Cyclosporine-A transport in isolated renal proximal tubular cells: Inhibition by calcium channel blockers

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages ]226-'1230

December 30, 1988

CYCLOSPORINE-A TRANSPORT IN ISOLATED RENAL PROXIMAL TUBULAR CELLS: INHIBITION BY CALCIUM CHANNEL BLOCKERS

Chandrasekharam N. Nagineni*, David B.N. Lee, Brijesh C. Misra and Norimoto Yanagawa

Division of Nephrology, VA Medical Center, Sepulveda, California 91343 and UCLA School of Medicine, Los Angeles, California 90024 Received November ii, 1988

We have studied the transport characteristics of cyclosporine A (CSA) in isolated rabbit renal proximal tubular cells (PTC). The uptake as well as efflux was very rapid and dependent on temperature. PTC accumulated CSA by several fold above the incubation medium concentration. Kinetic analysis yielded an apparent Km and Vmax values of 5.1 #M and 47 Pmoles/106 cells/min respectively. Calcium channel blockers verapamil or diltiazem, at concentrations (0.5-1.0 mM) that inhibited calcium uptake, reduced CSA uptake significantly. Other calcium transport modulators A23187 (5#M), trifluoroperazine (50#M) and ruthenium red (100/~M) induced anticipated changes in calcium uptake but had no effect on CSA uptake. These results suggest a close association or interaction between the calcium channels and the CSA transporting/binding sites on PTC membranes. ® 198~ Ac~demioPress, ~nc.

Cyclosporine A (CSA), a potent immunosuppressive agent(l), is being widely used to prevent graft rejection in organ transplantation(2). Nephrotoxicity associated with CSA administration has been observed in both experimental animals(3,4) and man(5).

The primary

site of injury in the kidney is the proximal tubules while glomeruli and distal tubules are less affected(6,7). Structural alterations induced by CSA in proximal tubular cells include vacuolization, degeneration of microvilli and mitochondria and dilatation of endoplasmic reticulum(6,8). Massive deposits of CSA within PTC has been reported in human renal allografts(9,10). Under culture conditions proximal tubular cells were shown to be more susceptible to CSA toxicity than cells from other segments of the nephron(l 1,12). Use of calcium channel blockers (CCB) simultaneous with CSA administration has been shown to amiliorate CSA nephrotoxicity in transplant recipients(13-15). However, the mechanisms of CSA transport in kidney and CCB protection of renal tubular damage are not known. Since proximal tubule is the major site of drug transport(16) and CSA toxicity, we have studied the nature of CSA transport and its interaction with CCB (verapamil and diltiazem) in freshly isolated rabbit renal proximal tubular cells (PTC). The results provide evidence for the interaction between CCB and CSA uptake in the kidney.

*To whom all correspondence should be addressed at: Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California 90024. 0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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MATERIALS AND METHODS

Chemicals: All chemicals were obtained f r o m Sigma Chemical Company, St. Louis, Missouri. [45Ca] calcium chloride w a s p u r c h a s e d from New England Nuclear, Boston, Massachusetts. Cyclosporine A and [3H] Cyclosporine A (specific activity, 9.5 c i / m m o l ) were gifts f r o m Dr. J.F. Borel, Sandoz Ltd, Switzerland. Cell preparation: PTC were prepared f r o m isolated proximal tubules of r a b b i t k i d n e y using an intracellular-like solution as described earlier(17). A f t e r isolation, cells were washed and suspended in uptake m e d i u m for at least 1 h o u r prior to uptake studies. The uptake m e d i u m (pH 7.4) has the following composition (mM): NaC1 137, KC1 5, CaC12 1.3, MgC12 0.5, MgSO 4 0.4, N a H C O 3 4, Hepes 10, glutamine 2, f l - h y d r o x y b u t y r a t e 1. Uptake and E f f l u x Studies: Stock solution of CSA (10raM) in ethanol was diluted just before use with an ethanol:tween 80 mixture. Final concentrations of ethanol (0.2%) and tween 80 (0.005%) in the incubation mixture had no e f f e c t on viability of cells. All transport studies of CSA and calcium were conducted as previously reported(17,18). Incubation tubes were constantly shaken at 60 cycles/rain for thorough mixing of the reaction components. Viability of cells (>90%) was routinely confirmed by the t r y p h a n blue dye exclusion method. O t h e r details are described in figure legends. RESULTS CSA (1 to 10#M) uptake by PTC was linear with the cell concentrations of 1 to 3 x 106 in a final incubation volume of 200 #L. Therefore, in all subsequent studies a b o u t 2 x 106 cells were used for each sample.

