ffets of ndothelin Reeptor Antagonist on Cylosporine-indued Vasoonstrition in solated Rat Renal Arterioles Diane M. Lanese and John D. Conger Department ofmediine, University of Colorado Health Sienes Center and Veterans Affairs Medial Center, Denver, Colorado 22 Abstrat Reent evidene suggests that the potent onstritor peptide, endothelin (T) has a mediating role in ylosporine A (CsA)- related renal vasoonstrition. However, the nature of the CsA-T interation and effet on the renal vasulature is unertain. The purpose of the present study was twofold: (a) to determine if CsA exposure aused diret loal release of T from the endothelium of the renal mirovasulature and (b) to determine if loally generated T has pararine effets on the underlying vasular smooth musle to indue vasoonstrition. xperiments were performed in isolated rat renal arterioles. First it was determined that both afferent arteriole (AA) and efferent arteriole (A) exhibited onentration-dependent dereases in lumen diameter to inreasing molar onentrations of CsA. The AA was more sensitive to the vasoonstritive effets of CsA than the A. Next, the bloking effet of a reently synthesized putative TA reeptor antagonist was verified in both the AA and A, where it was found that the yli peptide ylo D-Asp-L-Pro-D-Val-L-Leu-D-Trp totally inhibited the vasoonstrition observed with T addition. Finally, the role of loally stimulated T in CsA-indued vasoonstrition was tested by determining the effet of the TA reeptor antagonist on CsA-indued AA and A onstrition. n the AA the vasoonstritor effet of 111 M CsA was ompletely bloked by the TA reeptor antagonist. However, in ontrast to AA, 1 1 M CsA in A in the presene of the TA reeptor antagonist dereased A lumen diameter by a mean of 1% from baseline (.±.75,um vs 7.±.,tm, P <.5). This hange in lumen diameter was similar to that indued by CsA alone. These data suggest that CsA diretly onstrits renal mirovessels. This effet is mediated by T in the AA but not the A. (J. Clin. nvest. 1993. 91:21-219.) Key words: afferent * efferent * nephrotoxiity * vasular transplant ntrodution Cylosporine A (CsA)' is an effetive immunosuppressive agent; however, its linial effiay is ompliated by substantial nephrotoxiity. The exat mehanism for CsA-indued nephrotoxiity is unlear, but a growing body of evidene sug- Address orrespondene to John D. Conger, M. D., Veterans Affairs Medial Center ( 11 C), 155 Clermont Street, Denver, CO 22. Reeivedfor publiation September 1992 and in revisedform 3 November 1992. 1. Abbreviations used in thispaper: AA, afferent arteriole; All, angiotensin ; CsA, ylosporine A; A, efferent arteriole; T, endothelin; T- A, TA reeptor antagonist. The Journal of Clinial nvestigation, n. Volume 91, May 1993, 21-219 gests that the primary pathogeneti mehanism may be vasular ( 1-3). The vasular effets of CsA in animal models have inluded sustained vasoonstrition in both the pre- and postglomerular irulation (-9). While a diret effet has not been absolutely exluded, a number of physiologi agonists have been impliated as mediators of CsA-indued vasoonstrition. Reent evidene has supported a potentially important role for the potent onstritor peptide, endothelin (T), in the effets of CsA on vasular smooth musle (). The exat role played by T in CsA-indued vasoonstrition is unertain. A number of investigators have demonstrated an inrease in urinary T exretion in rats after aute CsA infusion (1, 11). The study of Bunhman et al. ( 12) showing onentration-dependent CsA indution of T synthesis in ultured endothelial ells would indiate that CsA may diretly stimulate inreased T ativity in the systemi or renal vasulature. On the other hand, the findings of Zimmerhakl et al. ( 13) suggest that T stimulation by CsA may be more omplex. Using the hydronephroti rat kidney, they showed that CsA-indued vasoonstrition was limited to the more proximal vessels, i.e., aruate and interlobular arteries. Thus, it is possible that the proximal vasoonstritor effet of CsA is either diret, or mediated by another agonist, and the inrease in T ativity is seondary to distal arterial ishemia, a potent stimulus of T release ( 1). n addition to the question regarding the mehanism of stimulation, there is also unertainty surrounding the mode and site of ation of T released by CsA