Role of IGFBP7 in Diabetic Nephropathy: TGF-β1 Induces IGFBP7 via Smad2/4 in Human Renal Proximal Tubular Epithelial Cells

Abstract

Tubular injury is one of the important determinants of progressive renal failure in diabetic nephropathy (DN), and TGF-β1 has been implicated in the pathogenesis of tubulointerstitial disease that characterizes proteinuric renal disease. The aim of this study was to identify novel therapeutic target molecules that play a role in the tubule damage of DN. We used an LC-MS/MS-based proteomic technique and human renal proximal epithelial cells (HRPTECs). Urine samples from Japanese patients with type 2 diabetes (n = 46) were used to quantify the candidate protein. Several proteins in HRPTECs in cultured media were observed to be driven by TGF-β1, one of which was 33-kDa IGFBP7, which is a member of IGFBP family. TGF-β1 up-regulated the expressions of IGFBP7 mRNA and protein in a dose- and time-dependent fashion via Smad2 and 4, but not MAPK pathways in HRPTECs. In addition, the knockdown of IGFBP7 restored the TGF-β1-induced epithelial to mesenchymal transition (EMT). In the immunohistochemical analysis, IGFBP7 was localized to the cytoplasm of tubular cells but not that of glomerular cells in diabetic kidney. Urinary IGFBP7 levels were significantly higher in the patients with macroalbuminuria and were correlated with age (r = 0.308, p = 0.037), eGFR (r = -0.376, p = 0.01), urinary β2-microglobulin (r = 0.385, p = 0.008), and urinary N-acetyl-beta-D-glucosaminidase (NAG) (r = 0.502, p = 0.000). A multivariate regression analysis identified urinary NAG and age as determinants associated with urinary IGFBP7 levels. In conclusion, our data suggest that TGF-β1 enhances IGFBP7 via Smad2/4 pathways, and that IGFBP7 might be involved in the TGF-β1-induced tubular injury in DN.

Fig 1

(A) IGFBP7 was secreted from HRPTECs exposed to TGF-β1 using an LC-MS/MS-based proteomic technique. HRPTECs were treated with D-glucose (30 mM), TGF-β1 (2.5 ng/ml) and/or metformin (10 mM) for 24 h. Samples were separated by 5%–20% SDS-PAGE and detected by Coomassie brilliant blue (CBB). Six bands were excised from the gel, and the digested peptides were separated by LC and analyzed by MS/MS as described in Materials and Methods. (B) LC-MS/MS, followed by a database search using MASCOT v.2.0 (Matrix Science). Identified peptides are shown in bold.

Fig 2. TGF-β1 induced IGFBP7 expression in HRPTECs.

HRPTECs were incubated for 48 h with the indicated concentrations of TGF-β1 in serum-free medium. IGFBP7 mRNA expression was determined by real-time RT-PCR (A). The expression of IGFBP7 was normalized to that of GAPDH in the same cell extracts. The expression of IGFBP7 protein in the cell lysates was evaluated by a Western blot analysis (B). The amounts of IGFBP7 were analyzed by western blotting. GAPDH was used as a loading control. The representative immunoblot shows the expression of IGFBP7. TGF-β1 dose-dependently enhanced the expressions of IGFBP7 mRNA (A) and protein (B) in the HRPTECs. The treatment of HRPTECs for the indicate times with 2.5 ng/ml TGF-β1 showed a time-dependent increase of IGFBP7 mRNA (C) and protein expression (D). Each bar represents means ± SD of three separate experiments. *p<0.05 vs. control, **p<0.01 vs. control.

Fig 3. IGFBP7 enhancement by TGF-β1 is dependent on Smad pathways.

(A) The effects of pretreatment of HRPTECs for 1 h with the Erk1/Erk2 inhibitor PD98059 (PD, 10 μM), the p38 MAPK inhibitor SB203580 (SB, 10 μM), or the JNK inhibitor SP600125 (SP, 10 μM), followed by the administration of TGF-β1 (2.5 ng/ml) for 48 h on the expression of IGFBP7 protein. “None” indicates no administration of the kinase inhibitor. These MAPK inhibitors did not affect the TGF-β1-induced IGFBP7 expression. (B) HRPTECs were transiently transfected with control, Smad2-, Smad3-, or Smad4-specific siRNAs (25 nM final concentration). Forty-eight hours after transfection, the HRPTECs were serum-starved for an additional 24 h and then stimulated with TGF-β1 (2.5 ng/ml) for 48 h, and a western blot analysis for IGFBP7 protein was performed. Lowering the expressions of Smad2 and Smad4 proteins reduced the TGF-β1-induced IGFBP7 expression. **p<0.01 vs. control, ^##p<0.01 vs. TGF-β1-treated HRPTECs.

