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Morphological evaluation of kidney following Cyclorsporine administration.


Cyclosporine (CsA) is extracted from Tolypocladium inflatum Gams, which is metabolized through the superfamily of hepatic isoenzymes P-450. CsA has a mean life of 6.4-8.7 h, although this varies among different individuals. Ninety percent of the drug is withdrawn through biliary excretion and only 6% appears unchanged in the urine. The exact mechanism of action of CsA is unknown; however, CsA has the ability to act on the immune system by blocking the biosynthesis of some lymphokines produced by T lymphocytes and interleukine-2 synthesis at the transcriptional level. It has been suggested that CsA acts by interacting with cytoplasmic membrane and activates the intracellular calcium pathway, or binds to cytoplasmic proteins (Parra, 2003). At toxic levels, CsA also has the ability to cause renal damage and histological changes that can affect the function of a transplanted kidney (Kahn, 1989, Wang, 1994, Bagnis, 1996, Hansen, 1996). Some of the typical signs of CsA usage include reduced glomerular filtration and changes in intrarenal hemodynamic function, which can start to occur after one week of usage (Tegzess, 1988, Kahan, 1989). Endothelial dysfunction and hypertension are common complications of calcineurin inhibitors, such a cyclosporine and Tacrolimus. Calcineurin inhibitors exert its effects on vasomotor tone. Oxidative stress induced by superoxide has been implicated as a cause of hypertension. NADPH oxidase is the main source of superoxide production in phagocytic and vascular cells. The [p22.sup.phox] subunit is involved in NADPH oxidase activation. Kidney and heart transplant patients that are treated with cyclosporine have been shown to have an increase in reactive oxygen species production, upregulation of the nitric oxide synthase gene expression, and nitrite/nitrate levels. Using [p22.sup.phox] as a marker of oxidative stress, there was not a significant difference between the group treated with cyclosporine or tacrolimus when compared to normotensive healthy controls (Calo, 2002).

In the early stages of CsA administration kidney damage can occur, which causes alterations in intrarenal hemodynamics related to afferent arteriolar vasoconstriction, causing a decrease in glomerular filtration rate, renal plasma flow, loss of proximal tubular cells brush border, proximal tubule dilatation, swelling, necrosis, and infiltration of white blood cells in the kidney cortex. Renal tubular toxicity induced by CsA can be acute with the appearance of oligoanuria, presence of atrophic tubules, and edema. In addition, CsA can induce a subacute syndrome with giant mitochondria, isometric vacuolization, and microcalcifications in proximal tubules. CsA causes an imbalance of the cellular oxidative status because of increased formation of free radicals, which can be attributed to degradation of membrane phospholipids. In in vitro studies, CsA has induced lipid peroxidation in rat kidney and liver microsomes. In vivo studies have demonstrated that lipid peroxidation induced by CsA was dose- dependent and paralleled the renal function, measured as decreased glomerular filtration rate, and renal blood flow, and increase renal vascular resistance. In the rat glomeruli treated with CsA, showed a dose dependent increase in enzyme activity.

Hypothesis A: Cyclosporine is a potent immunosuppressive agents that act on many cells of the body, including epithelial cells and may cause a decrease in the cell proliferation and increase markers for cell damage. Specific Aim : To evaluate kidney epithelial cells after exposure to various doses (low, medium, and high) of CsA and to measure changes in cellular proliferation, morphology and function with time.

Rationale: Renal damage caused by therapeutic treatment with CsA has been previously documented; however, the exact mechanism by which this drug causes nephrotoxicity has yet to be clarified. TGF-P induces the synthesis of numerous extra cellular matrix (ECM) proteins such as fibronectin and collagen; decreases matrix degradation by down regulating proteases inhibitors; enhances the expression of integrins on the cell surface; facilitates deposition of matrix; and is a major regulator in the healing process (Border, 1990). Previous findings in our laboratory showed increasing doses of Cort to RMKC caused a decrease in cell number and an increase in cellular damage (Vance, 2003).


The RMKEC cell line was obtained from the American Type Culture Collection (ATCC) and grown in 24 well plates on coverslips. Each group was treated with CsA 10n.g/ml, 25n.g/ml, or 50n.g/ml of cyclosporine respectively. After treatment and incubation, the supernatant were removed from the wells and the coverslips were mounted on a glass slide. The slides were stained with H&E stain and digitized using Image Pro.


