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

Background Podocyte injury plays a key role in the development of proteinuria. We previously found that the intracellular inositol 1, 4, 5-trisphosphate receptor (IP3R)-glucose-regulated protein 75 (Grp75)- voltage dependent anion channel 1 (VDAC1)-mitochondrial calcium uniporter (MCU) calcium axis contributes to podocyte injury in cultured mouse podocytes. Objective This study investigated whether the IP3R-Grp75-VDAC1-MCU calcium axis is involved in the development and improvement of proteinuria in nephropathy rats. Methods The expression of members of the IP3R-Grp75-VDAC1-MCU calcium axis in the renal cortex of a previously established adriamycin (ADR)-induced nephropathy rat model and cultured mouse podocytes was investigated by western blot analysis and immunohistochemical staining. The effects of ruthenium red (RR), an MCU inhibitor, oninteractions in the IP3R-Grp75-VDAC1-MCU calcium axis were investigated by in vitro co-immunoprecipitation assays. Results The overexpression and inhibition of members of the glomerular IP3R-Grp75-VDAC1-MCU calcium axis were accompanied by the development and improvement of proteinuria, respectively, in nephropathy rats. RR inhibited the upregulation of members of the IP3R-Grp75-VDAC1-MCU calcium axis induced by ADR and their interactions. Conclusions The IP3R-Grp75-VDAC1-MCU calcium axis is involved in proteinuria in ADR-induced nephropathy and can be inhibited by RR.

proteinuria, hypoalbuminemia, oedema and hypercholesterolemia. Podocytes have been confirmed to be the key cell type that maintains the function of the glomerular filtration barrier. Podocyte injury is the core mechanism that contributes to the development of proteinuria. However, as the mechanism of podocyte injury has not been fully elucidated and clinical drugs used to control proteinuria remain limited, further investigation is needed [1].
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 most important calcium reservoirs in podocytes. Charged calcium is transferred from the ER to the mitochondrial matrix in a process called mitochondrial-endoplasmic reticulum coupling. The inositol 1, 4, 5-trisphosphate receptor (IP 3 R)-glucose-regulated protein 75 (Grp75)-voltage dependent anion channel 1 (VDAC1)-mitochondrial calcium uniporter (MCU) axis is the molecular basis by which Ca 2+ transfer from the ER to mitochondria is regulated in mitochondrial-endoplasmic reticulum coupling. In the calcium axis, IP 3 R is a calcium release channel located at the ER membrane, and VDAC1 is located at the outer membrane of mitochondria. MCU, which is located at the inner membrane of mitochondria, is the final channel through which calcium enters the mitochondrial matrix. Grp 75 is a bridging protein that interacts with both IP 3 R and VDAC1 to form a complex [3,4]. Proper calcium transfer from the ER to the mitochondrial matrix is key to maintaining ATP production and normal mitochondrial function [5]. We previously confirmed that the upregulation of members in the IP 3 R-Grp75-VDAC1-MCU calcium axis contributes to mitochondrial calcium overload and podocyte apoptosis induced by adriamycin (ADR) and angiotensin II, respectively. 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. To clarify its involvement, this study investigated changes in the expression of members of the IP 3 R-Grp75-VDAC1-MCU calcium axis during the development and improvement of proteinuria and podocyte injury in ADR-induced nephropathy rats and cultured mouse podocytes. After the injection of ADR, 24 h urinary protein excretion in the rats increased significantly after 6 weeks. We previously confirmed that the injection of RR (R2751, Sigma), an MCU inhibitor, significantly improves proteinuria induced by ADR after 6 weeks [2]. As reported, the Sprague-Dawley rats were divided into four groups: a normal saline control group (Ctl, n = 6), an RR control group (RR, n = 6), an ADR group (ADR, n = 10), and an ADR plus RR group (ADR + RR, n = 6). All rats were sacrificed and harvested at the 6-week time point [2]. Stored renal tissues were used for this study.