exposure. Kon et al. () reported an attenuation of aute CsA-indued renal vasoonstrition and glomerular dysfuntion with renal arterial infusion of rabbit anti-porine endothelin antibody. Presumably, the moleular radius of suh an antibody was too large to permit signifiant extravasular movement into the subintimal or perivasular spae suggesting its primary effet likely involved the binding of irulating T. On the other hand, there is evidene that T released from endothelial ells ats primarily by a pararine mehanism, binding to reeptors on adjaent smooth musle ells ( 15, 1). Theoretially, suh a mehanism would not be affeted by an anti-t antibody with limited aess to the subintimal spae. Thus the extent to whih CsAindued T ats by an immediate pararine mehanism or by release into the irulation with subsequent binding to downstream smooth musle ell reeptors after gaining aess to the perivasular and subendothelial spaes is unresolved. The purpose of the present study was, first, to determine whether CsA exposure aused diret loal release of T from the endothelium of the renal mirovasulature in either pre- or postglomerular resistane vessels and, seond, to determine if loally generated T ould at in a pararine fashion on the underlying vasular smooth musle to indue vasoonstrition. The isolated renal arteriolar vessel tehnique was used to address these questions. As the initial step, CsA onentration related hanges in afferent (AA) and efferent arteriolar (A) lumen diameters were measured. Next, the effet of a new y- 21 D. M. Lanese and J. D. Conger
li pentapeptide TA reeptor antagonist on T-1-indued AA and A vasoonstrition was determined ( 17). Finally, the effet of the TA reeptor antagonist on CsA-indued AA and A onstrition was assessed. Methods The in vitro-isolated rat arteriole tehnique used in these experiments has been desribed in detail previously ( 1, 19). Sprague-Dawley rats (2-25 g) that had been given standard rat how and water ad lib. for 1 wk were anesthetized with methoxyflurane. The kidneys were removed through a midline abdominal inision and immediately plae in a VC disseting media of Krebs-Ringer biarbonate buffer (ph 7.) onsisting of (mm) NaCl 115, NaHCO3 25, K2PO 2.5, MgSO 1.2, CaC2 1., gluose 5.5, pyruvi aid 2., and 1 g/dl dialyzed BSA (fration V; Sigma mmunohemials, St. Louis, MO). The kidneys were deapsulated, biseted longitudinally, and the medulla removed. The tissue was mined to 1-3-mm fragments. Glomeruli were isolated by a sieving tehnique and AA and A attahed to individual glomeruli were transferred with a silionized Pasteur pipette to a temperature ontrolled hamber ( 1 -ml vol) on a mirosope stage. The glomerular end of the arteriole was aspirated into a pipette with a flute-shaped tip so that the glomerulus was ompressed as the diameter of the pipette narrowed. The mirovessels were annulated by aspirating the nonglomerular end into the tip of a holding miropipette and the lumen annulated with an inner perfusing miropipette with a minimal internal diameter of.2 gm. Perfusing pipettes of smaller diameter have been found to have signifiant tip resistane invalidating pressure estimates ( 19). The intraluminal pressure ofthe vessel was then set at a predetermined value with a syringe onneted through a pressure transduer (1Db; Statham nstruments, Oxnard, CA) and a series of stopoks to the luminal perfusion pipette. Pressure was reorded on a reorder (Statham). The intraluminal pressure was set at mmhg for AA and 3 mmhg for A. When plaed in the perfusion hamber under high power magnifiation (Xl, 1) AA and A were readily distinguished by their appearane, whih had been determined previously from isolated glomeruli found to have both arterioles attahed. The identifying riteria were detailed in earlier publiations ( 1, 19). The vessel was then perfused at 37C for 3 min before experimental manipulations with a perfusate idential to the previously desribed disseting medium. The bathing solution (referred to in this study as "standard" bathing medium) was also idential to the disseting medium exept that it did not ontain albumin. Albumin in the bathing solution was found to promote baterial growth, whih aused deterioration of the vessels. All bathing solutions were gassed with % 2/2% CO2 for 3 min before use. The bathing solution was ontinuously