At 4°C, practically no uptake or efflux was observed (Fig.l).

After

t e r m i n a t i o n o f reaction, cell associated CSA was f o u n d u n c h a n g e d up to 2 hours w h e n the tubes were left on ice before processing the cells for scintillation counting.

The uptake was rapid at

37°C reaching almost saturation by 3 rain with no f u r t h e r change up to 20 rain (Fig.lA).

At

25°C, the initial rates of uptake were significantly lower than at 37°C, but the same m a x i m u m uptake was attained in 10 min.

The rate of efflux was higher at 37°C than at 250C (Fig.lB).

In the presence of 5#M CSA, the efflux was a u g m e n t e d due to counter exchange of intracellular [3HI CSA with m e d i u m CSA. However, when [3H] CSA was added along with 5#M CSA, cell associated CSA was m a i n t a i n e d at steady state level.

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Figure 1: Effect of temperature on time course of CSA uptake(A) and efflux(B) by_ PTC. Incubation mixture in a final volume of 200#L of uptake medium contained 2 x 10b cells, 5#M CSA and 105 CPM of [3H] CSA. Cells were incubated at 37"C, 25°C or 4"C for the indicated periods and the reaction was terminated by adding 1 ml of ice-cold uptake medium. After 10 minutes of cooling on ice, cells were washed twice and pelleted by centrifugation at 1315 x G and solubilized in 0.5 ml of I% triton X-100 for scintillation counting. For efflux studies, cells were loaded with 5#M CSA and 105 CPM of [3H] CSA by incubation at 37°C for 10 minutes, washed and resuspended in uptake medium (CSA-free) at 4, 25 or 37"C. In the "37"C + CSA (5 #M)" study, the uptake medium contained 5#M CSA. Aliquots of cells were removed at different times and processed as above. Results are means -+ SEM for 3 experiments in duplicate with different cell preparations. 1227

Vol. 157, No. 3, 1988

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

70 A to.O,uM

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: A. Time course of CSA uptake by PTC at different medium concentrations of CSA. ells in 100#L of uptake medium were preincubated at 37°C for 5 min and 100LzL of uptake medium (at 37°C) containing different concentrations (0.1 - 20#M) of CSA and [3HI CSA was added. After indicated periods of incubation the uptake was terminated and cells processed for counting as described earlier. Results are means + SEM for 4 experiments in duplicate with different cell preparations. B. Double reciprocal plot of CSA concentration versus initial rate of CSA uptake. Uptake values at 30 seconds incubation time from the data presented in panel A were used.

Time course of CSA uptake at different concentrations of m e d i u m CSA (Fig.2A) indicate that accumulation of CSA in cells reaches plateau by about 3 minutes at all concentrations.

Cells concentrated CSA by 77 and 31 folds above the extracellular

concentrations of 1.0 and 10.0/~M respectively.

Since the uptake exhibits linearity up to 30

seconds, we have used the values at this time point to draw double reciprocal plots of CSA concentrations versus uptake (Fig.2B).

K m value of 5.1/zM and Vmax of 47 pmoles/106

cells/min were obtained from this plot as well as by linear regression analysis. We have also studied CSA uptake by PTC in the presence of various types of calcium transport modulators to examine possible interaction between calcium fluxes and CSA uptake by PTC.

Organic CCB verapamil and diltiazem significantly inhibited CSA uptake (Fig.3) at

concentrations that also inhibited calcium uptake (Fig.4). Calcium ionophore A23187 (stimulator of Ca ++- 2H + exchange), trifluoroperazine (inhibitor of Ca ++ d e p e n d e n t ATPase) and r u t h e n i u m red (inhibitor of Ca++-2Na + exchange) had no effect on CSA transport (Fig.3) at concentrations that altered calcium uptake by these cells (Fig.4). DISCUSSION Because of its k n o w n lipophilic nature, it has been proposed that CSA enters the cell by passive diffusion through partitioning into the lipid phase of plasma membrane(19). This hypothesis is supported by the observation that CSA uptake by PTC(18) or by h u m a n lymphocyte cell line, Raji(20), is i n d e p e n d e n t of metabolic energy or intracellularly-directed sodium gradient.

