Influence of genetic background on albuminuria and kidney injury in Ins2(+/C96Y) (Akita) mice

Abstract

Previous studies have shown that Akita mice bearing the Ins2(+/C96Y) mutation have significant advantages as a type I diabetes platform for developing models of diabetic nephropathy (DN; Gurley SB, Clare SE, Snow KP, Hu A, Meyer TW, Coffman TM. Am J Physiol Renal Physiol 290: F214-F222, 2006). In view of the critical role for genetic factors in determining susceptibility to DN in humans, we investigated the role of genetic background on kidney injury in Akita mice. To generate a series of inbred Akita mouse lines, we back-crossed the Ins2(C96Y) mutation more than six generations onto the 129/SvEv and DBA/2 backgrounds and compared the extent of hyperglycemia and renal disease with the standard C57BL/6-Ins2(+/C96Y) line. Male mice from all three Akita strains developed marked and equivalent hyperglycemia. However, there were significant differences in the level of albuminuria among the lines with a hierarchy of DBA/2 > 129/SvEv > C57BL/6. Renal and glomerular hypertrophy was seen in all of the lines, but significant increases in mesangial matrix compared with baseline nondiabetic controls were observed only in the 129 and C57BL/6 backgrounds. In F1(DBA/2 x C57BL/6)-Ins2(+/C96Y) mice, the extent of albuminuria was similar to the parental DBA/2-Ins2(+/C96Y) line; they also developed marked hyperfiltration. These studies identify strong effects of genetic background to modify the renal phenotype associated with the Ins2(C96Y) mutation. Identification of these naturally occurring strain differences should prove useful for nephropathy modeling and may be exploited to allow identification of novel susceptibility alleles for albuminuria in diabetes.

Fig. 1.

Blood glucose levels in male mice from ages 2 to 6 mo. The control curve represents a composite control of all nondiabetic animals. The Ins2^C96Y mutation caused sustained and robust hyperglycemia throughout the 4-mo experimental period in each of the strains tested (*P < 0.0001, unpaired t-test for all strains). There was no difference in the level of hyperglycemia among the 3 inbred Ins2^+/C96Y lines (P = 0.591, ANOVA).

Fig. 2.

Systolic blood pressure (SBP) measured by tail-cuff manometry in 6-mo-old male mice. There were significant differences in blood pressures among the strains of nondiabetic (Ins2^+/+) mice with similar levels of elevated SBP in the 129/SvEv-Ins2^+/+ and DBA/2-Ins2^+/+ animals compared with the C57BL/6-Ins2^+/+ group (‡P < 0.01, ANOVA). Compared with the respective nondiabetic controls, the Ins2^+/C96Y mutation was associated with an increase in blood pressure (*P < 0.04) in the C57BL/6, 129/SvEv, and F1 (DBA/2 x C57BL/6) (†P = 0.06) strains, but not the DBA/2 strain.

Fig. 3.

A: urinary albumin excretion in 6-mo-old male mice. Wild-type (Ins2^+/+) DBA/2 mice had significantly higher levels of albumin excretion than wild-type C57BL/6 animals (†P = 0.009), whereas albumin excretion rates in wild-type C57BL/6 and 129/SvEv mice were similar. On each of the 3 genetic backgrounds, the presence of the Ins^C96Y mutation was associated with a significant increase in albumin excretion (*P < 0.05 unpaired t-test for all strains). Among the three Akita strains, absolute levels of albumin excretion were highest in the DBA/2 line, intermediate in 129/SvEv group, and lowest in the C57BL/6 line. B: albumin-to-creatinine ratios were numerically increased in all strains except C57BL/6. †P < 0.0001 for DBA/2 WT vs. DBA/2-Ins2^Akita and F1(DBA/2 x C57BL/6) wild-type vs. F1(DBA/2 x C57BL/6) -Ins2^Akita.

Fig. 4.

Glomerular filtration rate (GFR) in 6-mo-old male mice. GFR was measured in C57BL/6, 129/SvEv, and F1(DBA/2 x C57BL/6) wild-type and Akita (Ins2^+/C96Y) mice. GFR was higher in wild-type 129/SvEv group compared with wild-type C57BL/6 and wild-type F1(DBA/2 x C57BL/6) mice (†P < 0.04). Also, diabetic 129/SvEv-Ins2^+/C96Y mice had higher GFR levels compared with C57BL/6-Ins2^+/C96Y mice (‡P = 0.0004). The levels of GFR were significantly higher in all groups of diabetic Akita mice compared with their respective wild-type controls (*P < 0.05).

Fig. 5.

Photomicrographs of periodic acid-Schiff-stained sections of mouse kidneys. Low-power views of kidneys from a wild-type 129/SvEv-Ins2^+/+ mouse (A) and a diabetic 129/SvEv-Ins2^+/C96Y animal (B). As seen in the representative section (B), abnormalities in the Akita mice were largely confined to the glomerulus without evidence of chronic tubulointerstitial disease. C–E: high-power views of glomeruli illustrating the scoring system used to quantitate mesangial pathology. C: representative normal glomerulus from a wild-type mouse, score “0.” D: glomerulus from an Akita mouse with minimal mesangial pathology, score “1.” E: moderate mesangial pathology in a glomerulus from and Akita mouse, score “2.”

Fig. 6.

Blood glucose, urinary albumin excretion, glomerular volumes, and mesangial scores for F1(DBA/2 x C57BL/6)Ins2^+/C96Y mice compared with the parental C57BL/6-Ins2^+/C96Y and DBA/2-Ins2^+/C96Y lines. A: F1(DBA/2 x C57BL/6)-Ins2^+/C96Y mice developed sustained and robust hyperglycemia at levels that were similar to the parental DBA/2 line but modestly increased compared with the C57BL/6 line (†P = 0.003, ANOVA). B: F1 animals developed a >6-fold increase in albuminuria compared with their littermate Ins2^+/+ controls. This level of albuminuria was significantly higher than in the C57BL/6-Ins2^+/C96Y animals (†P = 0.02), but was not different from the DBA/2-Ins2^{+/ C96Y} line. C: F1(DBA/2 x C57BL/6)Ins2^+/C96Y mice developed significant glomerular hypertrophy compared with nondiabetic F1 controls. The extent of their hypertrophy was similar to the DBA/2-Ins2^+/C96Y parental line and greater than the C57BL/6-Ins2^+/C96Y line. D: like the C57BL/6-Ins2^+/C96Y line, F1(DBA/2 x C57BL/6)Ins2^+/C96Y mice also developed significant mesangial expansion compared with wild-type littermates whereas the parental DBA/2-Ins2^{+/ C96Y} mice did not. *P < 0.05 between Akita (black bars) and wild-type (gray bars).