Fig 4. Knockdown of IGFBP7 restored the TGF-β1-induced EMT in HRPTECs.

(A) TGF-β1-induced an epithelial-mesenchymal-transition (EMT) in HRPTECs. HRPTECs were incubated with 2.5 ng/ml TGF-β1 for the indicated times, and then the extracted proteins were analyzed by western blotting using a mesenchymal marker, α-SMA and an epithelial marker, E-cadherin, respectively. TGF-β1 induced α-SMA expression and decreased the E-cadherin expressions in HRPTECs. (B) The knockdown of IGFBP7 attenuated the TGF-β1-induced α-SMA expression in the HRPTECs. HRPTECs were transfected with control or IGFBP7 siRNAs (25 nM final concentrations) and subsequently stimulated with TGF-β1 (2.5 ng/ml, 48 h). Lowering the expressions of IGFBP7 proteins reduced the TGF-β1-induced α-SMA expression, but failed to restore the TGF-β1-decreased E-cadherin expression. Values are means ± SD. **p<0.01 vs. control, ^##p<0.01 vs. TGF-β1-treated HRPTECs, ^†p<0.05, ^††p<0.01 vs. control siRNA-transfected HRPTECs, ^‡p<0.01 vs. control siRNA-transfected and TGF-β1-treated HRPTECs, n = 3. (C) HRPTECs were transfected with control or IGFBP7 siRNAs (25 nM final concentrations) and subsequently stimulated with TGF-β1 (2.5 ng/ml, 48 h). Lowering the expressions of IGFBP7 proteins attenuated the TGF-β1-increased mesenchymal marker vimentin expression and restored the TGF-β1-repressed epithelial marker ZO-1 expression and restored the TGF-β1-induced morphological changes. To confirm the first antibodies work, we performed immunocytochemistry without first antibodies as negative control.

Fig 5. IGFBP7 in human diabetic nephropathy.

(A) Colocalization of IGFBP7 and TGF-β1 in human renal tubular cells in renal biopsy form type 2 diabetic patients with overt nephropathy. IGFBP7 was localized to the cytoplasm of tubular cells but not that of glomerular cells (G) in diabetic kidney. The confocal microscopy analysis showed the presence of IGFBP7 and TGF-β1 in the proximal tubular cells as indicated by the merged images. The arrowheads indicate the partial colocalization of IGFBP7 and TGF-β1 in proximal tubules. (B) IGFBP7 expression in proximal tubular cells. Positive staining for CD-15 indicated the segment of proximal tubules. The proximal tubular cells which are characterized by a brush border and stained by CD15 showed the positive immunostaining for IGFBP7. The arrows indicate the IGFBP7-positive cells observed in CD-15 positive proximal tubules. (C) Western blot analysis of IGFBP7 immunoreactive forms in urine from patients with DN or without DN. Urine (10 μl) was processed by SDS-PAGE (10% gels) under reducing conditions and electroblotted onto PDF membranes. Lane 1, recombinant human IGFBP7 (200 ng/lane); lane 2, culture medium (10 μl); lane 3, urine sample from a patient with macroalbuminuria (Mac); lanes 5–7, individual urine samples from patients with normoalbuminuria (Nor); lanes 4, 8 and 9, individual urine samples with microalbuminuria (Mic). Arrow: 33 kDa IGFBP7, arrowhead: 25 kDa cleaved IGFBP7. (D) Increased urinary IGFBP7 levels in diabetic nephropathy. The urinary IGFBP7 levels for patients grouped according to different stages of nephropathy as having normoalbunuria (Nor), microalbuminura (Mic), or macroalbuminuria (Mac). Horizontal bars = medians, columns = interquartile ranges, and vertical bars = 95% CIs. *p<0.05, **p<0.01 by one-way ANOVA with a Tukey’s test.

Fig 6. Role of IGFBP7 in Diabetic Nephropathy: TGF-β1 Induces IGFBP7 via Smad2/4 in Human Renal Proximal Tubular Epithelial Cells.

Profibrotic TGF-β1 induces IGFBP7 expression in human renal proximal tubular cells via Smad 2/4. IGFBP7 is also involved in the expressions of epithelial marker ZO-1 and mesenchymal markers α-SMA and vimentin, suggesting the potential role of IGFBP7 in epithelial-mesenchymal transition (EMT) leading to renal tubulointersitital fibrosis.