pumped through the hamber at 1. ml/min. During experiments water-saturated gas (% 2/2% C2) was gently blown over the surfae of the hamber to maintain ph at 7. whih was measured frequently with a miroliter ph meter (Radiometer, Copenhagen, Denmark). The arterioles were observed at the above magnifiation with the use of a mirosope, an attahed amera and video monitor. Lumen diameter was measured diretly from the monitor sreen at various points along the vessel. Repeated measurements were made at these same points. Means ofthe measurements were alulated. xperiments were reorded on videotape. The viability of the vessels was assessed by the appearane of the intat wall and response to known vasoonstritor agonists of established poteny for this tehnique suh as angiotensin (All) and N. The following protools were arried out: (a) ffets of CsA and CsA vehile on AA and A. Control measurements of AA and A lumen diameters were made at 1-min intervals for 2 min. The standard bath was quikly hanged to a standard bath ontaining either a known molar onentration of CsA or an equivalent onentration of CsA vehile (Cremophor ). Beause of both the prolonged vasoonstritor effet and potential toxiity of repeated and umulative CsA exposure, eah vessel was exposed to only a single molar onentration of CsA. Lumen diameter was measured after 2 and min of inubation. (b) ffets oft antagonist on T-indued vasoonstrition ofaa and A. Conentration response urves to inreasing molar onentrations of T were previously determined by our laboratory ( 1). Using known near maximal onentrations of T for AA ( 1-1 M) and A ( 1-11 M), hanges in lumen diameter with T alone were ompared to hanges observed with T in the presene ofthe putative TA reeptor antagonist (Cylo D-Asp-L-Pro-D-Val-L-Leu-D-Trp; Peptides nternational n., Louisville, KY). The vasoonstritor inhibiting effet of the TA reeptor antagonist was tested by adding an exess onentration of the antagonist to the bathing medium ( l-7 M in the AA and 1- M in the A) after baseline lumen diameters were measured. Sine the TA reeptor antagonist had some agonist effet, a maximal onentration ofthe antagonist that aused a < 1% hange in baseline lumen diameter was hosen. Lumen diameter was again measured min after adding the antagonist. T was then added in AA and A at the same respetive onentrations as ited above in the presene ofthe antagonist. Lumen diameter was again measured after 2 and min of inubation. The bath was then exhanged several times with the standard bathing medium. As a postontrol step to assure vessel viability and reativity, near C5 All ( 1-' M in AA, 1-11 in A) was added to the bathing media and hanges in vessel lumen diameters were measured. Like T, the TA reeptor antagonist appeared to have a prolonged affinity for the reeptor and, as a onsequene, T ould not be used as a postontrol vasoonstritor. () ffets oftantagonist on CsA-indued vasoonstrition in AA and A. AA and A were bathed in standard media and baseline lumen diameters measured. The T antagonist was added to the bathing medium at the respetive onentrations for AA and A given above. The vessels were allowed to inubate for min and repeat lumen diameters measured. CsA ( 1-" M in AA and 1-" M in A) was added to the bathing medium in the presene of the T antagonist and lumen diameters measured at 2 and min. f the vessel failed to onstrit in response to the above experimental manipulation, then the bath was exhanged several times. After lumen diameter was determined to be stable by repeated measurements, All was added to the bath and hanges in vessel lumen diameter reorded to affirm vessel viability and reativity. Data analysis and alulations. Data are expressed as mean±sm. xept for the CsA onentration-response urves, the data are presented as atual lumen diameters in mirometers. CsA onentrationresponse data were presented as perent onstrition to establish C5 and maximal onstriting onentrations. Values for mean dose ofthe agent required to produe 5% of maximal ontration was defined as the C5. Two methods an be used to alulate the C5. The first method, reently reviewed by Meddings et al. (2), determines the C5 from best-fit urve nonlinear regression analysis. The seond method uses the means of the individual vessel dose response urve at 5% ontration. Both methods