Adriamycin plus RR group. a, compared with the Ctl group; b, compared with the RR group; c, compared with the ADR group. *, P < 0.05; **, P < 0.01. a Compared with the Ctl (n = 6) or RR (n = 6) groups, 24 h urinary protein levels increased significantly in the ADR group (n = 10) at 2, 4, and 6-weeks. Compared with the ADR group, 24 h urinary protein levels were decreased significantly in rats in the ADR + RR (n = 6) group at 2, 4, and 6 weeks. b and c Compared with the Ctl (n = 6) or RR (n = 6) groups, significantly increased podocyte foot process width was observed in rats in the ADR group (n = 6). Compared with the ADR group, podocyte foot process width was decreased significantly in rats in the ADR + RR group (n = 6)
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References

    1. Kemper MJ, Valentin L, van Husen M. Difficult-to-treat idiopathic nephrotic syndrome: established drugs, open questions and future options. Pediatr Nephrol. 2017;6:1–9. - PubMed
    1. Wieder N, Greka A. Calcium, TRPC channels, and regulation of the actin cytoskeleton in podocytes: towards a future of targeted therapies. Pediatr Nephrol. 2016;31:1047–1054. doi: 10.1007/s00467-015-3224-1. - DOI - PMC - PubMed
    1. Tharaux PL, Huber TB. How many ways can a podocyte die? Semin Nephrol. 2012;32:394–404. doi: 10.1016/j.semnephrol.2012.06.011. - DOI - PubMed
    1. Pedriali G, Rimessi A, Sbano L, Giorgi C, Wieckowski MR, Previati M, et al. Regulation of endoplasmic reticulum-mitochondria Ca2+ transfer and its importance for anti-Cancer therapies. Front Oncol. 2017;7:180. doi: 10.3389/fonc.2017.00180. - DOI - PMC - PubMed
    1. Marchi S, Patergnani S, Missiroli S, Morciano G, Rimessi A, Wieckowski MR, et al. Mitochondrial and endoplasmic reticulum calcium homeostasis and cell death. Cell Calcium. 2018;69:62–72. doi: 10.1016/j.ceca.2017.05.003. - DOI - PubMed

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