Preliminary observations on whole-ovary xenotransplantation as an experimental model for fertility preservation.

Ovarian tissue preservation and retransplantation is a promising strategy to restore fertility in cancer survivors. Ischaemia accompanying ovarian tissue grafting, however, can lead to significant follicle loss. Transplantation of the whole ovary by vascular anastomosis has been considered as an alternative to prevent widespread ischaemic damage. In this study, the feasibility and function of transplanting whole ovary with intact vasculature were evaluated, with the goal of developing a xenograft model for studies using donated human ovaries. Whole-swine ovaries with vascular pedicles were perfused and transplanted as intact ovaries by anastomosis into irradiated ovariectomized nude rats (n = 10). The observation period was between 1 and 4 weeks. Fresh swine ovaries served as controls (n = 10). Ovarian stroma and follicle populations were assessed through histological examination in both transplanted and control ovaries. Most of the transplanted whole ovaries (n = 6) maintained stromal quality and all preantral follicle classes were represented, although follicle numbers decreased compared with fresh control. Four transplanted ovaries were fibrotic after 1-4 weeks within the nude rat. Our results demonstrate transplantation of whole-pig ovary into nude rats is possible and support development of this xenograft model system for human studies.

Long-term gene therapy with thrombospondin 2 inhibits TGF-ß activation, inflammation and angiogenesis in chronic allograft nephropathy.

We recently identified Thrombospondin-2 (TSP-2) as a regulator of matrix remodelling and inflammation in experimental kidney disease by using TSP-2 null mice and successfully proved TSP-2 overexpression as a therapeutic concept in a short term glomerulonephritis model in the rat. In this current study, we investigated if long-term TSP-2 overexpression is also capable to ameliorate the progression of chronic kidney disease in the setting of the chronic allograft nephropathy F344-Lewis model in the rat. Two weeks after renal transplantation, two rat thigh muscles were transfected once only with either a TSP-2 overexpressing plasmid (n = 8) or a luciferase-expressing plasmid as control (n = 8). Rats were monitored for renal function, histological changes and gene expression in the graft for up to 30 weeks after transplantation. Unexpectedly, only in the TSP-2 treated group 2 rats died before the end of the experiment and renal function tended to be worsened in the TSP-2 group compared to the luciferase-treated controls. In addition, glomerular sclerosis and tubular interstitial injury as well as cortical fibronectin deposition was significantly increased in the TSP-2 treated kidneys despite reduced TGF-ß activation and marked anti-inflammatory (macrophages, T-cells and B-cells) effects in this group. Long-term TSP-2 therapy impaired repair of renal endothelium, as demonstrated by significant higher glomerular and peritubular endothelial rarefaction and reduced endothelial cell proliferation in the transplanted kidneys from TSP-2 treated rats compared to controls. This TSP-2 effect was associated with decreased levels of renal VEGF but not VEGF1 receptor. In conclusion, despite its anti-inflammatory and TGF-ß activation blocking effects, TSP-2 gene therapy did not ameliorate but rather worsened experimental chronic allograft nephropathy most likely via its anti-angiogenic properties on the renal microvasculature.

Injured kidney endothelium is only marginally repopulated by cells of extrarenal origin.

The role of bone marrow marrow-derived cells after kidney endothelial injury is controversial. In this study, we investigated if and to what extent extrarenal cells incorporate into kidney endothelium after acute as well as during chronic endothelial injury. Fischer F-344wt (wild type) rat kidney grafts were transplanted into R26-hPAP (human placental alkaline phosphatase) transgenic Fischer F-344 recipient rats to allow identification of extrarenal cells by specific antibody staining. A severe model of renal thrombotic microangiopathy was induced via graft perfusion with antiglomerular endothelial cell (GEN) antibody and resulted in eradication of 85% of the glomerular and 69% of the peritubular endothelium (GEN group). At week 4 after injury, renal endothelial healing as well as recovery of the kidney function was seen. Endothelial chimerism was evaluated by double staining for hPAP and endothelial markers RECA-1 or JG-12. Just 0.25% of the glomerular and 0.1% of the peritubular endothelium was recipient derived. In a second experiment, chronic endothelial injury was induced by combination of kidney transplantation with 5/6 nephrectomy (5/6 Nx group). After 14 wk, only 0.86% of the peritubular and 0.05% of the glomerular endothelium was of recipient origin. In summary, despite demonstration of extensive damage and loss as well as excellent regeneration, just a minority of extrarenal cells were incorporated into kidney endothelium in rat models of acute and chronic renal endothelial cell injury. Our results highlight that kidney endothelial regeneration after specific and severe injury is almost exclusively of renal origin.