yielded numbers that were not different from the other. n this study the seond method was used. Maximal onstrition was onsidered to be that onentration at whih there was no further derease in lumen diameter with addition of the next higher log onentration. Maximal onstrition in every vessel ourred at < 2,um absolute mean lumen diameter. Comparisons of sequential responses within a group were made by repeated measures ANOVA and Sheffe's method for individual omparisons (21 ). An unpaired t test was used to ompare hanges in lumen diameter to similar experimental maneuvers in separate study groups and between AA and A with the same experimental maneuvers. P <.5 was onsidered statistially signifiant. Results ffets of CsA and CsA vehile on AA and A. The CsA onentration response urves for the AA and A are shown in Figs. 1 and 2, respetively. n both the AA and A there was a ffet ofndothelin Antagonist on Constritor Response to Cylosporine 215
C ( C 1. - o~ 1 2 A (n = 2) was less sensitive than the AA to CsA with an C5 value of 3.1±.7 X 1-11 M (P <.5). The CsA vehile, applied in the diluent onentrations used with CsA in this study, had no effet on vessel lumen diameter in either the AA (n = ) or A (n = ). Reativity of these latter vessels was verified, however, by doumenting their response to C5 All. ffets of the T antagonist on T-indued vasoonstrition ofaa and A. The data for AA and A, respetively, are _1 /shown in Figs. 3 and. The mean lumen diameter of the AA before the addition of T alone was 1.12±1.2,m. Addition of l-1 M T resulted in a marked derease in lumen diameterto 2.75±1.,um (P <.5). When the TA reeptorantagonist was added to a separate group of AA (n = ), mean baseline lumen diameter did not hange (11.12±.3 vs 1.37±.7 um) and remained stable over min. When T was then added in the presene of the antagonist, no further hange in mean lumen diameter ourred (1.37±.7 vs 1.37±.51 grm). To determine vessel viability and reativity,,, All (11 M) was added to the bathing media after several -13-12 -11-1 -9 bath exhanges. Mean lumen diameter dereased to.±1.7 Cylosporine [log M] gm (P <.5). Qualitatively similar results were seen in the A. Baseline Figure 1. Cylosporine onentration response urve in the AA (n lumen diameter before T ( 1-" M) addition was 1.±2.3 = 2). The data are presented as perent derease in lumen diameter. itm (n = ). Addition oft resulted in a signifiant derease in There wats a onentration dependent derease in lumen diameter lumen diameter to 3.25±1.7 tim (P <.5). n a separate with inrleasing molar onentrations of CsA. The alulated C5 group of A in whih the TA reeptor antagonist was added value for CsA in the AA was.1±2. x 1-12 M. first, the mean baseline lumen diameter was 1. 17±.5 um (n = ). There was no signifiant hange in mean baseline lumen diameter with addition of the T antagonist ( 1.17±.5 vs 9.5±. gm). When T was then added in the presene of molar onentrations of CsA. The alulated C5 the antagonist, T-indued vasoonstrition was ompletely inhibited (9.5±. vs 9.3±.9 urm). Vessel viability was onentration-dependent derease in lumen diameter with inreasing value foircsa in the AA (n = 2) was.1±2. X l-2 M. The 1 k 12 - C: C Co C) a- k 2 k ( so. 1 2 * -13-12 -11-1 -9 - Cylosporine [log M] Figure 2. Cylosporine onentration response urve in the A (n = 2). The data are presented as perent derease in lumen diameter. There was a onentration-dependent derease in lumen diameter with inreasing molar onentrations of CsA. The A was less sensitive than the AA to the vasoonstritive effets of CsA (P <.5). The alulated C5 value was 3.1±.7 x 1" M. Baseline T Baseline T-A T-A Figure 3. ffets of 1-' M T alone and in the presene of T-A in the AA. The left side of the graph represents hanges in lumen diameter (Am) with T alone. (Right side) No signifiant hange in lumen diameter was seen with addition of T in the presene of T-A. Subsequent A addition resulted in a derease in lumen diameter (1.37±.7 vs.±1.7 Am, *P <.5). + T All 21 D. M. Lanese and J. D. Conger