Cyclosporine (CsA) is one of the most widely used drugs in organ transplantation. CsA administration following transplantation is associated with 1-year graft survival rates of approximately 90% (Harihran et al., 2000), but the major drawback is associated with renal vasoconstriction (English et al., 1987). Long term CsA use has been linked to structural changes in the kidney which leads to an irreversible decline in kidney function. Long term use is associated with interstitial fibrosis and tubular atrophy. The mechanism or events leading to CsA nephrotoxicity is currently unknown. Several studies suggest that CsA mode of action through an induction of apoptosis (Jennings 2007,). Baker et al., (2007) examined CsA on the viability of cultured proximal tubular epithelial cells by measuring cell death by apopotosis or necrosis. Their findings showed CsA at concentrations of < 10 ug/mL did not result in increased apoptotic behavior. Furthermore when CsA given at concentrations > 10ug/mL the results showed increased cell death. Jennings et al., (2007) also demonstrated that CsA can induce senescence in renal tubular epithelial cells. They showed CsA induced [H.sub.2][O.sub.2] production, which ultimately caused cell cycle arrest in the G0/G1 phase.


Irreversible cell damage can occur by electrophilic radicals that can react with oxygen giving rise to reactive oxygen species. ROS that have the ability to react with intracellular molecules, unsaturated fatty acids, and transmembrane proteins with oxidizable amino acids. The cellular changes that occurred resulted in alterations of ionic gradients, disruption of several membrane functions, and induced free radical production. Patients treated with CsA have a lower prevalence of bacterial and fungal infections, but a higher incidence rate of viral infections and pneumocystis carinii pneumonia. Renal dysfunction is the main complication associated with CsA treatment, which results in 30% of patients having moderate to severe kidney damage. The findings from our study indicate that the overall the administration of cyclosporine resulted in changes as early as 24 hours in comparison to the control. By 72 hours the group treated with cyclosporine displaced devastating morphological changes, which can ultimately result in kidney dysfunction in comparison to the control.


Calo, L., Davis, P., Giacon, B., Pagnin, E., Sartori, M., Riegler, P., et al., (2002). Oxidative stress in kidney transplant patients with calcineurin inhibitor-induced hypertension: Effect of ramipril. J Cardiovasc Pharmacol, 40(4), 625-631.

Hansen JM, Olsen NV, Leyssac PP.(1996) Renal effects of amino acids and dopamine in renal transplant recipients treated with or without cyclosporin A. Clin Sci (Lond). Oct; 91(4):489-96.

Hariharan S, Johnson CP, Bresnahan BA, Taranto SE, McIntosh MJ, Stablein D. (2000) Improved graft survival after renal transplantation in the United States, 1988 to 1996. N Engl J Med. Mar 2; 342(9):605-12.

Jennings P, Koppelstaetter C, Aydin S, Abberger T, Wolf AM, Mayer G, Pfaller W. (2007) Cyclosporine A induces senescence in renal tubular epithelial cells.

Am J Physiol Renal Physiol. Sep; 293(3):F831-8. Epub Jun 27.

Kahn D, Mazzaferro V, Cervio G, Venkataramanan R, Makowka L, Van Thiel DH, Starzl TE. (1989) Transplant Proc. Correlation between dose and level of cyclosporine after orthotopic liver transplantation. Feb; 21(1 Pt 2):2240-1

Parra, C., Conejo, G., Carballo, A., & De Arriba, G. (2003). Antioxidant nutrients protect against cyclosporine a nephrotoxicity. Toxicology, 15(189(1-2)), 99-111.

Tegzess AM, Doorenbos BM, Minderhoud JM, Donker AJ. (1988) Prospective serial renal function studies in patients with nonrenal disease treated with cyclosporine A. Transplant Proc. Jun; 20(3 Suppl 3):530-3.

Wang C, Salahudeen AK. (1994) Cyclosporine nephrotoxicity: attenuation by an antioxidant-inhibitor of lipid peroxidation in vitro and in vivo. Transplantation. Oct 27; 58(8):940-6.

Stacy Hull Vance, Michelle Tucci and Hamed Benghuzzi

University of Mississippi Medical Center, Jackson, Mississippi, USA
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Author:Vance, Stacy Hull; Tucci, Michelle; Benghuzzi, Hamed
Publication:Journal of the Mississippi Academy of Sciences
Article Type:Report
Geographic Code:1USA
Date:Apr 1, 2014
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