Amelioration of progressive renal injury by genetic manipulation of Klotho gene

Abstract

Klotho, an antiaging gene with restricted organ distribution, is mainly expressed in the kidney tubules; the mutant mice have shortened life span, arteriosclerosis, anemia, and osteoporesis, features common to patients with chronic renal failure. Conceivably, the reduction of the Klotho gene expression may contribute to the development of kidney failure; alternatively, its overexpression may lead to the amelioration of renal injury in an ICR-derived glomerulonephritis (ICGN) mouse model with subtle immune complex-mediated disease. To address this issue, four different strains of mice were generated by cross-breeding: ICGN mice without the Klotho transgene (ICGN), ICGN mice with the Klotho transgene (ICGN/klTG), wild-type mice with the Klotho transgene (klTG), and wild-type mice without the Klotho transgene (control). At 40 weeks old, the survival rate was approximately 30% in ICGN mice, and approximately 70% in the ICGN/klTG group. This improvement was associated with dramatic improvement in renal functions, morphological lesions, and cytochrome c oxidase activity but a reduction in beta-galactosidase activity (a senescence-associated protein), mitochondrial DNA fragmentation, superoxide anion generation, lipid peroxidation, and Bax protein expression and apoptosis. Interestingly, improvement was seen in both the tubular and glomerular compartments of the kidney, although Klotho is exclusively confined to the tubules, suggesting that its gene product has a remarkable renoprotective effect by potentially serving as a circulating hormone while mitigating the mitochondrial oxidative stress.

Fig. 1.

Kaplan–Meier curves reflecting survival for different strains of mice. In the ICGN group, the survival rate was ≈30% at 40 weeks of age, whereas it was restored to ≈70% in the ICGN/klTG group with the overexpression of the Klotho gene. In the control and klTG groups, the survival rate was >90%. ∗, P < 0.01 compared with the ICGN group (n = 20).

Fig. 2.

Representative kidney sections stained with periodic acid-Schiff from various strains of mice (A–D), reflecting glomerulosclerosis score (E) and the percentage of tubulointerstitial damage (F). In the ICGN group, the glomeruli were relatively large and hypercellular and had thickened loops (C, arrowhead) compared with the control and klTG groups (A and B). Also, tubular atrophy, thickening of the basement membranes, and influx of mononuclear cells was seen (C, double-headed arrow). (D) The glomerular hypercellularity and tubulointerstitial cellular infiltrates were notably lessened in the ICGN/klTG group with the overexpression of the Klotho gene. (E and F) A remarkable degree of glomerulosclerosis and tubulointerstitial damage was seen in the ICGN group, which was partially reversed in the ICGN/klTG group. (E) Results are given as the mean ± SEM. ∗, P < 0.05 compared with the control group; #, P < 0.05 compared with the ICGN group (n = 8). T, tubule.

Fig. 3.

Representative kidney sections exhibiting cellular senescence associated β-galactosidase activity in various strains of mice. An intense activity (blue staining) was observed in the kidney tubules of the ICGN group (C, arrowheads), which was notably reduced with the overexpression of the Klotho gene (D). A mild basal β-galactosidase activity was seen in the control and klTG groups (A and B). (E) Percentages of in β-galactosidase-positive areas were measured, and the results are presented as the mean ± SEM. ∗, P < 0.05 compared with the control group; #, P < 0.05 compared with the ICGN group (n = 8).

Fig. 4.

Representative kidney sections depicting mitochondrial changes (A and B) and cytochrome c oxidase activity (brown reaction product) (C–F) in various strains of mice. Mitochondria were smaller in the ICGN group compared with those of ICGN/klTG mice; however, the cristae were normal (arrows). An intense cytochrome c oxidase activity was present in the control and klTG groups (arrowheads). Activity was markedly reduced in the ICGN group, and it was largely restored in the ICGN/klTG group. The percentages of positive areas were measured, and the results are presented as the mean ± SEM (G). ∗, P < 0.05 compared with the control group; #, P < 0.05 compared with the ICGN group (n = 8).

Fig. 5.

Electrophoretogram of mtDNA isolated from renal tissues of various strains of mice. The mtDNA damage was evaluated by the long PCR technique. (A) In the ICGN mice, intensity of 8636-bp product was reduced compared with the 316-bp PCR product, suggesting damage to higher molecular DNA. The damage was restored in the ICGN/klTG strain of mice, indicating the Klotho gene being protective of mtDNA. (B) Densitometer readings of the ratio of 8636/316-bp band intensity are given as the mean ± SEM. ∗, P < 0.05 compared with the control group; #, P < 0.05 compared with the ICGN group (n = 8).

Fig. 6.

Representative kidney sections from various strains of mice depicting superoxide anion production (A and B) and lipid peroxidation (C and D), and urinary excretion of 8-hydroxy-deoxyguanosine (E). An intense UV fluorescence in samples of ICGN mice (A) was detected, indicating generation of superoxide anion by conversion of dihidroethidium to ethidium, which was drastically reduced in the ICGN/klTG group (B). Lipid peroxidation in glomerular and tubular cells of the ICGN mice was detected by employing anti-4-hydroxy-2-nonenal polyclonal antibody (C, arrowheads), but it was undetectable in the ICGN/klTG group (D). (E) The oxidative modification of nucleic acid was assessed by urinary excretion of 8-hydroxy-deoxy-guanosine (8OH-dG). The relatively high excretion was observed in the ICGN group, and it was reduced by 5- to 6-fold in the control, klTG, and ICGN/klTG groups. (E) Results are given as the mean ± SEM. ∗, P < 0.05 compared with the control group; #, P < 0.05 compared with the ICGN group (n = 8).

Fig. 7.

Immunoblot analyses of Bcl-2, Bax, α-tubulin (A–E) and extent of apoptosis (F) in renal tissues of various strains of mice. A decreased expression of Bcl-2, an antiapoptotic protein, and increased expression of Bax, a proapoptotic protein, was observed in ICGN mice; the expression was partially reversed by overexpression of the Klotho gene in the ICGN/klTG group (A, B, D, and E). The α-tubulin expression was similar in all of the strains (C). By TUNEL assay, a marked apoptosis was observed in the ICGN group, and it was suppressed by overexpression of the Klotho gene in the ICGN/klTG mice (F). A minimal degree of apoptosis was observed in the control and klTG groups. The results are given as the mean ± SEM (D–F). ∗, P < 0.05 compared with the control group; #, P < 0.05 compared with the ICGN group (n = 8).