The observed t e m p e r a t u r e - d e p e n d e n t rapid uptake of CSA by PTC in the

current study is also similar to that reported in murine t h y m o m a cell line, BW5147(21) and in Raji cells(20). However, there are differences in CSA transport between PTC and lymphocytes. The CSA efflux from PTC is more rapid (50% in 20 min) than in Raji cells (10% in 20 1228

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BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

140 120

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Figure 3: Effect of various calcium transport modulators on CSA uptake by PTC. 2 x 106 cells suspended in 100#L of uptake medium containing effector at the indicated concentration Sl,05 were preincubated for 5 rain at 37°C. 100/*L of uptake medium (37°C) containing 10/,M CSA, CPM of [3H] CSA and effector was added. After 3 rain of incubation, uptake was terminated and cells processed for scintillation counting as described. Effector and its concentration is indicated in each histogram. Results, expressed as percent of control, are means • SEM for 6 experiments in duplicate with different cell preparations. *P < 0.01; +P < 0.05; VP, verapamil; DZ, diltiazem; RR, ruthenium red; TFP, trifluoroperazine; A23187, calcium ionophore. Figure 4: Effect of various calcium transport modulators on calcium uptake by PTC. 2 x 106 cells, suspended in calcium free uptake medium containing effeetor, were preincubated for 5 min at 370C. 100#L of uptake medium (37 °) containing 1.3mM calcium chloride, l06 CPM of [45Ca] and effector was added. After 15 min of incubation at 37°C, the reaction was stopped and cells processed for counting. Results, expressed as percent of control, are means _+ SEM for 5 experiments in duplicate with different cell preparations. Abbreviations are as given for Figure 3.

min)(20).

Also, CSA accumulation in BW5147 (90 pmoles/106 cells)(21) is greater than that in

PTC (30 pmoles/106 cells) at 3#M extracellular concentration, These observations suggest d i f f e r e n c e s in m e m b r a n e and intracellular binding of CSA as well as t r a n s m e m b r a n e fluxes of CSA b e t w e e n PTC and lymphocytes. The presence of cyclophilin, a specific intracellular CSA b i n d i n g protein, has b e e n d e m o n s t r a t e d in a variety of cells with highest concentration in lymphocytes, kidney and brain(22). Binding of CSA to protein components of bile acid (cholate) transporter in hepatocytes(23) and close association of CSA binding sites to mitogenic receptors on lymphocyte m e m b r a n e ( 2 4 ) were reported.

In our preliminary studies, neither bile acids nor mitogens (con

A, PHA) were f o u n d to interfere with CSA uptake in PTC.

However, the observation that CCB

m a r k e d l y inhibits the uptake of CSA raises the possibility that in PTC CSA t r a n s p o r t / b i n d i n g sites and calcium channels are in close proximity so that interactions are facilitated.

It is

unlikely, however, that CSA occupies or transverses the same pathway as calcium because CSA (20/ztl) does not affect PTC calcium uptake(18).

The i n h i b i t o r y effect of CCB on CSA uptake is

also not a direct result of changes in Ca fluxes since the alteration in calcium fluxes b y other agents were without effect on CSA uptake. R e c e n t clinical trials suggest that CCB may amiliorate C S A - n e p h r o t o x i c i t y in transplant recipients without compromising immunosuppression(13-15,25).

The renal protective effect of

CCB is surprising because the circulating CSA levels were significantly higher in C C B - t r e a t e d patients than in control patients receiving similar dosage of CSA. The C S A - e l e v a t i n g effect of 1229