U) 12 r 1 k 2 : Baseline T * Baseline T-A T-A All Figure. ffets of 1-" M T alone and in the presene of T-A in the A. The left side of the graph represents hanges in lumen diameter (Mm) with T alone. As in the AA, the A showed no hange in lumen diameter with T in the presene of T-A. A aused a derease in lumen diameter from a baseline of 9.3±.9 Mm to 1.1±.92 Mm (*P <.5). by CsA, on the other hand, did not require T-TA interation. The results of this study, in part, onfirm the in vivo findings of Kon et al. () whih showed that infusion of an T antibody attenuated the renal vasopressor effets of CsA. n addition, an in vitro study by Takeda et al. ( 17) demonstrated that the TA reeptor antagonist bloked CsA-indued myosin light hain phosphorylation, a biohemial assessment of mesangial ell ontration in ulture. However, the model and design of the urrent study permitted examination of additional speifi questions regarding ylosporine A's vasular effets and the nature of the mediating role of T. These questions inluded the site ofvasular ation ofcsa, the potential importane of ishemia in CsA-indued T release, the possibility of a diret pararine effet of CsA-stimulated T on adjaent vasular smooth musle ells and the importane oft mediation in pre- and postglomerular arterial vessels. While the data of this study do not address the onstritor response of more proximal renal arterial vessels to CsA, they learly show that the immediate juxtaglomerular vasulature is diretly responsive to CsA at onentrations that are at least an order of magnitude lower than the generally aepted plasma therapeuti range in renal transplant patients (22). These results differ' from those of Zimmerhakl ( 13) who showed aruate and interlobular artery, but not arteriolar, onstrition with CsA infusion in the hydronephroti rat kidney. t is possible that there was a reative autoregulatory vasodilatation of the more distal vasulature assoiated with onstrition of aruate and interlobular arteries in the intat kidney that obsured a diret or mediated onstritor effet of CsA. The presonfirmed with All ( 1-11 M) whih dereased mean lumen diameter to 1.1±.92,um (P <.5). ffets of the T antagonist on CsA-indued vasoonstrition in the AA and A. Data are presented in Figs. 5 and. Mean baseline lumen diameter in the AA (n = ) was 13.2±2.23,m. No signifiant redution in lumen diameter ourred with addition ofthe T antagonist ( 12.75±2.12,um). A onentration of CsA (1" M) that aused a ±% derease in AA lumen diameter when added alone, produed no hange in lumen diameter when added in the presene of the T antagonist (13.33±2.2 Mum). However, All added to the bathing medium dereased lumen diameter to.75±2.37 Am (P <.5). Results in A ontrasted sharply from those observed in AA. Baseline lumen diameter ofa (n = 5) was.2±1.,m. Addition ofthe T antagonist alone resulted in an insignifiant derease in lumen diameter (7.±. Mum). When 1-" M CsA was then added in the presene ofthe T antagonist, mean lumen diameter dereased by 1±1% to.±.75 Mm (P <.5). This hange in lumen diameter was not different from the hange in lumen diameter that ourred with CsA alone (Fig. 2). Disussion The present study demonstrated that in isolated resistane arterial vessels, devoid of systemi and renal parenhymal neurohumoral influenes, CsA aused a onentration-dependent onstritor effet. Cylosporine A vasoonstrition in the AA required the mediation of TA reeptor ativation by assumed CsA-stimulated synthesis and release oft. Constrition ofa T 1-1 r 1 F 12 F 1 F 2 k Baseline T-A T-A All + CsA (1O-"M) Figure 5. Cylosporine (1-" M) and T-A in the AA. Baseline lumen diameter was 13.2±2.23 Mim, whih did not hange with addition of the T-A. n ontrast to the response to CsA alone, addition of 1-" M CsA in the presene of the T antagonist did not result in a derease in lumen diameter. However, A added to the bath dereased lumen diameter to.75±2.37,am. *P <.5. ffet ofndothelin Antagonist on Constritor Response to Cylosporine 217
