Effects and mechanisms of calycosin-preconditioned bone marrow derived mesenchymal stem cells on alleviating podocyte injury in mice model with chronic kidney disease

Abstract

Background: Chronic kidney disease (CKD) is a global public health problem with high morbidity and limited effective cures. B-cell translocation gene 2 (Btg2), belonging to the anti-proliferation gene family, is a suppressor gene whose mutation promotes lung and prostate cancer by inducing epithelial-mesenchymal transition (EMT) of the tumor cells. However, the role of Btg2 in CKD and whether it affects the EMT of kidney cells has not been reported. Bone marrow-derived mesenchymal stem cells (MSCs) show podocyte protective effects in CKD. Calycosin (CA), a phytoestrogen, is isolated from Astragalus membranaceus with a kidney-tonifying effect. It has been reported that CA preconditioning can enhance the protective effect of MSCs on renal fibrosis in unilateral ureteral occlusion (UUO) mice. However, the protective effect and underlying mechanism of CA-pretreated MSCs (MSCsCA) on podocytes in Adriamycin (ADR) induced focal segmental glomerulosclerosis (FSGS) mice remain unclear. Objectives: To explore the role of Btg2 in FSGS and whether it affects the podocytes’ EMT. Additionally, to investigate whether CA could enhance the podocyte protection of MSCs in FSGS induced by ADR in vivo and in vitro, and its potential mechanism related to Btg2. Methods: C57BL/6 mice or MPC5 cells were treated with ADR to detect the expression changes of Btg2 in FSGS. Podocyte-specific Btg2 knockout mice and Btg2-knockout or overexpressed MPC5 cells were used to explore the mechanism by which Btg2 affects podocyte EMT in FSGS. ADR was used to induce FSGS mice in vivo, and MSCs, CA, or MSCsCA were administrated to mice models for treatment, respectively. In vitro, ADR was used to stimulate MPC5 to induce injury, and the supernatant from MSCs, CA, or MSCsCA treated cells were collected to observe the protective effects on podocytes. The protective effect and possible mechanism of treatments on podocytes were observed by western blotting, immunohistochemistry, immunofluorescence staining, RT-PCR, Tunel staining, immunofluorescence staining, etc. Furthermore, we overexpressed Btg2 which is involved in cell apoptosis to evaluate whether MSCsCA mediated podocyte’s protective effect is associated with Btg2 inhibition in MPC5. Results: Btg2 promotes podocyte-mesenchymal transition in ADR-induced FSGS. In animal models, Btg2 was markedly upregulated by podocytes and correlated with progressive renal injury. Podocyte-specific deletion of Btg2 protected against the onset of proteinuria and glomerulosclerosis in ADR-treated mice along with inhibition of EMT markers such as α-SMA and vimentin while restoring epithelial marker E-cadherin. In cultured MPC5 podocytes, overexpression of Btg2 largely promoted ADR-induced EMT and fibrosis, which was blocked by disrupted Btg2. CA-pretreated MSCs enhanced the protective effect of MSCs against podocyte injury and the ability to inhibit podocyte EMT and apoptosis in ADR-induced FSGS mice and MPC5. The expression of Btg2 was up-regulated in ADR-induced FSGS mice and MPC5 and reversed after MSCsCA treatment, more significantly than MSCs or CA alone. When Btg2 was overexpressed in MPC5, MSCsCA could not fulfill its potential to inhibit podocyte EMT and apoptosis. Conclusion: Btg2 is pathogenic in FSGS and promotes podocyte EMT.CA-pretreated MSCs enhanced the protection of MSCs against ADR-induced podocyte EMT and apoptosis. The underlying mechanism may be related to MSCsCA targeted inhibition of the Btg2 in podocytes.