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Dendritic cells are key players in renal allograft rejection and have been identified as an intrinsic part of the kidney. Here we quantified and phenotyped the dendritic cell populations in well-defined biopsies of 102 patients with acute renal allograft rejection in comparison with 78 available pretransplant biopsies. There was a strong increase in BDCA-1(+) and DC-SIGN(+) myeloid, BDCA-2(+) plasmacytoid, and DC-LAMP(+) mature dendritic cells in rejection biopsies compared with the corresponding pretransplant tissue. Mature dendritic cells were mostly found in clusters of lymphoid infiltrate and showed a strong correlation with the Banff infiltrate score. The presence of both myeloid and plasmacytoid dendritic cell subsets in the kidney during acute rejection correlated with interstitial fibrosis and tubular atrophy. Importantly, the myeloid dendritic cell density at the time of acute rejection was an independent risk factor for loss of renal function after the first year. Thus, acute renal allograft rejection is characterized by an influx of myeloid and plasmacytoid dendritic cells, strongly associated with local damage in the graft. Hence, the density of myeloid dendritic cells during acute rejection could be an important risk factor for the long-term development of chronic changes and loss of graft function.

Background: In kidney transplantation, chronic allograft nephropathy (CAN) is the major cause of graft loss. Causes of CAN include chronic rejection and chronic cyclosporine A (CsA) nephrotoxicity. It is necessary to differentiate between these two entities in order to apply the appropriate therapeutic regimen for the individual patient, but this is hampered by the lack of discriminating functional and morphologic parameters. We investigated whether renal cortical mRNA levels for several matrix proteins can serve as discriminating parameters., Methods: Patients with chronic rejection (N= 19) and chronic CsA toxicity (N= 17) were selected by clinical and histologic criteria. Protocol biopsies without histologic abnormalities, taken at 6 months after transplantation from patients receiving CsA, were used as controls (N= 6). Total RNA was extracted from the renal biopsy tissue, and mRNA levels of transforming growth factor-beta (TGF-beta) and the extracellular matrix (ECM) molecules collagen Ialpha1, IIIalpha1, IValpha3, decorin, fibronectin, and laminin beta2 were measured by real-time polymerase chain reaction (PCR)., Results: In both patient groups, the mean collagen IValpha3 and fibronectin mRNA levels were significantly elevated compared to those in controls, whereas only in CsA toxicity were the laminin beta2 and TGF-beta mRNA levels significantly increased. The increase of laminin beta2 and TGF-beta mRNA levels was significantly higher in the CsA toxicity group than in the chronic rejection group (P < 0.001 and P= 0.004, respectively). Receiver-operating characteristic (ROC) curve analysis showed that with a 15.6-fold increase in laminin beta2 mRNA expression as cut-off point, the presence of CsA toxicity could be predicted with an 87% sensitivity and an 88% specificity., Conclusion: Renal laminin beta2 and TGF-beta mRNA levels can be used to differentiate between chronic rejection and chronic CsA toxicity in renal transplants. The method of mRNA quantification might be applicable as an additional diagnostic tool in clinical practice.

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.

Damage to the kidney generally elicits tissue repair mechanisms, but these processes themselves conversely may result in the progression of chronic renal disease. In a majority of patients chronic renal insufficiency progresses to a common histological end point, marked by the presence of a vast amount of scar tissue, that is, glomerulosclerosis and interstitial fibrosis. These lesions are the result of an excessive production of extracellular matrix (ECM) components. Studies on RNA expression in experimental kidney disease have shown that renal mRNA levels for ECM components and cytokines can function as prognostic tools. This suggests that mRNA levels potentially predict outcome and reaction to therapy in patients with renal diseases. Timely detection of molecular alterations could allow early therapeutic intervention that slows down or even prevents the development of sclerotic and fibrotic lesions. This review first provides a short introduction on mechanisms of initiation and progression of renal disease. Molecular techniques are available to identify renal RNA sequences potentially involved in disease progression. We discuss several molecular techniques that are being used in kidney research for quantitation and detection of mRNA. This is followed by a brief overview of investigation in experimental renal diseases, which reveal that alterations in tissue ECM mRNA levels precede histological damage and can function as predictors of clinical outcome. In particular, studies in human kidney biopsies that evaluate the prognostic value of mRNA levels with respect to renal function are examined, paying special attention to the pitfalls that potentially are encountered when interpreting the results of such studies. Then, we elaborate on ways of optimal exploitation of mRNA quantification as a prognostic tool. The potential and limitations of microarray technology in the search for genes specifically involved in progression of renal disease are reviewed, including RNA expression profiling and large-scale DNA mutation screening. Finally, the future utilities of microarray in nephrology and renal pathology are discussed.

Background: Investigation of the prognostic value of the expression of mRNA for extracellular matrix (ECM) components with respect to deterioration of kidney function in patients with renal disease requires an evaluation of the basal expression of ECM mRNA in healthy individuals and of the reliability of ECM mRNA measurements. In the current study, the collagen alpha 1(IV)/GAPDH (C4:G) and collagen alpha 1(I)/GAPDH (C1:G) mRNA ratios and the accumulation of collagen IV and collagen I protein were investigated in renal cortices of individuals of various age. Furthermore, we examined whether the C4:G mRNA ratio measured in a renal biopsy is representative of that in the rest of the kidney., Methods: To investigate the effect of age on collagen expression, kidneys obtained at autopsy from patients with a normal renal function (N = 18; age 19 to 92) were used. C4:G and C1:G mRNA ratios were measured by real-time polymerase chain reaction (PCR) analysis. Accumulation of collagen IV and collagen I protein was measured by quantitative image analysis on immunohistochemically stained sections. To determine whether the site at which a biopsy is taken affects the C4:G mRNA ratio, this ratio was measured in cortical biopsies taken from different locations from each of four kidneys: one without renal disease, one with diabetes mellitus type I, and two with diabetes mellitus type II. C4:G mRNA ratios were measured by using real-time PCR., Results: The C4:G mRNA ratio, but not the C1:G mRNA ratio or collagen IV protein accumulation, increased significantly with age (r = 0.55, P < 0.03). Collagen I protein accumulation increased with age (r = 0.85, P < 0.001) and correlated with the extent of interstitial fibrosis (r = 0.50, P < 0.05). The C4:G mRNA ratio did not differ significantly within a kidney., Conclusions: This report shows, to our knowledge for the first time, that in the aging, normally functioning human kidney, there is a dissociation between the levels of mRNA for collagen IV and collagen I and the accumulation of these proteins. The levels of mRNA for collagen IV in a single renal biopsy can be regarded as representative of those in the rest of the kidney. These observations should be taken into account when ECM mRNA levels are used for diagnostic purposes.