Western blot analysis
Western blot analysis was performed to detect target molecules in the renal cortex or cultured mouse podocytes. RIPA lysis buffer (89901, Thermo Scientific) containing protease inhibitor was used to extract total cellular proteins, and the proteins were boiled at 100℃ for 10 min After SDS-PAGE on 6%-15% gels, the proteins were transferred to Immuno-Bot PVDF membranes (Bio-Rad). The membranes were blocked with 5% BSA for 1 h and then incubated overnight at 4℃ with the primary antibodies anti-IP 3 R diluted 1:1000 (ab5804, Abcam), anti-Grp75

Co-immunoprecipitation assay
Co-immunoprecipitation assay was used to detect the effect of RR on the interactions between members of the IP 3 R-Grp75-VDAC1-MCU complex in cultured mouse podocytes, as we previously reported [2]. Podocyte lysates were collected and centrifuged, following which protein concentrations were determined. Coimmunoprecipitation was performed using a Thermo Scientific Pierce Co-IP Kit (26149, Thermo Fisher Scientific) according to the manufacturer's protocols.
Anti-Grp75 antibody (D13H4, #3593, Cell Signalling Technology) was used to capture proteins coupled in mitochondria/the ER, and normal rabbit IgG without antigenicity was used as a negative control. After co-immunoprecipitation, proteins pulled down by the anti-Grp75 antibody were analysed by western blotting. Lysates from both Ctl and ADR-treated podocytes without immunoprecipitation were used as positive controls (input).

Statistical analysis
Data are presented as the means ± SDs and were analysed using one-way AVONA and Student's t-tests. Differences for which P<0.05 were considered statistically significant.

VDAC1-MCU calcium axis in ADR nephropathy rats
Western blot analysis showed that, compared with the Ctl and RR groups, renal cortical expression of IP 3 R, Grp75, VDAC1 and MCU was significantly increased in rats in the ADR group at 6 weeks after the injection of ADR (Fig. 1) when proteinuria peaked.

VDAC1-MCU calcium axis in ADR nephropathy rats
The glomerular expression of molecules in the IP 3 R-Grp75-VDAC1-MCU calcium axis was analysed by immunohistochemical analysis. The ratio of the glomerular staining area to the total glomerular area was used to reflect the glomerular expression level of each molecule. In the Ctl group, glomerular staining for members of the IP 3 R-Grp75-VDAC1-MCU calcium axis was weak. No significant differences in the glomerular expression levels of each molecule were found between the Ctl and RR groups. Compared with the Ctl group, the glomerular expression of IP 3 R, Grp75, VDAC1 and MCU was significantly increased in the ADR group (Fig. 2). Similar findings were found by immunofluorescence staining for the glomerular expression of members of the IP 3 R-Grp75-VDAC1-MCU calcium axis (Fig. 3). In the ADR group, IP 3 R, Grp75, VDAC1 and MCU expression in the glomerulus co-localized with podocyte-specific podocin and synaptopodin molecules.

RR inhibited the increased glomerular expression of members of the IP 3 R-Grp75-VDAC1-MCU calcium axis in ADR rats
Compared with the ADR group, the renal cortical expression of IP 3 R, Grp75, VDAC1 and MCU was decreased significantly in the ADR+RR group at the 6-week time point, as shown by western blot analysis (Fig. 1). By immunohistochemical analysis, the glomerular expression of IP 3 R, Grp75, VDAC1 and MCU was shown to be significantly decreased in rats in the ADR+RR group compared with the ADR group (Fig. 2).
As shown by RT quantitative PCR, compared with the Ctl group of cultured mouse podocytes, the expression of IP 3 R, Grp75, VDAC1 and MCU was significantly increased in the ADR-treated cultured mouse podocytes. Compared with the ADRtreated podocytes, the expression of IP 3 R, Grp75, VDAC1 and MCU was significantly decreased in the ADR+RR-treated podocytes (Fig. 5).

