الفهرس 
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Abstract
Chronic kidney disease is a worldwide problem that leads to end-stage renal disease (ESRD). ESRD is known to be the 9th cause of death among other diseases. Also, it is ranking 17th among causes of disability.
Dialysis and transplantation are the therapeutic modalities for ESRD. However, hemodialysis may expose the patients to many hazards. Nowadays, transplantation has become the treatment of choice for ESRD.
The cornerstone of successful transplantation is the perfect introduction of immunosuppressive drugs. The use of immunosuppressive drugs should be monitored periodically due to the narrow safety margin of these drugs. Therapeutic drug monitoring is essential to avoid overdosing toxicity or under-dosing rejection.
The chemokine CXCL10 also known as interferon-inducible protein 10 is a promising non-invasive urinary marker for acute rejection. It may indicate the main candidates for the biopsy procedure which is the gold standard tool for defining the acute rejection.
The protocol regimen widely used is the combination of tacrolimus and mycophenolate mofetil (MMF). They have largely replaced cyclosporine and azathioprine together with prednisone which is considered the most prescribed treatment in developed countries.
The MMF is metabolized to its active form the mycophenolic acid (MPA). MPA has a selective, reversible inhibitory effect on the inosine monophosohate dehydrogenase (IMPDH II). This enzyme together with the uridine glucuronsonyl transferase (UGT1A9) are two key enzymes important for the drug action. IMPHD II is responsible for the proliferation of the T and B lymphocyte through the de novo pathway for the generation of guanosine. When IMPDH II is inhibited by the MPA, this results in arrest of the immune cells. UGT1A9 is the metabolizing enzyme responsible for the clearance of the drug through the glucuronidation of the active compound into its inactive 7‐O‐glucuronide.
Monitoring of MPA by high pressure liquid chromatography (HPLC) and enzyme-multiplied immunoassay technique is important to minimize the side effects such as anemia, leucopenia, gastro-intestinal upsets and urinary tract infection.
Several non genetic factors may lower the MPA level such asGIT disturbances and drug interactions. Many SNPs have been found in IMPDH II and UGT1A9 genes controlling the drug action and metabolism.
IMPDH II T3575C leads to increase in the IMPDH II enzyme activity and thus patients carrying this SNP should have higher dose of the drug to fulfill the complete immune response suppression and prevent the rejection.
UGT1A9 T275A increases the clearance of the MPA thus lowering its dose in the blood of the patients. This may predispose those patients expressing the mutant genotype to rejection.
Our study aimed to evaluate the association between T3757C and T275A polymorphisms of the IMPDH II and UGT1A9 genes, respectively and level of MPA among a sample of renal transplant Egyptian recipients.
This study was carried out on 50 Egyptian patients aged above 18 years who underwent kidney transplantation. All patients received full dose of the mycophenolate for at least 3 months post-transplantation. Pregnant women, simultaneous transplantation of any other organ, those not receiving MPA and patients with GIT problems have been excluded from the study. They were recruited from the Outpatient Clinic of Renal Transplantation Unit in Alexandria University Hospital.
The following polymorphisms were detected by 5′ nuclease allele discrimination assay using real-time PCR; IMPDH II (rs11706052T>C) SNP and UGT1A9 (rs6714486T>A) SNP. Rotorgene Q Real time PCR system was employed to obtain specific sequences amplification according to the following steps: 10µl of TaqMan® universal PCR master Mix (20X) was added followed by 0.5µl of specific assay mix (40X) (T275A, T3757TC) containing primers and probes. Then 1-20ng of extracted DNA and sterile water were added to complete a total volume of 20µl. The thermal profile was as such: An initial step at 95ºC for 10 minutes (hold), followed by 40 cycles of denaturing to 95ºC for 15 seconds and annealing/ extension at 60ºC for 1 minute.
- Measurement of the level of MPA was assessed by HPLC using ClinRep®HPLC Complete Kit for Mycophenolic Acid in plasma from RECIPE®.
- Measurement of the level of CXCL10 in urine using Quantikine®ELISA, Human CXCL10/IP-10 Immunoassay, was done.
The kidney rejection was significantly associated with variant genotypes CC and AA in both IMPDHII T3757C andUGT1A9 T275A (p<0.001andp=0.001, respectively).
Also, MPA AUC showed a statistical significant lower level among patients with graft failure with a mean of 45.7 ± 15.3 (p<0.001).
Urinary levels of CXCL10 were significantly higher in patients with allograft failure (132.70 ± 24.27 pg/ml) compared to those with normal kidney function (43.91 ± 31.70 pg/ml) at p<0.001. ROC curve for CXCL10 was done to predict kidney rejection at the cut-off value of106.58 pg/ml with highest sensitivity (80%), specificity (95.56%) and AUC was 0.987 (95% CI 0.959–1.00).
Patients expressing variant genotypes in both SNPs; IMPDH II T3757C and UGT1A9 T275A had lower MPA AUC with mean 42.46(×103) ± 17.07(×103) and 50.52(×103) ± 16.45(×103), respectively compared with mean of patients expressing wild genotypes 711.10(×103) ± 514.43(×103) and 874.56(×103) ± 483.82(×103), respectively (p<0.001).
We found a significant negative correlation between the CXCL10 and MPA AUC (p<0.001).
Multivariate linear regression was done considering (IMPDHII T3757Cand UGT1A9 T275A) as well as urea, creatinine, eGFR and CXCL10 as confounding factors while MPA AUC as dependent variable. UGT1A9 T275A was the strongest predictive parameters for lower MPA AUC followed by CXCL10 then IMPDHII T3757C and lowest one was creatinine (t= 6.562, 3.786, 3.757 and 3.725, p <0.001, <0.001, 0.001 and 0.001, respectively).
from the current study we concluded that renal transplant recipients should be genotyped before taking MPA to prevent renal allograft rejection. Graft could be monitored by urinary level of CXCL10 with high specificity and sensitivity.