IgG expressed by renal tubular epithelial cells in epithelial mesenchymal transformation and interstitial fibrosis in diabetic kidney disease

Abstract

Studies have reported that immunoglobulin G (IgG) "deposited" in the basement membrane of renal tubules is associated with tubulointerstitial damage in patients with diabetic kidney disease (DKD). Our previous study found that renal tubular epithelial cells (RTECs) can express and secrete IgG (RTEC-IgG) which may be associated with fibrosis. The present study aimed to explore the role of RTEC-IgG in renal tubulointerstitial fibrosis (TIF) in DKD. The results showed that RTEC-IgG expression was up-regulated in the renal tubulointerstitium of DKD patients and was associated with worse kidney function, more severe anemia, and higher interstitial fibrosis and tubular atrophy (IFTA) scores, and positively correlated with tubular epithelial mesenchymal transition (EMT) and TIF. IgG expression was also enhanced in the renal tubulointerstitium of DKD mice, which was positively correlated with TIF. High glucose induced an over expression of IgG in human renal tubular epithelial cells, and knockdown of IgG with siRNA relieved the expression of α-smooth muscle actin (SMA), collagen IV, fibronectin, and transforming growth factor (TGF)-β1 under high glucose conditions. In conclusion, our study suggests that RTEC-IgG is involved in the development of DKD by promoting EMT and interstitial fibrosis via TGF-β1.

Keywords: Diabetic kidney disease; epithelial mesenchymal transition; immunoglobulin G; renal tubular epithelial cell; renal tubulointerstitial fibrosis.

Figure 1.

RTEC-IgG expression in renal tubulointerstitium and it’ s relation with EMT and TIF in DKD patients. RTEC-IgG expression (identified by RP215) was significantly elevated and positively correlated with EMT and TIF in DKD patients. (A and B) Immunohistochemistry staining for RTEC-IgG (identified by RP215) expression in renal tubulointerstitium of NC and DKD patients. Scale bars, 50 µm and 10 µm. (C) Correlation analysis between the RP215 and α-SMA positive area of DKD. (D) Correlation analysis between the RP215 and Col IV positive area of DKD. Data are presented as the mean ± SEM (NC, n = 6, DKD, n = 60). *p < 0.05 vs. NC. RTEC-IgG, IgG expressed by renal tubular epithelial cell; DKD, diabetic kidney disease; EMT, epithelial mesenchymal transition; TIF, tubulointerstitial fibrosis; NC, normal control.

Figure 2.

IgG Expression in renal tubular epithelial cells and it is relation with TIF in DKD model mice. IgG expression in renal tubular epithelial cells was significantly elevated and positively correlated with TIF in DKD model mice. (A and B) Immunohistochemistry staining for IgG expression in renal tubulointerstitium of Sham and DKD mice. Scale bars, 25 µm and 10 µm. (C) The blood glucose level in Sham and DKD mice. (D) Urine albumin to creatinine ratio (UACR) in Sham and DKD mice. (E) Serum creatinine (sCr) in Sham and DKD mice. (F) HE, Masson and immunohistochemistry staining of Col IV in renal tubulointerstitium from Sham and DKD mice. (G) Area of tubulointerstitial fibrosis of Sham and DKD mice assessed by Masson staining. (H) Immunohistochemistry analysis of Col IV expression in renal tubulointerstitium of Sham and DKD mice. (I) Correlation analysis between the IgG and Col IV positive area of DKD mice. Data are presented as the mean ± SEM. n = 6 mice per group. *p < 0.05, ****p < 0.0001 vs. Sham mice. RTEC-IgG, IgG expressed by renal tubular epithelial cell; DKD, diabetic kidney disease; TIF, tubulointerstitial fibrosis. HE, hematoxylin and eosin.

Figure 3.

RTEC-IgG in the regulation of hyperglycemia-induced EMT and ECM deposition via TGF-β1 in HK-2 cells. α-SMA, Col IV, FN and TGF-β1, induced by high glucose, were accompanied by higher RTEC-IgG and were alleviated by knock-down of RTEC-IgG with siR-IgG in HK-2 cells. (A, B) Western blot analysis of the protein levels of RTEC-IgG (identified by RP215), α-SMA, Col IV, FN and TGF-β1 in NG or HG after siR-NC. (C) qPCR analysis of the mRNA levels of IgG, α-SMA, FN and TGF-β1 in HG after siR-NC or siR-IgG. (D, E) Western blot analysis of the protein levels of RTEC-IgG (identified by RP215), α-SMA, Col IV, FN and TGF-β1 in HG after siR-NC or siR-IgG. Data are presented as the mean ± SEM. n = 3. *p < 0.05 vs. NG + siR-NC. **p < 0.01 vs. NG + siR-NC. ^#p < 0.05 vs. HG + siR-NC. ^##p < 0.01 vs. HG + siR-NC. ^###p < 0.001 vs. HG + siR-NC. NG + siR-NC, transfected with siR-NC and treated with 5.56 mM glucose. HG + siR-NC, transfected with siR-NC and treated with 30 mM glucose. HG + siR-IgG, transfected with siR-IgG and treated with 30 mM glucose. RTEC-IgG, IgG expressed by renal tubular epithelial cell; EMT, epithelial mesenchymal transition; ECM, extracellular matrix; NC, normal control.