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Cyclosporine A alters expression of renal microRNAs: New insights into calcineurin inhibitor nephrotoxicity.

ABSTRACT: Calcineurin inhibitors are powerful immunosuppressants that revolutionized organ transplantation. However, non-immune effects of the calcineurin inhibitor, such as cyclosporine A (CsA), have significantly hindered their use. Specifically, nephrotoxicity, which is associated with tubulointerstitial fibrosis, inflammation, and podocyte damage, affects up to half of all transplant patients. Calcineurin is involved in many aspects of kidney development and function; therefore, mechanisms of CsA-induced nephrotoxicity are complex and not yet fully understood. MicroRNAs are short non-coding RNAs that regulate protein-coding RNA expression through post-translational repression of target messenger RNAs. MicroRNA dysregulation is known to be involved in kidney diseases including fibrosis. In this study, we compared the renal microRNA expression profiles between mice that received CsA (20 mg/kg) or vehicle daily for six weeks. The results demonstrate that CsA induces significant changes in renal microRNA expression profile. We used combined criteria of False Discovery Rate (?0.1), fold change (?2) and median signal strength (?50) and identified 76 differencially expressed microRNAs. This approach identified microRNAs previously linked to renal fibrosis that includes let-7d, miR-21, miR-29, miR-30, miR-130, miR-192, and miR-200 as well as microRNAs that have not been reported to be related to nephrotoxicity or immunosuppression. Pathway analysis of microRNA/mRNA changes highlights the Wnt, TGF-?, mTOR, and VEGF pathways. The mRNA expression profiles were compared in the same samples. The change of mRNA and microRNA profiles showed close correlations. To validate that the observed microRNA and mRNA expression level changes in mice kidney tissue were directly related to CsA treatment, the expression change induced by CsA treatment of three microRNAs (miR-21, miR-186, and miR-709) and three mRNAs (BMPR1a, SMURF1 and SMAD7) were compared in HEK293 cell line. A similar trend of expression level change was induced by CsA treatment in all selected microRNAs and mRNAs in the in vitro cell model. These data provide a roadmap for future work to study the role of the known and novel candidate microRNAs in the mechanism of nephrotoxicity and their further therapeutic potential.

SUBMITTER: Gooch JL

PROVIDER: S-EPMC5393575 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Cyclosporine A alters expression of renal microRNAs: New insights into calcineurin inhibitor nephrotoxicity.

Gooch Jennifer L JL King Clayton C Francis Cynthia E CE Garcia Paul S PS Bai Yun Y

PloS one 20170417 4

Calcineurin inhibitors are powerful immunosuppressants that revolutionized organ transplantation. However, non-immune effects of the calcineurin inhibitor, such as cyclosporine A (CsA), have significantly hindered their use. Specifically, nephrotoxicity, which is associated with tubulointerstitial fibrosis, inflammation, and podocyte damage, affects up to half of all transplant patients. Calcineurin is involved in many aspects of kidney development and function; therefore, mechanisms of CsA-indu ...[more]

