Sildenafil Citrate Enhances Renal Organogenesis Following Metanephroi Allotransplantation into Non-Immunosuppressed Hosts

Abstract

In order to harness the potential of metanephroi allotransplantation to the generation of a functional kidney graft on demand, we must achieve further growth post-transplantation. Sildenafil citrate (SC) is widely known as a useful inductor of angiogenesis, offering renoprotective properties due to its anti-inflammatory, antifibrotic, and antiapoptotic effects. Here, we performed a laparoscopic metanephroi allotransplantation after embedding sildenafil citrate into the retroperitoneal fat of non-immunosuppressed adult rabbit hosts. Histology and histomorphometry were used to examine the morphofunctional changes in new kidneys 21 days post-transplantation. Immunofluorescence of E-cadherin and renin and erythropoietin gene expression were used to assess the tubule integrity and endocrine functionality. After the metanephroi were embedded in a 10 µM SC solution, the new kidneys' weights become increased significantly. The E-cadherin expression together with the renin and erythropoietin gene expression revealed its functionality, while histological mature glomeruli and hydronephrosis proved the new kidneys' excretory function. Thus, we have described a procedure through the use of SC that improves the outcomes after a metanephroi transplantation. This study gives hope to a pathway that could offer a handsome opportunity to overcome the kidney shortage.

Keywords: kidney; metanephros; organogenesis; regenerative medicine; transplantation.

Figure 1

Experimental design. Metanephroi were recovered, embedded in 5 µL drops of sildenafil citrate (0, 10, 30 µM), and laparoscopically transplanted into non-immunosuppressed hosts. The immunological response, recovery rate (kidneys recovered/metanephroi transplanted), and the new kidneys weight and function (excretory and endocrine) were assessed (figure created with BioRender.com).

Figure 2

White blood cell counts after metanephroi allotransplantation: (A) total white blood cells; (B) lymphocytes; (C) eosinophils; (D) monocytes.

Figure 3

Development of new kidney recovery 21 days after metanephroi allotransplantation with or without sildenafil citrate (SC). (A) Nascent kidney from metanephroi without SC treatment. Arrows indicate the growing metanephroi. (B) Developing kidney from metanephroi treated with the 10 µM SC solution. Asterisk indicates neoangiogenesis. Arrows indicate the growing metanephroi. (C) Developing kidney from metanephroi treated with the 30 µM SC solution. Asterisk indicates neoangiogenesis. Arrows indicate the growing metanephroi. (D–F) New kidneys recovered from transplanted metanephroi (D) without SC treatment, (E) with the 10 µM SC solution, and (F) with the 30 µM SC solution. Scale bars: 2 cm.

Figure 4

Kidney weight recovery 21 days after metanephroi allotransplantation embedding in sildenafil citrate (0, 10 and 30 µM). The control kidney originated from a neonatal rabbit (1 week-old).

Figure 5

Histology of developing kidneys with or without sildenafil citrate. (A) Micrograph showing the glomerulus details (original magnification, ×400) and the outer renal cortex (original magnification, ×40) of the control kidney originating from a neonatal rabbit (1 week-old). (B–D) Micrograph showing the glomerulus details (original magnification, ×400) and the outer renal cortex (original magnification, x40) of a new kidney after metanephroi transplantation: (B) without sildenafil citrate (SC) treatment, (C) with a 10 µM SC solution, and (D) with a 30 µM SC solution.

Figure 6

Immunofluorescence and confocal microscopy assay for the detection of tubular integrity marker E-Cadherin. (A) Micrograph showing tubular integrity of the control kidney originating from a neonatal rabbit (1 week-old). (B–D) Micrograph tubular integrity of the new kidney after metanephroi transplantation: (B) without sildenafil citrate (SC) treatment, (C) with a 10-µM SC solution, and (D) with a 30-µM SC solution. Original view: × 20.

Figure 7

Renal gene expression levels of the renin and erythropoietin in the new kidney recovery 21 days after metanephroi allotransplantation embedding in sildenafil citrate (0, 10, and 30 µM), and the kidneys originated from a neonatal rabbit (1 week-old, control group).