Cyclosporine A Nephrotoxicity: Downregulation of Multiple Drug Resistance-Associated Protein 2 (MRP2) by Upregulated miRNA133a: Its Importance in Renal Transplantation and Treatment of Proteinuric Disease Such as FSGS

Background: Multiple drug resistance-associated protein 2 (MRP2) is regarded as transport protein for xenobiotics such as cyclosporine A (CyA). We have shown that MRP2 is downregulated though miRNA133a binding to its 3`UTR region, induced by endothelin-1 (ET-1) via the ETB-receptor in minimal change nephrosis (MCN) and membranous nephropathy (MN). The status in focal-segmental sclerosis, (FSGS) where CyA is a treatment option, is still unknown. The mechanism of CyA nephrotoxicity in renal transplantation is also still elusive. In particular, co-morbidities/clinical parameters, the morphological extent of MR2 damage and the involvement of miRNA133a leading to nephrotoxicity have not been investigated.

Aims: To analyze co-morbidities, the role of ET- 1 and miRNA133a for development of CyA renal toxicity and in FSGS.

Methods: Analysis of co-morbidities/clinical factors in 230 biopsies from 124 patients. In 35 CyA patients (tubular damage, arteriolar damage, no damage; plus 5 cases of so- called null biopsies as controls), and in FSGS (n=8) immune histology and subsequent morphometry of MRP2 expression was performed and a qRT-PCR for the detection of miRNA133a was analysed. Human proximal tubular cell culture was used for studying the effect of ET-1 alone or in combination with CyA on MRP2 and miRNA133a levels. MiRNA levels were further determined in the ETB-receptor knock-out rat (ETBR sl/sl; wt vs. ko -/- ETBR) after 5/6 nephrectomy as a model for FSGS.

Results: No single clinical parameter was clearly responsible for CyA nephrotoxicity. Morphometric analysis revealed an increasing degree of damage: from no CyA toxicity, to arteriolar CyA toxicicity, to combined arteriolar and tubular toxicity; the maximum degree was seen in tubular toxicity. The effect could be studied in human proximal tubular cell culture, where the combination of ET-1 and CyA showed a higher effect of tubular damage than ET- alone. CyA induced mostly tubular but also arteriolar damage by downregulating the mrp2 gene via the upregulation of miRNA133a. In FSGS, with only a small difference to normal conditions observed by immune histology, by qRT-PCR Mrp2 was downregulated, while miRNA133a was clearly upregulated. Under proteinuric conditions of renal ablation, ET-BR knock-out animals were protected from miRNA133a upregulation.

Conclusion: Downregulation of MRP2 by miRNA133a is an important mechanism of CyA nephrotoxicity and in FSGS. The intensity of cytotoxicity may increase if FSGS is treated with CyA. Potential urinary analysis as detection assay and treatment options by an ETB-receptor antagonist are discussed. The therapeutic potential in steroid resistant syndrome such as in African pediatric FSGS cases is outlined.