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CCB may be mediated through an inhibitory effect on CSA-degradation by competing for the cytochrome P-450 enzyme system(26) or through interference with CSA pharmacokinetics. Other agents known to elevate circulating CSA levels by competition for the P-450 enzyme system exacerbate, rather than amiliorate CSA-nephrotoxicity(27). Our finding that CCB blocks cellular uptake of CSA supports a pharmacokinetic basis for the observed elevation in CSA levels. A redistribution of CSA from the intracellular to the extracellular compartment would increase circulating levels of CSA while reducing CSA accumulation in renal proximal tubular cells and thereby amilioration of nephrotoxicity. ACKNOWLEDGEMENTS This work was supported by grants from the American Heart Association of the greater Los Angeles Affiliate, the Shu-Ho Foundation and the Veterans Administration Medical Research Service. Dr. Misra was a research fellow of the National Kidney Foundation of Southern California. We thank Ms. Beverly Bardi for typing the manuscript. REFERENCES

1. Borcl, J.F. (1981) Triangle I0; 97-105. 2. Beveridge, T. (1983) Transplant. proc. 15, 433-437. 3. Whiting, P.H., Thomson, A.W., Blair, J.T. and Simpson, J.G. (1982) Br. J. Exp. Pathol. 63, 88-94. 4. Thiel, G. (1986) Clin. Nephrol. 25, 5205-5210. 5. Hamilton, D.U., Evans, D.B. and Thiru, S. (1982) In Cyclosporine A (D.J.G. White, Ed.), pp 393-412, Elsevier Biomedical, NY. 6. Blair, J.T., Thomson, A.W., Whiting, P.H., Davidson, R.J.L. and Simpson, J.G. (1982) J. Pathol. 138, 163-178. 7. Racuson, C.L., Kane, B.C. and Solez, K (1987) Renal Failure 10, 29-37. 8. Mihatsch, M.J., Ryffel, B., Hermle, M., Brunner, F.P. and Thiel, G. (1986) Clin. Nephrol. 25, 52-58. 9. Willebrand, E.V. and Hayzy, P. (1983) Lancet 2, 189-192. 10. Kolbeck, P.C., Wolfe, J.A., Burchette, J. and Sanfilippo, F. (1987) Transplantation 43, 218224. 11. Becker, G.M., Gandolfi, A.J. and Nagle, R.B. (1987) Res. Commun. Chem. Pathol. Pharmacol. 56, 277-280. 12. Trifillis, A.L. and Kahng, M.W. (1988) Transplant. proc. 20, 717-721. 13. Wagner, K., Albrecht, S. and Neumayer, H-H. (1986) Transplant. proc. 18, 510-516. 14. Wagner, K., and Neumayer, H-H. (1987) Transplant proc. 19, 1353-1357. 15. Wagner, K., Henkel, M. Heinemeyer, G. and Neumayer, H-H. (1988) Transplant. proc. 20, 561-568. 16. Irish III, J.M. and Grantham, J.J. (1981) In The Kidney, 2nd Ed. (B.M. Brenner and F.C. Rector, Eds), pp 619-649, Saunders, Philadelphia. 17. Nagineni, C.N., Leveille, P.J., Lee, D.B.N. and Yanagawa, N. (1984) Biochem. J. 223, 353358. 18. Nagineni, C.N., Misra, B.C., Lee, D.B.N. and Yanagawa, N. (1987) Transplant. proc. 19, 1358-1362. 19. Legrue, S.J., Friedman, A.W. and Kahan, B.D. (1983) J. Immunol. 131, 712-718. 20. Fabre, I., Fabre, G., Lena, N. and Cano, J-P. (1986) Biochem. Pharmacol. 35, 4261-4266. 21. Merker, M.M. and Handschumacher, R.E. (1984) J. Immunol. 132, 3064-3070. 22. Handschumacher, R.E., Harding, M.W., Rice, J,, Drugge, R.J., and Speicher, D.W. (1984) Science 226, 544-547. 23. Zeigler, K. and Frimmer, M. (1986) Biochem. Biophys. Acta 855, 147-156. 24. Ryffel, B., Gotz, V. and Heuberger, B. (1982) J. lmmunol. 129, 1978-1982. 25. Kohlhaw, K., Woniquit, K., Frei, V., Oldhaber, K., Neumann, K. and Pichlmayer, R. (1988) Transplant. proc. 20, 572-574. 26. Renton, K.W. (1985) Biochem. pharmacol. 34, 2549-2553. 27. Jensen, C.W.B., Flechner, S.M., Van Buren, C.T., Frazier, O.H., Cooley, D.A., Lorber, M.I. and Kahn, B.D. (1987) Transplantation 43, 263-270. 1230