Protective effect of L-glutamine against diabetes-induced nephropathy in experimental animal: Role of KIM-1, NGAL, TGF-β1, and collagen-1

Figures & data

Table 1. Primer sequences for KIM-1, NGAL, TGF-β, collagen-1, and β-actin.

Gene	Primer sequences (5′–3′)	Length (bp)
KIM-1	5′-ACTCCTGCAGACTGGAATGG-3′ 5′-CAAAGCTCAGAGAGCCCATC-3′	212
NGAL	5′-GATGTTGTTATCCTTGAGGCCC-3′ 5′-CACTGACTCACGACCAGTTTGCC-3′	230
TGF-β	5′-GTTCTTCAATACGTCAGACATTCG-3′ 5′-CATTATCTTTGCTGTCACAAGAGC-3′	309
Collagen-1	5′-GAGCGGAGAGTACTGGATCG-3′ 5′-GGTTCGGGCTGATGTACCAG-3′	218
β-actin	5′-AGGCATCCTGACCCTGAAGTAC-3′ 5′-TCTTCATGAGGTAGTCTGTCAG-3′	764

Table 2. Effect of LG on STZ-induced alterations in body weight, blood glucose level, food intake, water intake, urine output, and relative kidney weight in diabetic rats.

Treatment	Body weight (g)	Blood glucose level (mg/dL)	Food intake (g)	Water intake (mL)	Urine output (mL)	Kidney/body weight
N	239.00 ± 6.25	115.8 ± 6.42	22.00 ± 3.33	35.83 ± 3.07	9.17 ± 1.19	2.39 ± 0.41
DC	200.7 ± 7.54#	261.2 ± 4.9#	68.00 ± 3.37#	111.3 ± 2.47#	44.85 ± 0.68#	5.47 ± 0.33#
Sita (5)	229.2 ± 3.2*^,$	157.2 ± 16.31*^,$	31.67 ± 3.42*^,$	47.83 ± 2.44*^,$	16.99 ± 1.17*^,$	2.91 ± 0.23*^,$
LG (250)	203.2 ± 4.67	241.2 ± 12.94	58.17 ± 2.4	99.33 ± 3.55	41.15 ± 0.74	4.65 ± 0.3
LG (500)	217.2 ± 6.23*^,$	206.4 ± 8.15*^,$	43.83 ± 3.03*^,$	69.33 ± 2.23*^,$	30.69 ± 1.51*	4.07 ± 0.22*
LG (1000)	226.2 ± 7.66*^,$	179.6 ± 15.5*^,$	36.00 ± 2.97*^,$	57.67 ± 2.5*^,$	23.86 ± 0.71*^,$	3.18 ± 0.17*^,$
Per se	235.2 ± 3.89	139.8 ± 14.15	22.83 ± 2.7	41.00 ± 2.32	13.96 ± 1.35	2.38 ± 0.24

Results are represented as mean ± SEM, (n = 5) data were analyzed by One-way ANOVA followed by Tukey’s multiple range test for each parameter separately.

# p < .05 as compared with normal group.

* p < .05 as compared with diabetic control (DC) group.

$ p < .05 as compared with each other. N: normal group; DC: diabetic control group; Sita (5): sitagliptin (5 mg/kg, p.o.)-treated group; LG (250): L-glutamine (250 mg/kg, p.o.)-treated group; LG (500): L-glutamine (500 mg/kg, p.o.)-treated group; LG (1000): L-glutamine (1000 mg/kg, p.o.)-treated group.

Table 3. Effect of LG on STZ-induced alterations in serum and urine biochemical parameters in diabetic rats.

Treatment		BUN (mg/dL)	Uric Acid (mg/dL)	Creatinine (mg/dL)	Albumin (g/dL)	Creatinine clearance (mL/min)
N	Serum	10.37 ± 1.67	1.99 ± 0.24	0.2 ± 0.02	4.01 ± 0.41	1.57 ± 0.17
	Urine	34.65 ± 1.50	3.51 ± 0.16	48.48 ± 1.32	0.57 ± 0.06
DC	Serum	63.02 ± 1.99#	5.46 ± 0.2#	0.75 ± 0.02#	11.97 ± 0.69#	0.44 ± 0.05#
	Urine	5.42 ± 0.86#	1.71 ± 0.16#	10.68 ± 1.28#	2.00 ± 0.14#
Sita (5)	Serum	16.46 ± 2.81*^,$	2.41 ± 0.19*^,$	0.34 ± 0.02*^,$	7.01 ± 0.46*^,$	1.53 ± 0.14*^,$
	Urine	31.01 ± 1.99*^,$	3.21 ± 0.19*^,$	43.02 ± 1.03*^,$	0.96 ± 0.04*^,$
LG (250)	Serum	59.49 ± 1.53	5.24 ± 0.18	0.68 ± 0.03	10.84 ± 0.85	0.57 ± 0.07
	Urine	10.47 ± 2.81	2.07 ± 0.15	13.35 ± 1.18	1.85 ± 0.08
LG (500)	Serum	40.66 ± 1.5*^,$	4.72 ± 0.21*	0.51 ± 0.02*^,$	8.81 ± 0.51*	1.18 ± 0.14*^,$
	Urine	17.46 ± 1.66*^,$	2.69 ± 0.24*^,$	27.45 ± 1.29*^,$	1.76 ± 0.06*
LG (1000)	Serum	23.46 ± 1.66*^,$	3.41 ± 0.25*^,$	0.36 ± 0.01*^,$	7.59 ± 0.7*^,$	1.62 ± 0.04*^,$
	Urine	27.47 ± 1.53*^,$	3.14 ± 0.2*^,$	35.49 ± 1.48*^,$	1.28 ± 0.07*^,$
Per se	Serum	9.66 ± 2.34	2.23 ± 0.16	0.26 ± 0.02	5.37 ± 0.78	1.91 ± 0.25
	Urine	33.92 ± 1.48	3.43 ± 0.17	48.79 ± 1.37	0.54 ± 0.05

Results are represented as mean ± SEM, (n = 5) data were analyzed by One-way ANOVA followed by Tukey’s multiple range test for each parameter separately.

