DIFFERENTIAL PROTECTIVE EFFECTS OF CALCINEURIN INHIBITORS IN AN IN VITRO MODEL OF HUMAN TUBULAR EPITHELIAL CELLS UNDER OXIDATIVE STRESS.

 

Dr CE Nolan1, Dr AG Jardine2, Mr JLR Forsythe3 and Dr PG Shiels1.

1 University Department Surgery, Western Infirmary, Glasgow G11 6NT, 2 Dept. Medicine and Therapeutics, Western Infirmary, Glasgow G11 6NT, 3 Dept Transplant Surgery, Edinburgh Royal Infirmary, Edinburgh EH3 9YW.

 

Calcineurin inhibitors are established immunotherapies used in the prevention of allograft rejection. However, they have also been highlighted as potential contributors in the development of chronic allograft nephropathy (CAN), the leading cause of allograft failure in the first post transplant decade. More recent investigation into their properties however, suggests a novel function of these compounds, unrelated to their immunosuppressive qualities. They have been shown to be protective in conditions of ischaemia, and therefore, it was decided to investigate potential roles of these compounds in ameliorating transplant ischaemia/reperfusion injury.

 

Within the kidney, tubular epithelial cells may be most vulnerable to ischaemic damage and have previously been shown to preferentially sustain damage in a rat model of chronic allograft dysfunction. This apparent preferential susceptibility of epithelial cells to cellular damage is in keeping with observations from cloned animals, whereby those derived from epithelial cells consistently exhibit shorter telomeres. This is indicative of an inability to completely mitigate oxidative damage, which subsequently manifests in accelerated features of cellular senescence. Tubular epithelial cells may also be significant modulators of oxidative stress within the human kidney, in that the uric acid transporter, URAT-1, resides within them. Uric acid is the primary human anti-oxidant, and defects of this transporter result in reactive oxygen species -mediated tubular cell death. For this reason, we have elected to investigate the putative protective effects of calcineurin inhibitors in human tubular epithelial cells exposed to oxidative stress.

 

Cultures of primary human tubular epithelial cells grown in DMEM F12 medium / 20%FCS were exposed to 75mM H2O2 in the presence and absence of physiological concentrations of either cyclosporin A or tacrolimus. These cultures were investigated for markers of oxidative stress and senescence, and gene expression profiles were identified using Real Time quantitative RT-PCR (TaqMan).

 

As expected, the addition of H2O2 resulted in increased lipofuscin levels within the tubular cells, although the presence of tacrolimus appeared to ameliorate this. In addition, tacrolimus lowered the percentage of cells expressing b galactosidase at pH 6, a known senescence associated bio-marker. Both calcineurin inhibitors were effective in reducing expression of DNA damage response genes XRCC5, p21, and SIRT2 after exposure to H2O2. Although this represents preliminary data, it is significant that tacrolimus appears to be exerting a protective effect in tubular epithelial cells exposed to oxidative stress. Further work is required to more fully characterise this exciting observation, and assess a therapeutic role in ischaemia/reperfusion injury.

 

DIFFERENTIAL PROTECTIVE EFFECTS OF CALCINEURIN INHIBITORS IN AN IN VITRO MODEL OF HUMAN TUBULAR EPITHELIAL CELLS UNDER OXIDATIVE STRESS.

 

Dr CE Nolan1, Dr AG Jardine2, Mr JLR Forsythe3 and Dr PG Shiels1.

1 University Department Surgery, Western Infirmary, Glasgow G11 6NT, 2 Dept. Medicine and Therapeutics, Western Infirmary, Glasgow G11 6NT, 3 Dept Transplant Surgery, Edinburgh Royal Infirmary, Edinburgh EH3 9YW.

 

Mailing address for contact:

 

Dr PG Shiels

University Dept Surgery

Western Infirmary

44 Church St

Glasgow G11 6NT

Scotland

Tel       0141 211 2762

Fax      0141 211 1972

Email    P.Shiels@clinmed.gla.ac.uk