.. -a 1 r 2 Baseline T-A T-A CsA (1-11) Figure. Cylosporine ( 1- M) and T-A in the A. Lumen diameter remained stable with addition of the T-A. Addition of 1" M CsA resulted in a derease in lumen diameter to.±.75 Atm (*P <.5), a deline similar to that found with CsA alone. ent study annot exlude suh a possibility. However, the study of Kon et al. () showing hanges in efferent, in addition to afferent, arteriolar resistane with intravenous CsA infusion in a miropunture study would support the importane of diret arteriolar effets of CsA in the in vivo setting. The possibility that ishemia may have stimulated T release with exposure to CsA was suggested by the finding of proximal arterial onstrition in the study by Zimmerhakl ( 13 ). T release and inreases deteted in the urine ould have ome from the endothelium of the distal renal vasulature as the onsequene of redued perfusion. shemia is a known potent stimulus of T release ( 1, 23). The present findings do not support suh a mehanism. Rather, they indiate that CsA's stimulation of endothelin ours diretly without interposed ishemia. The isolated vessel system is adequately oxygenated and not part of a more omplex vasular network. Thus, the indiret evidene of T stimulation with the TA reeptor antagonist, partiularly in AA, would indiate that ishemia is not an important element ofcsa-indued T ativity. The finding of inreased immunoreative T in the supernatant of ultured endothelial ells exposed to CsA ( 12) also would support the diret T stimulation of CsA. The relative roles ofumulative irulating T and the pararine effet of loally generated T in the vasoonstritor effet of CsA, at least in the AA, were not diretly determined in this study. However, the finding of omplete inhibition of the CsAindued response using a low moleular weight TA reeptor bloker in an isolated vessel with negligible luminal flow would suggest that the pararine effet of T is substantial. Thus, while the T antibody results reported by Kon et al. () would argue that irulating T attenuation modifies the vasoonstritor effet of CsA, the effet of adjaent endothelial T on underlying smooth musle likely ontributes signifiantly to the inreased resistane of the small renal arterial vasulature. The failure of TA blokade to inhibit the onstritor effet of CsA in A, as opposed to AA, was not antiipated. The preliminary studies showing that the TA reeptor antagonist at the onentration used ompletely bloked the effet of a near maximal onstriting onentration ofexogenously added T in efferent arterioles would indiate that the TA reeptor inhibition was pharmaologially effetive and adequate. Therefore, it is likely that there is either a diret effet ofcsa in the A that does not require T, or CsA stimulates another mediating agent in this vessel. t is interesting to note the results with T antibody in the miropunture study of Kon et al. () suggested that the T-mediating role of CsA vasoonstrition might have been different in AA and A. The redution in afferent arteriolar resistane with T antibody infusion was, in fat, twofold greater than the observed deline in efferent arteriolar resistane suggesting that antagonism of T had a greater impat on CsA-indued tone in AA. Other physiologi agonists have been impliated in the renal vasoonstritor effets ofcsa inluding All, ateholamines, platelet-ativating fator, and thromboxane A2 (7, 2-29). Additional experiments will be required to determine mediated and diret effets ofcsa in the postglomerular vasulature. Aknowledgments These studies were supported by National nstitute of Diabetes, Digestive and Kidney Diseases grant RO DK 129, and National Researh Servie award 5 S32 DK 11-2. Referenes 1. Shnabel, F. R., R. B. Wait, P. Aaronson, and K. U. Kahng. 199. ffet of ylosporine A administration on vasular reativity in the rabbit. Transplant. Pro. 21:91-921. 2. Bossaller, C., U. Forstermann, R. Hertel, C. Olbriht, V. Reshke, and. Flek. 199. Cylosporin A inhibits endothelium-dependent vasodilatation and vasular prostaylin prodution. ur. J. Pharmaol. 15:15-19. 3. Rego, A., R. Vargas, M. L. Foegh, and P. W. Ramwell. 19. ffet of ylosporine A treatment on vasular reativity of the rat thorai aorta. Transplant. Pro. 2:572-577.. Curtis, J. J., and S. A. Laskow. 19. Cylosporine-indued nephrotoxiity: pathophysiology of dereased blood flow. Transplant. Pro. 2:5-53. 5. Sullivan, B. A., L. J. Hak, and W. F. Finn. 195. Cylosporine nephrotoxiity: studies in laboratory animals. Transplant. Pro. 17:15-15.. Kon, V., M. Sugiura, T. nagami, B. R. Harvie,. hikawa, and R. L. Hoover. 199. Role of endothelin in ylosporine-indued glomerular dysfuntion. Kidney nt. 37:17-191. 7. Barros,. J., M. A. Boim, H. Ajzen,. L. Ramos, and N. Shor. 197. Glomerular hemodynamis and hormonal partiipation on ylosporine nephrotoxiity. Kidney nt. 32:19-25.. Kaskel, F. J., P. Devarajan, L. A. Arbeit, and L. C. 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