Since its discovery (Yanagisawa et al, 1988) endothelin (ET) has been characterised as a highly vasoactive and mitogenic peptide. Besides its physiological actions, endothelin has been suggested to play a pathophysiological role in various diseases and after organ transplantation (Watschinger and Sayegh, 1996). Endothelin-1 (ET-1), one of the three isoforms of ET, is secreted at numerous sites in the kidney, where it acts in a paracrine or autocrine fashion on ET receptors of target cells. ET-1 regulates many aspects of renal function such as renal blood flow, glomerular filtration rate, and sodium and water transport at different nephron sites (Rubanyi and Polokoff, 1994).
Endothelin-1 exerts its effects by high affinity binding to G protein-coupled cell surface receptors ETA and ETB (Aral et al., 1990; Sakuri et al., 1990; Martin et al., 1990). Endothelin A receptors, present on vascular smooth muscle cells, bind ET-1 with higher affinity than endothelin-2 and endothelin-3 (Lin et al., 1989; Hosoda et al., 1992), and mediate the potent vasoconstrictive effects of ET-1. Endothelin B receptors have similar affinities to all three isoforms of ET (Lin et al., 1989; Martin et al., 1990) and mediate both vasoconstrictive and vasodilatory effects of ET-1.
The various cytokines and growth factors, which are released as a result of the interstitial and vascular inflammatory changes occurring during renal allograft rejection, may be responsible for ET-1 upregulation within the allograft. Thus ET-1 is a likely contributor to the vascular damage and haemodynamic changes associated with acute vascular rejection.
Abnormal proliferation of the vascular myocytes underlying endothelial cells is an important pathological change observed during chronic rejection of an allograft. Thus, the mitogenic action of ET-1 and other growth factors on endothelial cells, fibroblasts and smooth muscle cells may be involved in the pathogenesis of these processes (Hirata et al., 1989b; Bobik et al., 1990; Leppaluoto and Ruskoaho, 1992).
To investigate the association between preproEndothelin-1 (PPET-1), ETA and ETB mRNA levels and renal allograft rejection, quantitive RT-PCR assays were developed to measure levels of these mRNAs in small amounts of renal biopsy tissue. Direct tissue measurement of PPET-1, ETA and ETB mRNA levels may help in the elucidation of the intra-renal effects of kidney transplantation on the human renal endothelin system.
I developed a quantitative competitive RT-PCR assay for analysis of PPET-1 mRNA levels. Using this assay I found no significant difference in the amounts of PPET-1 mRNA present in renal biopsies from patients with or without renal allograft rejection. Furthermore, PPET-1 mRNA levels did not significantly correlate with age, sex, type of rejection, type of immunosuppressive therapy, blood cyclosporine level, serum creatinine, or systolic or diastolic blood pressure, at the time of biopsy. Thus I conclude that in my hands, using my assay the amount of PPET-1 mRNA does not correlate with renal allograft rejection.
In addition, I used specific primer pairs for the amphfication of human PPET-1, ETA and ETB mRNAs with equivalent amplification efficiencies. The amplified products were found to be homologous to their predicted nucleotide sequences, indicating amplification of the specific gene products. To assess comparative levels of these mRNAs in renal biopsies from groups of transplant and non-transplant patients, an endogenous house keeping gene, HPRT, was also amplified to control for the presence of initial template, the efficiency of reverse transcription and to normalise for variations in RNA measurement between samples. However some inter-individual variability was observed in the ratio between PPET-1 and HPRT in the control kidney samples. All this considered analysis of renal biopsies from patient groups with different rejection states suggests no correlation between renal transplant rejection and the expression of PPET-1, ETA and ETB mRNAs. In addition no significant correlation was observed between PPET-1, ETA or ETB mRNA levels in the biopsies and any of the clinical parameters analysed.
Pre-transplant sensitisation to organ-specific antigens and vascular endothelial cell antigens may arise in the same way as anti-MHC antibodies, through rejection of a prior graft, pregnancy or by cross-reaction with infectious agents, or may be the result of circulating immunogenic vascular endothelial cells transfused along with blood (Sbarbati et al, 1991; Bouvier et al., 1970). Preliminary studies have indicated that the incidence of preformed anti-endothelial cell antibodies (AECA) pre-transplant, is often greater than 7% of patients awaiting a kidney graft. (Cerilli and Brasile, 1988).
The hypothesis to be tested was that conserved autoantibodies reactive with vascular endothelial cell antigens, are important markers of renal allograft rejection.
I developed a whole cell ELISA, using glutaraldehyde-fixed human umbilical vein endothelial cells (HUVEC) as substrate, to investigate the level of anti-endothelial cell antibody reactivity in the pre-transplant sera from renal transplant recipients some of whom had subsequent episodes of rejection.
The HUVEC ELISA was used to screen several groups of sera for AECA reactivity. No significant AECA reactivity was detected in pre-transplant sera from patient who either had subsequent rejection of their grafts or patients who showed no evidence of graft rejection. However significant AECA reactivity was detected in the sera of patients with primary sclerosing cholangitis (PSC), but no significant AECA reactivity was detected in sera from alcoholics with liver disease or patients with IgG myeloma.
Immunoblot analysis showed that the reactivity of the pooled positive control sera detected by the HUVEC ELISA, correlated with reactivity to HUVEC proteins, as well as B-cell proteins.
My results suggest that there is no association between renal allograft rejection and the presence of AECA reactivity detected by ELISA using glutaraldehyde fixed HUVEC as substrate. However AECA was detected in serum samples from patients with PSC. Further study of AECA frequency, nature and relation to renal transplantation and vascular disease is needed, to elucidate their pathogenic role, if any, of AECA.
This thesis suggests that intra-graft levels of PPET-1, ETA or ETB mRNA cannot be used as markers of rejection in human renal transplantation. Furthermore AECA reactivity in pre-transplant sera assayed by ELISA using glutaraldehyde fixed HUVEC as substrate, cannot be used as a marker for subsequent rejection episodes in human renal transplantation.