Irradiation prolongs survival of Alport mice

Abstract

Alport syndrome is a hereditary nephropathy that results in irreversible, progressive renal failure. Recent reports suggested that bone marrow transplantation (BMT) has a beneficial, short-term effect on renal injury in Alport (Col4a3(-/-)) mice, but its long-term effects, especially with regard to survival, are unknown. In this study, Alport mice received a transplant of either wild-type or Col4a3(-/-) bone marrow cells. Surprising, laboratory evaluations and renal histology demonstrated similar findings in both transplanted groups. Transplanted cells accounted for >10% of glomerular cells at 8 wk, but type IV collagen alpha3 chains were not detected in glomerular basement membranes of either group by immunofluorescence or Western blot analysis, although Col4a3 mRNA in the kidney could be amplified by reverse transcription-PCR in knockout mice that received a transplant of wild-type bone marrow. Both transplanted groups, however, survived approximately 1.5 times longer than untreated knockout mice (log rank P < 0.05). These data suggested that irradiation, which preceded BMT, may have conferred a survival benefit; therefore, the survival time of knockout mice was assessed after sublethal irradiation (3, 6, and 7 Gy) without subsequent BMT. A strong positive correlation between irradiation dosage and survival time was identified (P < 0.0001). In conclusion, the improved survival observed in Alport mice that received a transplant of wild-type bone marrow might be primarily attributed to as-yet-unidentified effects of irradiation.

Figure 1.

Survival time. (A) Survival times of WT to KO BMT and KO to KO BMT mice improved significantly in comparison with untreated KO mice (mean survival time 125 ± 4 and 135 ± 5 versus 82 ± 1 d; log rank test for trend, P < 0.05), whereas there was no significant difference of the mean survival times between the two BMT mouse groups (z score = 1.48; P < 0.05). (B) The survival times of the irradiated KO mice without BMT were 100 ± 2, 108 ± 4, and 105 ± 11 d in 3, 6, and 7 Gy, respectively. The positive correlation between the irradiation dosage and survival time was strongly significant (hazard ratio 0.553, 95% confidence interval [CI] 0.466 to 0.656; P < 0.0001). The data of the KO group were repeated in A and B.

Figure 2.

Renal function at 7 and 11 wk of age. (A) The BUN and Cr levels of KO mice increased significantly in comparison with those of the WT mice at 11 wk of age (#P < 0.05). The BUN and Cr levels of two BMT mouse groups (each n = 5) significantly improved in comparison with those of untreated KO mice (n = 5) at 11 wk of age (*P < 0.05); no difference in the BUN and Cr levels was found between the two BMT mouse groups. (B) At 7 wk of age, the BUN level of 3 Gy KO mice (n = 5) was elevated in comparison with that of 6 Gy KO mice (n = 5; **P < 0.05). The BUN and Cr levels of 3 and 6 Gy KO mice significantly improved in comparison with those of the untreated KO mice (n = 5) at 11 wk of age (*P < 0.05). The data of the KO group were repeated in A and B.

Figure 3.

Periodic acid-Schiff stain and sclerotic index. (A) The sclerotic indices of KO and WT to KO BMT mice significantly increased in comparison with the WT group at 7 wk of age (#P < 0.05). The sclerotic indices of KO, WT to KO BMT, and KO to KO BMT significantly increased in comparison to the WT group at 11 wk of age (#P < 0.05). Although the sclerotic indices of the two BMT mice groups significantly improved in comparison with KO mice at 11 wk of age (*P < 0.05), there was no significant difference between the two BMT mouse groups. (B) There was no significant difference among the three groups receiving radiation at 7 wk of age. The sclerotic indices of 3 and 6 Gy KO significantly improved in comparison with those of the KO mice at 11 wk of age (*P < 0.05). The data of the KO group were repeated in A and B.

Figure 4.

Masson-Trichrome stain and fibrotic index. (A) The fibrotic index of KO mice significantly increased in comparison with the WT group at 7 wk of age (#P < 0.05). The fibrotic indices of KO, WT to KO BMT, and KO to KO BMT mice significantly increased in comparison with the WT group at 11 wk of age (#P < 0.05). Although the fibrotic indices of the two BMT mouse groups significantly improved in comparison with the KO mice (*P < 0.05), no significant difference was observed between the two BMT mouse groups at 11 wk of age. (B) There was no significant difference among the three groups receiving radiation at 7 and 11 wk of age. The data of the KO group were repeated in A and B.

Figure 5.

(A and B) Immunofluorescence for type IV collagen α1 through 6 chains at 7 wk of age (A) and at 11 wk of age (B). At 7 and 11 wk of age, α1 and 2 staining in the GBM was positive in all groups. At 7 and 11 wk of age, α3 and 4 staining in the GBM was positive only in the WT mice. α3 staining was done with two different mAb (H31 and H32). α5 staining in the GBM was positive in all groups at 7 and 11 wk of age. Although α6 staining was negative in the GBM of WT mice, it was positive in the other three groups at 7 and 11 wk of age.

Figure 6.

Reverse transcription–PCR analysis. At 11 wk of age, a 361-bp product of Col4a3 representing exons 46 through 48 was detected in the WT group. A 1458-bp product representing the same region of Col4a3 with an inserted neo cassette was detected in the KO, WT to KO BMT, and KO to KO BMT groups. Although the 361-bp band of Col4a3 was weaker than that of the WT group, a 361-bp band of Col4a3 was detected in the WT to KO BMT group. The Gapdh levels were similar among the four groups.

Figure 7.

Western blot analysis. (A) Type IV collagen α1 and 2 chains were detected as a monomer and a dimer in all four groups. Type IV collagen α3 and 4 chains were detected only in the WT group. The appearance of type IV collagen α5 chains was most strongly detected in the WT group and most weakly in the KO group. The signal in the two BMT groups was somewhat stronger than in the KO group. Although the type IV collagen α6 chain was hardly detected in the WT group, the signal was stronger in the kidney samples from the three KO groups. D, noncollagenous domain 1 (NC1) dimer (molecular mass of approximately 44 to 50 kD); M, NC1 monomer (molecular mass of approximately 24 to 28 kD). (B) The full-length type IV collagen α3 chain with a molecular mass of approximately 180 kD was detected only in the total kidney lysate from the WT mice. α-Tubulin was used as a loading control.