Protective effects of leflunomide on renal lesions in a rat model if diabetic nephropathy

Figures & data

Table 1. Primer sequences for quantitative real-time PCR.

Gene	Primer sequences	Product size (bp)	Tm (°C)	Accession number
CD68	5′-AATGTGTCCTTCCCACAAGC-3′ (sense)	109	60	NM_001031638
	5′-TGCTTGTATTTCCGCAACAG-3′ (sense)
OPN	5′-GATCGATAGTGCCGAGAAGC-3′ (sense)	111	60	M14656.1
	5′-TGAAACTCGTGGCTCTGATG-3′ (sense)
TGF-β1	5′-GGGACTCTCCACCTGCAA-3′ (sense)
	5′-TGGCGAGCCTTAGTTTGG-3′ (antisense)	98	60	NM_021578.2
	5′-TGGCGAGCCTTAGTTTGG-3′ (antisense)
α-SMA	5′-TGTGCTGGACTCTGGAGATG-3′ (sense)	148	62	NM_031004.2
	5′-GAAGGAATAGCCACGCTCA-3′ (antisense)
GAPDH	5′-CTCCCATTCTTCCACCTTTG-3′ (sense)	196	60	NG_028301.2
	5′-AGGGCCTCTCTCTTGCTCTC-3′ (antisense)

Figure 1. Body weight and kidney weight of rats from various groups. DM, diabetes mellitus group; V, vehicle; Ben, benazepril; LEF, leflunomide. Values are mean ± SEM; n = 8; *p < 0.05 versus control, #p < 0.05 versus vehicle-treated DM rats.

Table 2. The urine 24-h volume, 24-h urine protein and microalbumin.

	Urine 24-h volume (mL)		24-h urine protein (mg)		Urine microalbumin (µg/24 h)
Group	4 weeks	8 weeks	4 weeks	8 weeks	4 weeks	8weeks
Control	18.3 ± 2.6*	20.5 ± 3.1*	8.4 ± 2.4*	9.5 ± 2.0*	98.7 ± 26.8*	108.4 ± 29.4*
DM-V	26.8 ± 3.3	28.3 ± 4.3	38.3 ± 6.2	37.9 ± 5.8	575.3 ± 89.7	604.1 ± 103.3
Ben	22.9 ± 3.3*	25.2 ± 3.5	32.7 ± 8.1*	32.3 ± 3.2*	456.6 ± 65.1*	481.3 ± 74.8*
LEF-low	23.8 ± 3.8*	25.2 ± 3.2	22.7 ± 4.4*	23.8 ± 3.2*	408.9 ± 62.7*	394.3 ± 48.2*
LEF-high	20.2 ± 4.1*	20.4 ± 3.0*	15.8 ± 6.2*	18.4 ± 5.3*	382.2 ± 58.4*	375.7 ± 53.2*

Values are mean ± SEM; n = 8.

*p < 0.05 versus DM-V.

DM, diabetes mellitus group; V, vehicle; Ben, benazepril; LEF, leflunomide.

Figure 2. Blood creatinine and urea nitrogen (BUN) of rats from various groups. DM, diabetes mellitus group; V, vehicle; Ben, benazepril; LEF, leflunomide. Values are mean ± SEM; n = 8; *p < 0.05 versus control, #p < 0.05 versus vehicle-treated DM rats.

Figure 3. Representative hematoxylin & eosin staining (A) and PAS staining (B) in the kidney of rats from various groups. DM, diabetes mellitus group; V, vehicle; Ben, benazepril; LEF, leflunomide; Magnification = 400×.

Figure 4. Renal α-SMA expression in diabetic rats treated with vehicle, benazepril or leflunomide for 8 weeks. (A) Representative images showing α-SMA staining in the kidney (Magnification = 400X). (B) α-SMA staining intensity quantified using ImageJ. (C) Renal α-SMA mRNA levels determined by real-time PCR. DM, diabetes mellitus group; V, vehicle; Ben, benazepril; LEF, leflunomide. Values are mean ± SEM; n = 8; *p < 0.05 versus control, #p < 0.05 versus vehicle-treated DM rats.

Figure 5. Renal CD68 expression in diabetic rats treated with vehicle, benazepril or leflunomide for 8 weeks. (A) Representative images showing CD68 staining in the kidney (Magnification = 400X). (B) CD68 staining intensity quantified using ImageJ. (C) Renal CD68 mRNA levels determined by real-time PCR. DM, diabetes mellitus group; V, vehicle; Ben, benazepril; LEF, leflunomide. Values are mean ± SEM; n = 8; *p < 0.05 versus control, #p < 0.05 versus vehicle-treated DM rats.

Figure 6. Renal TGF-β1 expression in diabetic rats treated with vehicle, benazepril or leflunomide for 8 weeks. (A) Representative images showing TGF-β1 staining in the kidney (Magnification = 400X). (B) TGF-β1 staining intensity quantified using ImageJ. (C) Renal TGF-β1 mRNA levels determined by real-time PCR. DM, diabetes mellitus group; V, vehicle; Ben, benazepril; LEF, leflunomide. Values are mean ± SEM; *p < 0.05 versus control, #p < 0.05 versus vehicle-treated DM rats.

Figure 7. Renal OPN expression in diabetic rats treated with vehicle, benazepril or leflunomide for 8 weeks. (A) Representative images showing OPN staining in the kidney (Magnification = 400X). (B) OPN staining intensity quantified using ImageJ. (C) Renal OPN mRNA levels determined by real-time PCR. DM, diabetes mellitus group; V, vehicle; Ben, benazepril; LEF, leflunomide. Values are mean ± SEM; * p < 0.05 versus control, #p < 0.05 versus vehicle-treated DM rats.