Low-dose rapamycin does not impair vascular integrity and tubular regeneration after kidney transplantation in rats

Abstract

mTOR inhibitors offer advantages after kidney transplantation including antiviral and antitumor activity besides facilitating low calcineurin inhibitor exposure to reduce nephrotoxicity. Concerns about adverse effects due to antiproliferative and antiangiogenic properties have limited their clinical use particularly early after transplantation. Interference with vascular endothelial growth factor (VEGF)-A, important for physiologic functioning of renal endothelial cells and tubular epithelium, has been implicated in detrimental renal effects of mTOR inhibitors. Low doses of Rapamycin (loading dose 3 mg/kg bodyweight, daily doses 1.5 mg/kg bodyweight) were administered in an allogenic rat kidney transplantation model resulting in a mean through concentration of 4.30 ng/mL. Glomerular and peritubular capillaries, tubular cell proliferation, or functional recovery from preservation/reperfusion injury were not compromised in comparison to vehicle treated animals. VEGF-A, VEGF receptor 2, and the co-receptor Neuropilin-1 were upregulated by Rapamycin within 7 days. Rat proximal tubular cells (RPTC) responded in vitro to hypoxia with increased VEGF-A and VEGF-R1 expression that was not suppressed by Rapamycin at therapeutic concentrations. Rapamycin did not impair proliferation of RPTC under hypoxic conditions. Low-dose Rapamycin early posttransplant does not negatively influence the VEGF network crucial for recovery from preservation/reperfusion injury. Enhancement of VEGF signaling peritransplant holds potential to further improve outcomes.

Figure 1

Effect of Rapa on vascular integrity early after kidney transplantation. (a, b) Positive area in the cortex (a) and medulla (b) of transplanted kidneys from vehicle or Rapa treated rats stained by immunohistochemistry for rat endothelial cell antigen (RECA)-1. n = 4. (c) Representative photomicrographs for RECA-1 immunohistochemistry 7 days posttransplant with vehicle or Rapa treatment. (d) Total positive area in kidney transplants from vehicle or Rapa treated rats stained by immunohistochemistry for endothelial nitric oxide synthase (eNOS). n = 4–5. (e) Quantification of eNOS mRNA in whole transplanted kidneys by real-time PCR after treatment with vehicle or Rapa. n = 4–5. *P < 0.05, **P < 0.01.

Figure 2

Renal function and albuminuria during the first week after kidney transplantation. Plasma creatinine (a) and urea (b) concentrations in transplanted rats receiving vehicle or Rapa. n = 5–7. (c) Albumin concentrations measured in urine samples collected over 24 h in metabolic cages. n = 4–5. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

The vascular endothelial growth factor (VEGF) network in kidney grafts from rats receiving vehicle or Rapa. Quantitative real-time PCR on days 1–7 (a) and immunohistochemistry on day 7 (b) for VEGF-A. Representative photomicrographs are shown. mRNA expression of the VEGF receptors 1 (VEGF-R1; c) and 2 (VEGF-R2; d) and the VEGF co-receptor neuropilin-1 (e) as assessed with quantitative real-time PCR. n = 4–6. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Influence of hypoxia and mTOR inhibition by Rapa on the vascular endothelial growth factor (VEGF) network in rat renal proximal tubular cells (RPTC). RPTC were incubated with Rapa under normoxic or hypoxic conditions. Expression of VEGF-A mRNA was analyzed by quantitative real-time PCR after 4 h (a) and after 8 h (b). Results were normalized to vehicle control (0 nM Rapa) at normoxia (set to 1.00). Normoxia n = 7–8, hypoxia n = 4. (c) VEGF-A protein measured with an enzyme-linked immunosorbent assay (ELISA) in cell culture supernatants after 24 h normalized to total protein content. n = 9. (d) Expression of VEGF receptor 1 (VEGF-R1) mRNA quantified by real-time PCR after 8 h normalized to vehicle control (0 nM Rapa) at normoxia (set to 1.00). Normoxia n = 6, hypoxia n = 5. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Effect of Rapa on proliferation of tubular cells after kidney transplantation. Percentage of tubular cells positive for proliferating cell nuclear antigen (PCNA) in cortex (a) and medulla (b) during the first week after kidney transplantation as seen with immunohistochemistry. n = 3. (c) Representative photomicrographs for PCNA immunohistochemistry 2 days posttransplant with vehicle or Rapa treatment.

Figure 6

Effect of Rapa on proliferation and metabolic activity of rat renal tubular cells in vitro under normoxic and hypoxic conditions. Rat renal proximal tubular cells (RPTC) were incubated with Rapa under normoxic or hypoxic conditions for 24 h. (a) Proliferation was assessed as incorporation of BrdU. Results are normalized to vehicle control (0 nM Rapa) for each experiment. Normoxia n = 6, hypoxia n = 4. (b) Metabolic activity was measured as conversion of MTT to formazan. Results are normalized to vehicle control (0 nM Rapa) for each experiment. Normoxia n = 4, hypoxia n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.