PMID: 28414804

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Project description:Chronic calcineurin inhibitor (CNI) nephrotoxicity is a major drawback in current immunosuppressive regimens. Pathology includes vascular and tubulointerstitial alterations. Although still commonly in use, cyclosporine A (CsA) is increasingly exchanged for tacrolimus (Tac) for a more favorable clinical outcome. Data on their differential pathogenetic effects in the kidney are, however, still scarce. We asked how CsA and Tac may affect adverse renal responses differentially. Methods- Wistar rats were divided into groups receiving cyclosporineÊ AÊ (n=4, target trough plasma level 3 mg/ml) and the respective vehicle (n=4, saline), or tacrolimus (Tac, FK506, n= 6, target trough plasma level 3.5 ng/ml) and the respective vehicle (n=6, DMSO/PEG500), via subcutaneously implanted osmotic minipumps for 28 days. Clinical parameters from plasma and urine samples were controlled. Animals were prepared for high-end morphological analysis, elective immunostaining, and high-throughput technology. Large scale electron microscopy (EM), confocal, stimulated emission-depletion (STED) and 3D-structured illumination (SIM) microscopy were used for pathology. Protein biochemistry, transcriptome, proteome, and phosphoproteome technologies were used to identify gene expressional differences. Results - In rats, CsA and Tac produced common as well as distinct alterations in glomeruli and tubulointerstitium. Both drugs caused cortical fibrotic foci with sclerotic glomeruli and damaged tubules. With CsA, glomerular damage was milder than with Tac. Proximal tubules showed widespread dysmorphic lysosomes with peripheral LAMP1 signal as well as autophagic and mitophagic vacuoles along with high apoptosis rate and diminished albumin uptake; megalin-dependent endocytosis was causally involved in the changes, and lysosomal exocytosis was sharply stimulated at the luminal cell pole. KIM1 signal was moderate. With Tac, these changes were far less pronounced, but the incidence of focal tubular necrosis along with intense KIM-1 signal was high. High throughput analysis showed differential changes between CsA and Tac with almost no overlap between the respective spectra. CsA caused upregulation of components of the unfolded protein response and apoptosis, whereas Tac resulted in RAS-associated stimulation and vascular deterioration. Conclusions- CNI nephrotoxicity presented with widely differing pathogenetic characteristics upon chronical CsA vs. Tac treatments. Beyond its known vascular effects, CsA markedly affected proximal tubular integrity, whereas Tac was primarily affecting vascular/glomerular components. Both medications produced comparable degrees of nephron loss and fibrosis. Results may serve to better adjust immunosuppressive treatment combinations in transplant patients.

Project description:The use of calcineurin inhibitor (CI) immunosuppressants has significantly improved the early allograft survival rate in organ transplantation. However, CI therapy has been associated with chronic nephrotoxicity, which limits their long-term utility. In order to understand the mechanisms of the toxicity, we analyzed the gene expression changes that underlie the development of CI immunosuppressant-mediated nephrotoxicity, in male Sprague-Dawley (SD) rats dosed daily with cyclosporine (CsA), FK506 or rapamycin (Rapa) for 1 to 28 days. We identified a group of genes, whose expression in rat kidney is quantitatively correlated with CI-induced kidney injury as observed in changes in blood urea nitrogen (BUN) levels and kidney histopathology. These genes include both up-regulated genes, such as Ren1 and Klks3, and down-regulated genes, such as Calb1, Egf, NCC, and kidney specific Wnk1 (KS-Wnk1). Using the down-regulated genes alone we successfully predicted CI immunosuppressant-mediated kidney injury in rats following 7 days of treatment. Among these genes are two mechanism-related genes, NCC and KS-Wnk1, both of which are involved in the sodium transport in the distal nephrons. The down-regulation of both genes at the mRNA and protein level in rat kidney following CI treatment was confirmed by quantitative RT-PCR and immunohistochemical staining, respectively. We hypothesize that decreased expression of NCC may cause reduced sodium chloride reabsorption in the distal tubules, and contribute to the prolonged activation of the Renin-Angiotensin-System (RAS), a demonstrated contributor to the development of CI-induced nephrotoxicity in both animal models and clinical settings. Therefore, NCC and KS-Wnk1 could potentially be used as biomarkers for early detection and prevention of CI-related nephrotoxicity in clinical practice. Male SD rats approximately 5-7 weeks old were maintained on certified rodent chow and dosed i.p. with olive oil, CsA (CAS # 59865-13-3, 2.5 or 25 mg/kg/day), FK506 (CAS # 104987-11-3, 0.6 or 6 mg/kg/day), or Rapa (CAS # 53123-88-9, 1 or 10 mg/kg/day) (LC Laboratories, Woburn, MA) for 1, 7, 14 or 28 consecutive days. Blood was collected at the terminal necropsy (24 hours after 1, 7, 14 or 28 days of treatment) for clinical chemistry analysis. Each kidney was cut in half (one cross section and the other longitudinal). One half of the left kidney and one half of the right kidney were used for histopathological evaluation and the remaining portions of the kidney were processed for gene expression analysis. Total RNA was isolated from rat kidney using QIAGEN RNeasy kits (QIAGEN Inc., Valencia, CA) following manufacture’s instruction. The rat genome array (RAT 230 2.0) GeneChip (Affymetrix, Santa Clara, CA) was used for gene expression profiling.

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Cyclosporine A alters expression of renal microRNAs: New insights into calcineurin inhibitor nephrotoxicity.

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Cyclosporine A alters expression of renal microRNAs: New insights into calcineurin inhibitor nephrotoxicity.

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