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Sirolimus Does Not Reduce Receptor-Mediated Endocytosis of Albumin in Proximal Tubule Cells

2007; Wolters Kluwer; Volume: 83; Issue: 1 Linguagem: Inglês

10.1097/01.tp.0000240055.49572.07

1534-6080

Ellein Mreich, Jason D. Coombes, Gopala K. Rangan,

Organ Donation and Transplantation

An increasing number of clinical reports indicate that the conversion of renal transplant patients with chronic allograft dysfunction to sirolimus is associated with the development of de novo proteinuria or an exacerbation of preconversion proteinuria (1). The vast majority of these studies are case reports or uncontrolled case series, and it is difficult to precisely determine the pathogenesis of the proteinuria from these data (1). It has been hypothesized that the postconversion proteinuria is largely due to the withdrawal of calcineurin inhibitors (2). On other hand, because of the rapid onset of the proteinuria coinciding with the commencement of the drug, others have postulated that sirolimus, at least in part, has direct effects on glomerular structure/permeability (3) as well as the handling of filtered plasma proteins by proximal tubule cells (PTCs) (4). With regard to the latter, in the normal kidney, approximately 6 to 8 g of albumin escapes into the tubular lumen, but the majority is reabsorbed in the proximal tubule via receptor-mediated endocytosis (RME) (5) and a postglomerular retrieval pathway (6). Based on clinical (4) and other experimental observations (7, 8), we tested the hypothesis that sirolimus reduces RME of albumin in PTCs. To test this hypothesis, opossum kidney (OK)-derived PTCs (kindly provided by Prof. Carol Pollock) (9) were grown to confluence and quiescence was induced by growth in serum-free media for 24 hr. Exposure to differing concentrations of sirolimus (0.1, 1, 10, 25 ng/ml) for 24 hrs did not alter PTC morphology (by light microscopy) or cell cytotoxicity (as assessed by LDH leakage), when compared to the vehicle (dimethylsulfoxide [DMSO]) (data not shown). To determine the effect on RME, the uptake of Texas red (TR)– labeled albumin was undertaken. PTCs were treated with serum-free media, DMSO, sirolimus (0.1, 1, 10, and 25 ng/ml) or latrunculin A (1.5 μmol/L) for 24 hrs. Latrunculin A disrupts the actin microfilament network, thus abolishing albumin uptake (8, 9). As expected, TR-albumin uptake was suppressed by latrunculin A at 4 and 24 hrs (Fig. 1). TR-albumin uptake was increased in the 10 ng/mL sirolimus group compared to DMSO (P=0.008) at 24 hrs only (Fig. 1). No other changes were present at the other concentrations. We next examined the effect of sirolimus on the in vivo expression of the microtubule marker, α-tubulin. In polarized epithelial cells, an intact cytoskeleton and microtubule system is required for RME of albumin and postendocytic processing (10, 11). If sirolimus were to alter tubule cell protein handling, one would expect to see changes in the expression of α-tubulin (11). For this experiment, archival tissue from a previous study was used (8). Immunoperoxidase staining was performed and quantitated as previously described (11). In the present study, α-tubulin was localized in a filamentous pattern in tubule cells, and was greater in intensity in distal more than PTCs. In PON rats, sirolimus did not alter the pattern and expression of cortical α- tubulin (Fig. 1). In conclusion, these data suggest that proteinuria associated with sirolimus is not due inhibition of RME or postendocytic processing of albumin in PTCs.FIGURE 1.: (A) The effect of sirolimus on receptor-mediated endocytosis of albumin in opossum kidney (OK)-derived proximal tubule cells, as determined by the uptake of Texas-red albumin normalized to protein concentration. Data expressed as mean±SEM (n=4–6). *P<0.05 when compared to 0.1% DMSO. (B) Immunohistochemistry for α-tubulin in rat kidney cortex at day four in saline+DMSO (vehicle), protein-overload (PON)+DMSO and PON+sirolimus groups (×400). (C) Quantitative morphometric analysis of α-tubulin immunohistochemistry in the groups (n=6–8 each). Data expressed as mean±SEM.ACKNOWLEDGMENTS The authors thank Prof. Carol Pollock, Ms. Tanya S. Polhill, and Ms. Veronica A. Stevens (Department of Renal Medicine, Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, Sydney) for providing the OK cells, and assistance with cell culture and the Texas-red albumin uptake assay. Ellein Mreich Department of Renal Medicine Kolling Institute of Medical Research University of Sydney Royal North Shore Hospital Sydney, Australia Jason D. Coombes Gopala K. Rangan Centre for Transplant and Renal Research Westmead Millennium Institute The University of Sydney at Westmead Hospital Sydney, Australia