# p < .05 as compared with normal group.

* p < .05 as compared with diabetic control (DC) group.

$ p < .05 as compared with each other. N: normal group; DC: diabetic control group; Sita (5): sitagliptin (5 mg/kg, p.o.)-treated group; LG (250): L-glutamine (250 mg/kg, p.o.)-treated group; LG (500): L-glutamine (500 mg/kg, p.o.)-treated group and LG (1000): L-glutamine (1000 mg/kg, p.o.)-treated group.

Table 4. Effect of LG on STZ-induced alterations in renal oxidonitrosative stress and hydroxyproline level in diabetic rats.

Treatment	SOD (U/mg of protein)	GSH (μg/mg of protein)	LPO (nM/mg of protein)	NO (μg/mL)	Hydroxyproline (μg/mg protein)
N	15.21 ± 1.42	11.76 ± 0.63	5 ± 0.52	229.6 ± 25.05	0.79 ± 0.05
DC	3.87 ± 0.5#	2.54 ± 0.58#	24.73 ± 0.64#	655.3 ± 24.16#	2.09 ± 0.17#
Sita (5)	15.38 ± 0.66*^,$	10.07 ± 0.62*^,$	10.32 ± 0.71*^,$	294.7 ± 30.26*^,$	1.12 ± 0.07*^,$
LG (250)	6.84 ± 0.72	2.82 ± 0.41	22.01 ± 0.45	607.9 ± 23.57	1.92 ± 0.06
LG (500)	11.65 ± 0.53*^,$	5.42 ± 0.85*^,$	16.75 ± 0.98*^,$	524.1 ± 29.99*	1.61 ± 0.06*
LG (1000)	14.12 ± 1.51*^,$	9.35 ± 0.81*^,$	13.19 ± 1.57*^,$	445.6 ± 25.72*^,$	1.2 ± 0.07*^,$
Per se	15.76 ± 1.14	11.12 ± 0.57	5.414 ± 0.9	246.3 ± 30.85	0.73 ± 0.08

Results are represented as mean ± SEM, (n = 5) data were analyzed by One-way ANOVA followed by Tukey’s multiple range test for each parameter separately.

# p < .05 as compared with normal group.

* p < .05 as compared with diabetic control (DC) group.

$ p < .05 as compared with each other. N: normal group; DC: diabetic control group; Sita (5): sitagliptin (5 mg/kg, p.o.)-treated group; LG (250): L-glutamine (250 mg/kg, p.o.)-treated group; LG (500): L-glutamine (500 mg/kg, p.o.)-treated group; LG (1000): L-glutamine (1000 mg/kg, p.o.)-treated group.

Figure 1. (A) Effect of LG on STZ-induced alterations in renal KIM-1, NGAL, TGF-β, and Collagen-1 mRNA expression in diabetic rats. (B) Quantitative representation of the mRNA expression of KIM-1, (C) NGAL, (D) TGF-β, and (E) Collagen-1. Results are represented as mean ± SEM, (n = 4) data were analyzed by One-way ANOVA followed by Tukey’s multiple range test for each parameter separately. #p < .05 as compared with normal group, *p < .05 as compared with diabetic control (DC) group and $p < .05 as compared with each other. N: normal group; DC: diabetic control group; Sita (5): sitagliptin (5 mg/kg, p.o.)-treated group; LG (250): L-glutamine (250 mg/kg, p.o.)-treated group; LG (500): L-glutamine (500 mg/kg, p.o.)-treated group; LG (1000): L-glutamine (1000 mg/kg, p.o.)-treated group. Lane 1: Ladder 1000 bp; Lane 2: mRNA expression of normal group; Lane 3: mRNA expression of diabetic control group; Lane 4: mRNA expression of sitagliptin (5 mg/kg, p.o.) treated group; Lane 5: mRNA expression of L-glutamine (250 mg/kg, p.o.) treated group; Lane 5: mRNA expression of L-glutamine (500 mg/kg, p.o.) treated group; Lane 7: mRNA expression of L-glutamine (1000 mg/kg, p.o.) treated group and Lane 8: mRNA expression of per se group.

Figure 2. (A) Correlation analysis between blood glucose level and KIM-1, (B) NGAL, (C) TGF-β as well as (D) Collagen-1 mRNA expression in diabetic rats.

Figure 3. Effect of LG on STZ-induced alterations in kidney histology. Photomicrograph of sections of kidney of (A) normal, (B) diabetic control rats, (C) Sitagliptin (5 mg/kg, p.o.) treated rats (C), LG (250 mg/kg, p.o.) treated rats (D), LG (500 mg/kg, p.o.) treated rats (E) and LG (1000 mg/kg, p.o.) treated rats. H & E staining at 100×. Increase in thickening of the basement membrane (black arrow), necrosis (white arrow) and inflammatory infiltration (red arrow).

Figure 4. Effect of LG on STZ-induced alterations in kidney histology. Photomicrograph of sections of kidney of (A) normal, (B) diabetic control rats, (C) Sitagliptin (5 mg/kg, p.o.) treated rats (C), LG (250 mg/kg, p.o.)-treated rats (D), LG (500 mg/kg, p.o.)-treated rats (E) and LG (1000 mg/kg, p.o.)-treated rats. MT staining at 40×. Fibrosis (Black arrow).