Discussion
Podocytes play a key role in maintaining the glomerular filtration barrier and preventing the development of proteinuria. Different mechanisms of podocyte injury have been reported [11]. We previously investigated the mechanism of podocyte injury with a focus on intracellular calcium regulation by the IP 3 R-Grp75-VDAC1-MCU calcium axis, which is involved in mitochondrial-ER coupling. The IP 3 R-Grp75-VDAC1-MCU calcium axis regulates calcium transfer from the ER to the mitochondrial matrix. Proper calcium transfer into the mitochondrial matrix is a prerequisite for mitochondrial ATP production [12]. We previously found that the upregulation of members of the IP 3 R-Grp 75-VDAC1-MCU axis in cultured mouse podocytes contributed to podocyte injury induced by ADR and that antagonists to members of this axis inhibited mitochondrial calcium overload and podocyte apoptosis. We also found that the inhibition of MCU by RR significantly improved podocyte injury and proteinuria in ADR-induced nephropathy rats [2]. To the best of our knowledge, whether the IP 3 R-Grp75-VDAC1-MCU axis is involved in the development and improvement of proteinuria is unclear. Considering the crucial role played by podocytes in the development of proteinuria, we hypothesized that the abnormal expression of IP 3 R-Grp75-VDAC1-MCU axis members is involved in the development of proteinuria. In this study, we used tissues from a previously established ADR nephropathy rat model to investigate this hypothesis, which revealed several findings.
First, we found that the upregulation of members of the IP 3 R-Grp75-VDAC1-MCU calcium axis was involved in the development of proteinuria in ADR-induced nephropathy rats. At 6 weeks after ADR injection, when urinary protein peaked, as reported previously [2], the renal cortical expression of members of the IP 3 R-Grp75-VDAC1-MCU calcium axis in ADR-induced nephropathy rats was increased significantly. By immunofluorescence staining with the podocyte marker molecules podocin and synaptopodin, we found that glomerular staining for members of the IP 3 R-Grp75-VDAC1-MCU calcium axis was very weak in normal control rats but obvious in ADR nephropathy rats. Glomerular staining for IP 3 R-Grp75-VDAC1-MCU axis molecules showed their expression in podocytes. The glomerular expression of molecules in the IP 3 R-Grp75-VDAC1-MCU calcium axis was further semi-quantitated by immunohistochemical staining, which showed a significant increase in glomerular staining for IP 3 R-Grp75-VDAC1-MCU calcium axis molecules in ADR-induced nephropathy rats. This phenomenon has not been reported and suggests that the increased expression of members of the IP 3 R-Grp75-VDAC1-MCU axis is involved in the mechanism of proteinuria. This result is consistent with our previous finding that the increased expression of members of this axis is involved in the mechanism of podocyte injury in cultured mouse podocytes.
Second, based on our previous finding that the MCU inhibitor RR significantly decreased proteinuria and foot process effacement in ADR-induced nephropathy rats [2], we investigated the relationship between the expression of members of the IP 3 R-Grp75-VDAC1-MCU calcium axis and improved proteinuria in the same rat model. We found that the glomerular expression levels of members of the IP 3 R-Grp75-VDAC1-MCU calcium axis decreased significantly with improvements in proteinuria in rats in the ADR + RR group. The results of this study support the involvement of molecules in the IP 3 R-Grp75-VDAC1-MCU calcium axis in proteinuria.
In addition, RR was found to regulate members of the IP 3 R-Grp75-VDAC1-MCU calcium axis. We further explored the effect of RR on these molecules in cultured mouse podocytes and found that RR significantly downregulated their expression both in protein and mRNA levels. In addition, co-immunoprecipitation experiments revealed that RR decreased interactions between members of the IP 3 R-Grp75-VDAC1 complex. The exact molecular mechanism by which RR regulates the expression of members of the IP 3 R-Grp75-VDAC1-MCU calcium axis is unclear. RR is an MCU inhibitor that can inhibit calcium influx into the mitochondrial matrix [13], which may have a negative regulatory effect on the IP 3 R-Grp75-VDAC1-MCU calcium axis.
In conclusion, this study clearly reveals that changes in the expression of members

Ethics approval and consent to participate
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). The expression of members of the IP3R-Grp75-VDAC1-MCU calcium axis in cultured mouse po The mRNA expression of members of the IP3R-Grp75-VDAC1-MCU calcium axis in cultured mo