Efficacy of antisense monocyte chemoattractant protein-1 (MCP-1) in a rat model of mesangial proliferative glomerulonephritis

Figures & data

Figure 1. Integration of exogenous MCP-1 cDNA into the mesangial cells of rats. (A) The neo^r sequence of DNA on transfected pLXSN vector in mesangial cells by PCR; (B) the MCP-1 fragment of DNA amplified by PCR in mesangial cells.

Figure 2. Growth of exogenous mesangial cells in the rat kidney. Paraffin sections of renal tissue in the BC and AM groups were detected by in situ hybridization. The distribution of cells with positive expression of the neo^r gene was mainly in the glomeruli. There was no expression of positive cells in kidney tubules, renal interstitium, or blood vessels. ISH ×400. (A) BC group; (B) AM group. Black arrows indicate positive cells expressing the neo^r gene.

Figure 3. Time-dependent changes in 24-h proteinuria in rats of each experimental group. Urinary protein was measured in 24-h urine samples were collected at one, four, and seven days after treatment with saline or Thy-1.1 antibody. Results represent the mean ± SD of measurements from eight rats in each group. Groups: SO, sham-operation; TG, Thy-1 glomerulonephritis; MC, non-transfected normal rat mesangial cell; BC, pLXSN empty vector or blank control; AM, antisense MCP-1 transfection. All measurements at Day 4 and 7 in the TG, MC, BC, and AM groups were significantly (p < 0.05) higher than those at the corresponding time point in the SO group. The decrease in levels of 24-h urinary protein on the seventh day was greater (p < 0.05) in the AM group than those of the other nephritis groups.

Table 1. Comparison of kidney lesion scores among different groups (mean ± SD).

	SO	TG	MC	BC	AM
N	8	8	8	8	8
Cell number	38.6 ± 2.6	49.7 ± 2.9^a	52.7 ± 3.1^a	53.6 ± 3.7^a	47.3 ± 3.7^abc
Glomerular lesion score	0.25 ± 0.46	4.25 ± 1.03^a	4.50 ± 1.06^a	4.00 ± 0.75^a	3.37 ± 0.74^abd
Kidney tubule lesion score	1.87 ± 0.64	1.62 ± 0.74	1.87 ± 0.83	1.62 ± 0.74	1.62 ± 0.74

^aSignificantly different (p < 0.05) from the corresponding value in the SO group.

^bSignificantly different (p < 0.05) from the corresponding value in the MC group.

^cSignificantly different (p < 0.01) from the corresponding value in the BC group.

^dSignificantly different (p < 0.05) from the corresponding value in the TG group.

Figure 4. Expression of MCP-1 and TGF-β₁ detected by immunohistochemical staining. (A) Negative controls for immunohistochemical staining. IHC ×400. (B) Expression of MCP-1 in positive cells of renal tissue. (C) Expression of TGF-β1 in positive cells of renal tissue. IHC ×400. Black arrows indicate cells exhibiting positive expression in glomeruli.

Figure 5. Percentage of positive cells showing expression of MCP-1 (A) and TGF-β1 (B) in each experimental group. Renal tissue from each experimental group was stained with the appropriate antibody, the SABC immunohistochemical kit, and DAB color reagent. The percentage of positively stained cells was counted under normal light microscopy in 20 glomeruli from each group at seven days after administration Results represent the mean ± SD of measurements from eight animals per group. Groups: SO, sham-operation; TG, Thy-1 glomerulonephritis; MC, non-transfected normal rat mesangial cell; BC, pLXSN empty vector or blank control; AM, antisense MCP-1 transfection. Panel A shows that the number of positive MCP-1 cells in the TG, MC, BC, and AM groups were significantly (p < 0.05) higher than those in the SO group. The number of positive MCP-1 cells in the AM group was significantly (p < 0.01) lower than that in the MC group, but there was no significant difference between the TG and BC groups (p > 0.05). Panel B shows that the number of positive TGF-β1 cells in the TG, MC, BC, and AM groups was significantly (p < 0.05) higher than that in the SO group, but there was no significant difference among the TG, MC, BC, and AM groups.

Figure 6. mRNA expression of MCP-1 (A), TGF-β1 (B), and CCR2 (C). mRNA levels were determined by semi-quantitative RT-PCR. GAPDH was used as a loading control. Groups: SO, sham-operation; TG, Thy-1 glomerulonephritis; MC, non-transfected normal rat mesangial cell; BC, pLXSN empty vector or blank control; AM, antisense MCP-1 transfection.

Figure 7. Comparison of mRNA expression of MCP-1, TGF-β1, and CCR2 among different groups and the correlation between mRNA levels of MCP-1 and CCR2. Panels A–C represent the alteration of mRNA expression of MCP-1, TGF-β1, and CCR2 in each experimental group, respectively. Results represent the mean ± SD of measurements from eight animals per group. All measurements in the TG, MC, BC, and AM groups were significantly (p < 0.05) higher than those at the corresponding time point in the SO group. All measurements in the TG, MC, and BC groups were significantly (p < 0.05) higher than those at the corresponding time point in the AM group. Panel D shows the correlation between mRNA expression of MCP-1 and CCR2, illustrating a parallel increase in both levels. Groups: SO, sham-operation; TG, Thy-1 glomerulonephritis; MC, non-transfected normal rat mesangial cell; BC, pLXSN empty vector or blank control; AM, antisense MCP-1 transfection.