Promotion of chemerin in rat diabetic kidney disease through enhancement of TGF-β1/Smads/CTGF pathway

Abstract

Objective: Although increasing evidence shows that the adipokine chemerin is involved in diabetic kidney disease (DKD), it is still unclear whether the chemerin acts as a critical element in renal function through the signaling pathways of transforming growth factor β1/Smads/connective tissue growth factor (TGF-β1/Smads/CTGF) in the context of DKD. Therefore, we sought to determine the role of chemerin and TGF-β1/Smads/CTGF signaling pathway in the development and/or progression of DKD.

Methods: We used rat renal mesangial cells (RMCs) and a DKD rat model as study subjects. RMCs and rats were randomly separated into different groups and transfected with the constructed chemerin expression vector pcDNA™ 3.1 (+)-chemerin. Rat renal function and inflammatory cytokines were assessed after treatment with chemerin or CCX832 (ChemR23 antagonist). Real time polymerase chain reverse transcription (RT-QPCR) was used to detect the mRNA expressions of TGF-β1, Smad2, Smad4, and CTGF. Western blot was performed to determine protein expression for semiquantitative analysis.

Results: In in vitro studies, the mRNA and protein levels of TGF-β1, Smad2, Smad4, and CTGF were significantly increased in the groups of high glucose and chemerin as compared to the normal control and normal glucose groups, most notably in the high glucose chemerin group (all P<0.05). In vivo studies revealed that the mRNA and protein levels of TGF-β1, Smad2, Smad4, and CTGF were higher in the DKD group and the normal chemerin group than in the normal control group and the blocking receptor group, while appearing the highest in the DKD chemerin group (all P<0.05). Moreover, kidney/body weight ratio, urea, creatinine, and urine protein were increased, and the weight and endogenous creatinine clearance rate decreased in the DKD group and the normal chemerin group (all P<0.05). These changes were more pronounced in the DKD chemerin group. At the same time, blood glucose, triglycerides (TGs), and total cholesterol (TC) in the blocked receptor group was lower than those in the DKD group and the DKD chemerin group (all P<0.05). In contrast to those in the normal control group and blocked receptor group, tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1 showed higher concentrations in the DKD group and the normal chemerin group. This result was more pronounced in the DKD chemerin group (all P<0.05).

Conclusion: Chemerin may play a role in DKD by enhancing the signaling pathways of TGF-β1/Smads/CTGF transduction either in vitro or in vivo. Moreover, high glucose accelerates kidney injury by activating fibrotic pathways.

Keywords: Diabetic kidney disease; chemerin; connective tissue growth factor; transforming growth factor β1/Smads.

Figure 1

The features of the pcDNA™ 3.1 (+) vector. The complete sequences for pcDNA™ 3.1 (+) were downloaded from www.invitrogen.com.

Figure 2

General index, renal function, and inflammatory factors in different rat groups (n=8). Kidney/body weight ratio (B), urea (F), creatinine (G), and urine protein (I) were higher and the weight (A), Ccr (H) were fewer in the DKD group and normal chemerin group than in the normal control group and blocking receptor group. This result was more pronounced in the DKD chemerin group. The blood glucose (C), TG (D), TC (E) of the blocking receptor group were lower than those in the DKD group and the DKD chemerin group. In comparison with those in the normal control group and blocking receptor group, the levels of IL-1 (J) and TNF-α (K) were significantly higher in the DKD group and normal chemerin group. This result was more pronounced in the DKD chemerin group. *P<0.05, ^NSP>0.05. TG: triglyceride, TC: total cholesterol, Ccr: endogenous creatinine clearance rate; DKD: diabetic kidney disease.

Figure 3

Comparative renal histopathology in different groups of rat kidneys (n=8). (A) The normal control group showed normal renal tubules and glomeruli. Representative sections (stain indicated) from the other four groups of rat kidneys tissues show varying degrees of tubular and glomerular damage evidenced by glomerulosclerosis, tubulointerstitial fibrosis (H&E, PAS, PASM+Massson), and accumulation of protein casts in the tubular lumen (PAS; 200×). (B and C) Standardized semiquantitative methods were used to generate indexes of glomerular injury (B) and tubulointerstitial injury (C). The blocked receptor group had significantly reduced both glomerular and tubulointerstitial injury, while the DKD chemerin group was the worst. *P<0.05. DKD: diabetic kidney disease. The arrows point to the typical location of tubular and glomerular damage.

Figure 4

Concentrations of Chemerin in rat and RMCs. RMC: renal mesangial cells.

Figure 5

Relative mRNA levels of genes and protein expression in the TGF-β1/Smads/CTGF pathway in different groups of rat kidneys (n=8). The mRNA and protein expression levels of TGF-β1 (A, E), Smad2 (B, F), Smad4 (C, G), and CTGF (D, H) in the TGF-β1/Smad/CTGF pathway showed no remarkable distinction between the normal control group and the blocking receptor group; nevertheless, these levels were higher in the DKD group, the normal chemerin group, and the DKD chemerin group than those in the normal control group, especially in the DKD chemerin group. **P<0.01. *P<0.05; compared with the normal control group, ^NSP>0.05. DKD: diabetic kidney disease.

Figure 6

Relative mRNA levels of genes and protein expression in the TGF-β1/Smads/CTGF pathway in different groups of rat mesangial cells (n=6). The mRNA and protein expression levels of TGF-β1 (A, E), Smad2 (B, F), Smad4 (C, G) and CTGF (D, H) in the TGF-β1/Smad/CTGF pathway showed no remarkable distinction between the normal control group and the blocked receptor group; however, the expression levels in the high glucose group, the chemerin group, and the high glucose chemerin group were notably higher than in the blank control group, especially in high glucose chemerin group. **P<0.01, *P<0.05; compared with the blank control group, ^NSP>0.05. TGF-β1: transforming growth factor β1; CTGF: connective tissue growth factor.

Figure 7

Possible mechanism whereby chemerin administration may affect the TGF-β1/Smads/CTGF pathway to enhance ECM production, which is the key to promote and develop DKD. Three receptors of chemerin: ChemR23, CCRL2, GPR1. TGF-β1: transforming growth factor β1; CTGF: connective tissue growth factor; ECM: extracellular matrix.