Urinary mRNA for the Diagnosis of Renal Allograft Rejection: The Issue of Normalization.

Urinary messenger RNA (mRNA) quantification is a promising method for noninvasive diagnosis of renal allograft rejection (AR), but the quantification of mRNAs in urine remains challenging due to degradation. RNA normalization may be warranted to overcome these issues, but the strategies of gene normalization have been poorly evaluated. Herein, we address this issue in a case-control study of 108 urine samples collected at time of allograft biopsy in kidney recipients with (n = 52) or without (n = 56) AR by comparing the diagnostic value of IP-10 and CD3ε mRNAs-two biomarkers of AR-after normalization by the total amount of RNA, normalization by one of the three widely used reference RNAs-18S, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Hypoxanthine-guanine phosphoribosyltransferase (HPRT)-or normalization using uroplakin 1A (UPK) mRNA as a possible urine-specific reference mRNA. Our results show that normalization based on the total quantity of RNA is not substantially improved by additional normalization and may even be worsened with some classical reference genes that are overexpressed during rejection. However, considering that normalization by a reference gene is necessary to ensure polymerase chain reaction (PCR) quality and reproducibility and to suppress the effect of RNA degradation, we suggest that GAPDH and UPK1A are preferable to 18S or HPRT RNA.

Fibrosis progression according to epithelial-mesenchymal transition profile: a randomized trial of everolimus versus CsA.

Markers of epithelial-mesenchymal transition (EMT) may identify patients at high risk of graft fibrogenesis who could benefit from early calcineurin inhibitor (CNI) withdrawal. In a randomized, open-label, 12-month trial, de novo kidney transplant patients received cyclosporine, enteric-coated mycophenolate sodium (EC-MPS) and steroids to month 3. Patients were stratified as EMT+ or EMT- based on month 3 biopsy, then randomized to start everolimus with half-dose EC-MPS (720 mg/day) and cyclosporine withdrawal (CNI-free) or continue cyclosporine with standard EC-MPS (CNI). The primary endpoint was progression of graft fibrosis (interstitial fibrosis/tubular atrophy [IF/TA] grade increase ≥1 between months 3-12) in EMT+ patients. 194 patients were randomized (96 CNI-free, 98 CNI); 153 (69 CNI-free, 84 CNI) were included in histological analyses. Fibrosis progression occurred in 46.2% (12/26) CNI-free EMT+ patients versus 51.6% (16/31) CNI EMT+ patients (p = 0.68). Biopsy-proven acute rejection (BPAR, including subclinical events) occurred in 25.0% and 5.1% of CNI-free and CNI patients, respectively (p < 0.001). In conclusion, early CNI withdrawal with everolimus initiation does not prevent interstitial fibrosis. Using this CNI-free protocol, in which everolimus exposure was relatively low and administered with half-dose EC-MPS, CNI-free patients were overwhelmingly under-immunosuppressed and experienced an increased risk of BPAR.

Ofloxacin: new applications for the prevention of urinary tract infections in renal graft recipients.

BACKGROUND: Urinary tract infections (UTIs), the most common form of bacterial infection in kidney transplant recipients, recently have been demonstrated to be detrimental for long-term graft outcome. Therefore, reinforcing antibiotic prophylaxis might be vital, in addition to basic hygiene recommendations, surgical care, and prophylaxis by trimethoprim-sulfamethoxazole. METHODS: In 2006, a Legionella pneumophila contamination of our department's water pipes meant that all the patients undergoing renal transplantation underwent a 1-month regimen of ofloxacin (OFLO) (200 mg every other day). We took this opportunity to measure the incidence of UTI, including acute pyelonephritis (APN), in 100 consecutive patients transplanted before (n = 50) and after (n = 50) this treatment decision was reached. We also studied the antimicrobial resistance profiles in our department and in the rest of the hospital. RESULTS: No patient developed Legionnaire's disease. A dramatic decrease in the incidence of UTI (-63%) was also seen in patients undergoing OFLO treatment. Logistic regression analysis demonstrated that the use of OFLO was independently associated with a reduction in UTI (odd ratio [OR] = 0.31%, 95% confidence interval [CI] 0.11-0.84, P = 0.02) and APN (OR = 0.21%, 95% CI 0.07-0.98, P = 0.045). This protection was sustained during the whole first year post transplantation. As for resistance rates, we observed a decrease in the susceptibility of Pseudomonas aeruginosa to ciprofloxacin in our nephrology department, compared with that observed in the rest of the hospital. The incidence of multi-resistant bacteria was stable. DISCUSSION: Our unintentional extension of prophylactic antibiotherapy with OFLO gave rise to a dramatic decrease in the 1-year incidence of UTI and APN in kidney recipients. Emergence of resistant strains is, however, a major concern.

Risk factors for early epithelial to mesenchymal transition in renal grafts.

Epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells (TECs) may participate in the pathogenesis of renal fibrosis. We performed a prospective study of EMT markers in protocol biopsies obtained 3 months after engraftment from 56 patients who received deceased donor kidneys and who had stable renal function. The presence of EMT was examined, and quantified by immunohistochemical staining for vimentin and translocation of beta-catenin to the cytoplasm. EMT status was defined as the presence of EMT markers in > or = 10% of TECs. EMT features were virtually absent in implantation biopsies, whereas 41% of the grafts were EMT-positive in the absence of advanced chronic allograft nephropathy. Thirteen patients (23%) had borderline changes or acute rejection. EMT features were more frequent in these patients than in those with normal kidney grafts (vimentin expression, p = 0.003; beta-catenin translocation, p = 0.002). EMT in grafts corresponded with elevated serum creatinine of the donor before the recovery of kidney (p = 0.02) and longer cold ischemia time (p = 0.02). In contrast, the donor age had no influence on the expression of EMT markers. These results suggest that EMT is an early and frequent phenomenon in kidney transplants that could be triggered by immunological and/or ischemic tubular injury.