Molecular comparison of calcineurin inhibitor-induced fibrogenic responses in protocol renal transplant biopsies.

The calcineurin inhibitor cyclosporine (CsA) induces a fibrogenic response that may lead to scarring of the renal allograft. This study investigated whether tacrolimus, a novel calcineurin inhibitor, exerts fibrogenic effects to a similar extent. Sixty patients were enrolled in a randomized study: 29 received CsA, and 31 received tacrolimus. Patients were subjected to tailored exposure-controlled calcineurin inhibitor regimens. Protocol biopsies were obtained at the time of transplantation and 6 and 12 mo after transplantation. Cortical TGF-beta and collagens alpha1(I) and alpha1(III) mRNA steady-state levels were determined with real-time PCR. The extent of protein deposition of TGF-beta, alpha-smooth muscle actin, and interstitial collagens in the renal cortex was quantified with computer-assisted image analysis. The extent of interstitial collagen deposition measured with Sirius red and the accumulation of alpha-smooth muscle actin and TGF-beta protein after 6 and 12 mo were similar for both immunosuppressive regimens. mRNA levels of TGF-beta and collagens alpha1(I) and alpha1(III) were not significantly different in the treatment groups either. It is concluded that the fibrogenic response in renal allografts is similar in patients who receive CsA-based regimens and patients who receive tacrolimus-based regimens.

No difference in degree of interstitial Sirius red-stained area in serial biopsies from area under concentration-over-time curves-guided cyclosporine versus tacrolimus-treated renal transplant recipients at one year.

Interstitial fibrosis is the main characteristic of chronic allograft nephropathy and long-term graft failure. Cyclosporin (CsA) is thought to be more fibrogenic than tacrolimus. In a prospective, randomized, multicenter trial using a calcineurin-sparing regimen, renal interstitial volume was compared in CsA- and tacrolimus-treated renal transplant recipients by image analysis of Sirius red (SR)-stained cortical areas in protocol biopsies obtained at 6 mo (n = 94) and 12 mo (n = 97) after transplantation. Immunosuppression consisted of CsA or tacrolimus, CD25 mAb, mycophenolate mofetil, and prednisolone. CsA therapy increased the 6-mo risk for subclinical rejection. The prevalence of subclinical rejection was 38.8% in the CsA-treated and 15.2% in the tacrolimus-treated patient group (P = 0.012). Strikingly, no difference in the degree of interstitial SR-stained area was detectable between the two treatment groups. In particular, previous subclinical rejection episodes did not influence the degree of interstitial volume. Also, no difference in GFR occurred at 1 yr, when the mean GFR mounted 63 ml/min. No significant differences in the degree of interstitial SR-stained area could be observed at 6 and 12 mo between CsA- and tacrolimus-treated renal transplant recipients. Although CsA-treated patients developed significantly more subclinical rejections at 6 mo, this did not influence the degree of SR staining or the change in renal function at 1 yr.

Expression of surfactant protein-C, S100A8, S100A9, and B cell markers in renal allografts: investigation of the prognostic value.

The intent of this study was to identify genes of which expression during acute rejection is associated with progression to chronic allograft nephropathy using gene expression profiling. Ten patients who had graft loss through chronic allograft nephropathy (progression [PR] group) and 18 patients who had stable graft function over time (nonprogression [NP] group) were studied. Rejection severity and extent of infiltrating leukocytes in acute rejection biopsies were similar for both groups. Microarray analysis and real-time PCR validation showed that surfactant protein-C (SP-C), S100 calcium-binding protein A8 (S100A8), S100A9, and beta-globin levels distinguished the two groups. Relationship between expression of B cell markers and prognosis was also examined. Location in the graft of the protein and mRNA expression of candidate genes was investigated. The prognostic value of mRNA transcripts was tested in an independent cohort of 43 rejection biopsies. mRNA and protein expression of S100A8 and S100A9 in infiltrating cells was significantly higher in the NP group compared with the PR group. Expression of SP-C was four-fold higher in the PR group and was detected in glomeruli. No association between B cell clusters and outcome was found. In the second group of acute rejection biopsies, SP-C mRNA levels predicted renal function course beyond 6 mo in multivariate analysis. Relatively high expression of S100A8 and S100A9 during acute rejection is associated with a favorable prognosis, and high SP-C expression is associated with an unfavorable prognosis. Messenger RNA transcripts complement the biopsy in the prediction of graft function deterioration.

Improvement of extraction and processing of RNA from renal biopsies.

BACKGROUND: Assessment of mRNA levels has the potential to predict renal outcome. The objective of this study was to optimize several steps in the protocol for obtaining cDNA from routine clinical kidney biopsy material. METHODS: RNA integrity was compared between different methods for extraction of RNA, synthesis of cDNA, and storage of renal tissue. Hereby, RNAlater, an RNA-preserving compound, was tested for implementation in a protocol for renal biopsies that combines routine histology and RNA expression studies. Gel electrophoresis and real-time polymerase chain reactions (PCR) were outcome parameters for assessment of RNA integrity. RESULTS: The Trizol method rendered higher RNA yields from fresh renal tissue than the NP40 method and RNeasy spin columns. RNA yields were not affected when renal tissue was stored at -70 degrees C for up to 2 months in phosphate-buffered saline (PBS). cDNA levels obtained using avian myeloblastosis virus (AMV) reverse transcriptase (RT) were at least twice as high as those obtained with Sensiscript and Superscript RT. RNAlater maintained RNA integrity in whole renal cortex stored at 4 degrees C for up to 3 months. Dissection of small biopsies in RNAlater rendered similar RNA yields in comparison with dissection in PBS, but the yield of glomeruli from the cortices was 50% lower (P < 0.005). Integrity of RNAlater-treated tissue, evaluated by light microscopy and immunofluorescence, was diminished. CONCLUSION: This study shows optimization of several steps in the protocol for extraction and handling of RNA in renal cortical tissue. RNA extraction and cDNA synthesis can be optimized by the use of the Trizol method and AMV RT, respectively. RNAlater is beneficial for preserving RNA integrity in whole renal cortex during storage and processing, but is not suitable for implementation in routine diagnostic histologic stainings combined with RNA expression studies in dissected biopsy material.