Improved renal ischemia tolerance in females influences kidney transplantation outcomes

Abstract

Experimentally, females show an improved ability to recover from ischemia-reperfusion injury (IRI) compared with males; however, this sex-dependent response is less established in humans. Here, we developed a series of murine renal ischemia and transplant models to investigate sex-specific effects on recovery after IRI. We found that IRI tolerance is profoundly increased in female mice compared with that observed in male mice and discovered an intermediate phenotype after neutering of either sex. Transplantation of adult kidneys from either sex into a recipient of the opposite sex followed by ischemia at a remote time resulted in ischemia recovery that reflected the sex of the recipient, not the donor, revealing that the host sex determines recovery. Likewise, renal IRI was exacerbated in female estrogen receptor α-KO mice, while female mice receiving supplemental estrogen before ischemia were protected. We examined data from the United Network for Organ Sharing (UNOS) to determine whether there is an association between sex and delayed graft function (DGF) in patients who received deceased donor renal transplants. A multivariable logistic regression analysis determined that there was a greater association with DGF in male recipients than in female recipients. Together, our results demonstrate that sex affects renal IRI tolerance in mice and humans and indicate that estrogen administration has potential as a therapeutic intervention to clinically improve ischemia tolerance.

Figure 1. Female hormonal environment protects against renal IRI.

(A) BUN measurements after 28 minutes of renal IRI in female mice (F 28 min; n = 10) showed renal injury equivalent to that in male mice following 15 minutes of renal IRI (M 15 min; n = 11; P = 0.36, by 2-way ANOVA); 28-minute IRI was unsurvivable in male mice (M 28 min, *n = 4); dashed line shows the baseline BUN level. (B) Neutered female mice (Neu F; n = 5) had impaired renal function after warm IRI compared with that seen in intact females (Int F; n = 5; P < 0.01, by 2-way ANOVA). Neutered male mice (Neu M; n = 5) tolerated IRI with impairment similar to that in neutered females, but worse function than in intact females (P < 0.01, by 2-way ANOVA). (C) Female kidneys transplanted into female recipients (F→F; n = 5) and male kidneys transplanted into female recipients (M→F; n = 5) had similar impairment of renal function when subjected to 25 minutes of warm IRI. Female kidneys transplanted into male recipients (F→M; n = 5) had decreased survival (2 of 5), with significantly higher BUN levels compared with those of either of the other 2 groups (P < 0.0001, by 2-way ANOVA). (D) The F→M transplanted kidneys had significantly more fibrosis on Sirius red staining at 28 days than did the transplanted kidneys in either of the other 2 groups (P < 0.001, by 1-way ANOVA). (E) H&E, trichrome, and Sirius red staining (original magnification, ×20) showed increased fibrosis in the F→M cohort compared with that observed in the F→F and M→F cohorts. Error bars indicate the mean ± SEM.

Figure 2. Role of estrogen in murine renal IRI.

(A) ERα-KO mice (n = 5) had elevated BUN levels after warm IRI compared with levels in WT B6 controls (n = 5; P < 0.01, by 2-way ANOVA). This injury was universally lethal in ERα-KO mice by 96 hours after injury, as denoted by the asterisk. (B) Female mice treated with supplemental estrogen (1 mg/kg at 16 hours and 1 hour before IRI; n = 5) and subjected to warm IRI had lower post-injury BUN levels compared with levels in vehicle-treated controls (n = 5; P < 0.01, by 2-way ANOVA). (C) Sirius red fibrosis scoring at 28 days was significantly decreased in the estrogen-treated arm (P = 0.01, by 2-tailed Student’s t test). (D) H&E, trichrome, and Sirius red staining (original magnification, ×20) showed decreased fibrosis at 28 days in the estrogen-treated cohort. (E) Hormonally intact male mice treated with high-dose supplemental estrogen (10 mg/kg at 16 hours and 1 hour before IRI; n = 5) and subjected to warm IRI had equivalent post-injury BUN levels compared with those of vehicle-treated controls (n = 5; P = 0.98, by 2-way ANOVA). (F) Neutered male mice treated with high-dose supplemental estrogen (10 mg/kg at 16 hours and 1 hour before IRI; n = 5) and subjected to warm IRI had lower post-injury BUN levels compared with those of vehicle-treated controls (n = 4; P = 0.08, by 2-way ANOVA). (G) Post-injury administration of estrogen to intact females (10 mg/kg at surgical closing and at 24 hours) resulted in a nonsignificant trend toward improved post-injury BUN levels compared with those of vehicle-treated controls (n = 5 per group; P = 0.14, by 2-way ANOVA). Error bars indicate the mean ± SEM.

Figure 3. Sex environment affects a transplant model of cold renal IRI.

(A) Murine kidneys transplanted with an 8-hour period of cold ischemia from female donors into female recipients (F→F; n = 5) had superior renal function after native nephrectomy compared with that seen in male donor kidneys transplanted into female recipients (M→F; n = 5; P < 0.05, by 2-way ANOVA) and had a trend toward improved function compared with that of female donor kidneys transplanted into male recipients (F→M; n = 6) and male donor kidneys transplanted into male recipients (M→M; n = 6). CIT, cold ischemia time. (B) Renal fibrosis on Sirius red staining showed a trend toward increased fibrosis in the M→F cohort that was significant compared with the fibrosis observed in all other cohorts (P < 0.001, by 1-way ANOVA). Error bars indicate the mean ± SEM.

Figure 4. Size discrepancy in human renal transplantation.

Deceased donor renal transplants (n = 46,691) were stratified into deciles on the basis of donor/recipient BW ratio and analyzed for association with DGF. The extremes of size mismatch on both ends of the curve were associated with increased DGF risk (bars). The magnitude of these effects on DGF was smaller than recipient sex effects in every decile except the smallest donor/recipient ratio (circles).

Figure 5. Graft survival by sex and DGF occurrence.

Male and female renal transplant recipients who did not develop DGF had equivalent graft survival (P = 0.20). Both male and female renal graft recipients with DGF showed decreased graft survival compared with that of recipients of the same sex without DGF (P < 0.01). Female renal transplant recipients with DGF had decreased graft survival compared with that of male recipients with DGF (P = 0.002).