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

Abstract

BackgroundThe 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.MethodsThe 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.ResultsIncreased 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.ConclusionsThis 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.

Involvement of the IP3R-Grp75-VDAC1-MCU calcium axis in proteinuria in adriamycin-induced nephropathy rats

“…Antagonists of this calcium axis were shown to prevent podocyte apoptosis [2]. We also found that the MCU inhibitor ruthenium red (RR) significantly improved proteinuria and podocyte foot process effacement in ADR-induced nephropathy rats [2]. Based on the above findings, we hypothesized that the IP 3 R-Grp75-VDAC1-MCU calcium axis is involved in the development of proteinuria.…”

“…All protocols were approved by the Institutional Animal Care and Use Committee of Peking University First Hospital (number: 11400700229305). This study used a previously reported model [2]. ADR (D1515, Sigma, Santa Clara, California, USA) was successfully used to induce a nephropathy model in Sprague-Dawley rats (Beijing Vital River Laboratory Animal Technology Co. Ltd.) as previously 4 reported.…”

“…The 2-ΔΔCT method was used to calculate the relative expression of target genes to GAPDH [10]. The primers used were as followed: University, China) were cultured as we previously reported [2]. The podocytes were cultured at 33°C in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin, and 10 U/ml recombinant mouse interferon-γ (IFN-γ) to induce proliferation and transferred to culture media lacking IFN-γ at 37°C to differentiate for 10-14 days [2].…”

“…The primers used were as followed: University, China) were cultured as we previously reported [2]. The podocytes were cultured at 33°C in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin, and 10 U/ml recombinant mouse interferon-γ (IFN-γ) to induce proliferation and transferred to culture media lacking IFN-γ at 37°C to differentiate for 10-14 days [2]. The podocytes were pre-treated with the MCU inhibitor RR for 60 min before the addition of 0.5 μg/ml ADR for 24 h [2].…”

“…Previously, we investigated the mechanism of podocyte injury with a focus on the intracellular calcium axis [2]. The endoplasmic reticulum (ER) and mitochondria are the two main and 3 most important calcium reservoirs in podocytes.…”