Bortezomib and sirolimus inhibit the chronic active antibody-mediated rejection in experimental renal transplantation in the rat.

BACKGROUND: To date, no effective immunosuppressive standard for the prevention or treatment of alloantibody production in acute and chronic rejection of renal transplants has been established. Alloantibody formation has been recognized in the well-established rat model of Fischer to Lewis renal transplantation. We used this renal allotransplantation model to test the effectiveness of sirolimus (SRL), bortezomib (BZ) or their combination in an already established humoral rejection situation. METHODS: After 3 weeks, transplanted rats were treated either with placebo (P), SRL, BZ or combination of SRL and BZ. Rats were monitored for donor-specific alloantibodies as well as humoral responses in the spleen and bone marrow, renal function and histological changes in the graft. RESULTS: In all three treatment arms, glomerular, tubulointerstitial and vascular changes of chronic antibody-mediated rejection were ameliorated compared to the P group. Production of alloantibodies against components of glomerular basement membrane was reduced in all three treatment arms. The humoral response was strongly reduced, as shown by decreased numbers of IgG-secreting cells, plasma cells and partially B cells in all treatment groups with a trend of SRL/BZ combination being most effective. Infiltration of the graft with inflammatory cells like cytotoxic T cells, T helper cells, B cells and macrophages was efficiently blocked by BZ and the SRL/BZ combination and, except for B cells, by SRL. CONCLUSIONS: BZ and SRL represent promising drugs with anti-humoral activity in the situation of an already established chronic humoral or mixed alloimmune response after renal transplantation.

Long-term treatment of sirolimus but not cyclosporine ameliorates diabetic nephropathy in the rat.

BACKGROUND: Not just de novo induction of diabetes mellitus, but also the progression of diabetic nephropathy may be enhanced under immunosuppressive therapy after organ transplantation. We evaluated whether sirolimus (SRL) or cyclosporine A (CsA) therapy would be a superior immunosuppressant in streptozotocin-induced diabetic nephropathy. METHODS: Diabetes was induced by intravenous injection of streptozotozin (60 mg/kg body weight) in 26 male Sprague-Dawley rats. Eight days after diabetes induction, animals were divided into three groups, which were treated with placebo (n=8), SRL (n=9), or CsA (n=9). Six nondiabetic placebo-treated rats were included as controls. RESULTS: After 19 weeks of diabetes, SRL significantly decreased fibrosis as assessed by periodic acid Schiff staining and by specific extracellular matrix proteins such as fibronectin and laminin at messenger RNA and protein level compared with the diabetic placebo group. SRL ameliorated renal inflammation, glomerular hypertrophy, and podocyte loss as indicated by morphometric and immunohistological analysis. SRL lowered expression and activity of glomerular transforming growth factor-beta1/2 and vascular endothelial growth factor, all of which are considered central cytokines in the pathogenesis of diabetic nephropathy. In contrast, calcineurin phosphatase inhibition through CsA did not ameliorate any of the features of diabetic nephropathy compared with placebo treatment but slightly aggravated glomerular fibrosis without affecting transforming growth factor-beta1/2 or vascular endothelial growth factor. CONCLUSION: Compared with CsA, SRL by anti-inflammatory, antifibrotic, and podocyte-protective effects clearly seems to be the superior treatment of prevention or amelioration of diabetic nephropathy in the rat.