Calcineurin inhibitors cause renal afferent activation in rats: a novel mechanism of cyclosporine-induced hypertension

Abstract

Inhibition of calcineurin-mediated signaling in T lymphocytes is a major mechanism of cyclosporine A (CsA)-induced immunosuppression, and previous rat studies have suggested that inhibition of calcineurin-mediated signaling in central neuronal pools involved in blood pressure regulation plays an important role in causing acute CsA-induced hypertension. However, a central neural mechanism is difficult to reconcile with other data suggesting that CsA-induced hypertension is due to activation of renal and other subdiaphragmtic visceral afferents that reflexively increase efferent sympathetic nerve activity. Accordingly, we now have revised our hypothesis to suggest that CsA stimulates renal afferents by a calcineurin-dependent process. To test this new hypothesis, in anesthetized rats we recorded arterial pressure and multifiber afferent renal nerve activity from the cut distal end of the renal nerve before, during, and after intravenous infusion of either CsA (5 mg/kg over 20 min, n 8), FK506 (0.15 mg/kg, n = 7), another potent calcineurin inhibitor that is structurally unrelated to CsA, or rapamycin (0.15 mg/kg, n = 4), a structural analog of FK506 that has no effect on calcineurin. We found that renal afferent discharge was increased markedly by intravenous FK506, as well as CsA, but unaffected by rapamycin (or vehicle), indicating calcineurin mediation. After infusion of either calcineurin inhibitor, afferent renal nerve activity remained elevated for up to 2 h, paralleling the prolonged increase in blood pressure. Thus, the major new conclusion of this study is that, in contrast to what has been assumed previously, calcineurin inhibitors enhance sympathetic neurotransmission by a novel action localized to visceral sensory nerve endings rather than to nerve cell bodies or central synapses. In the rat, calcineurin-dependent activation of renal afferents appears to be the primary mechanism producing the large blood-pressure–raising effect of CsA. Because the data suggest that the major side-effect of CsA and FK506—hypertension—is inexorably linked to calcineurin inhibition in extralymphoid tissue, development of agents that selectively inhibit calcineurin only in T lymphocytes could eliminate this important secondary form of hypertension.

Cyclosporine A (CsA), calcineurin, FK506, rapamycin, renal afferent nerve activity, blood pressure

The immunosuppressive drug cyclosporine A (CsA) has emerged as an important new cause of secondary human hypertension,¹ but the underlying pathogenic mechanisms remain enigmatic. Renal, vascular, and neuronal mechanisms all have been implicated.²

In rats, intravenous CsA produces an acute form of hypertension that models acute CsA-induced hypertension in patients.^3–6 There is abundant evidence, at least in the rat, that the acute hypertensive response to CsA is sympathetically mediated.^3,5–7 The elevated blood pressure is accompanied by wide-spread increases in efferent sympathetic nerve activity (SNA) that persist for hours after a single intravenous dose of CsA; it is also abrogated by chemical or surgical sympathectomy or ganglionic blockade but unaffected by angiotensin-converting enzyme inhibition or endothelin blockade.^3–7 However, our own experiments produced seemingly contradictory conclusions as to whether the sympathoexcitatory action of CsA is best explained by a peripheral reflex or a central neural mechanism.

On one hand, the sympathetic activation is tightly linked to inhibition of calcineurin,⁵ the calcium-dependent phosphatase that is a known cellular target for CsA both in lymphoid and extralymphoid tissue.⁸ For example, SNA and blood pressure also are increased with FK506, another calcineurin inhibitor, but unaffected by rapamycin, a structural analog of FK506 that has no effect on calcineurin.⁵ Inhibition of neuronal calcineurin in vitro has been shown to enhance excitatory amino acid neurotransmission by both pre- and postsynaptic sites of action.^9–12 Thus, our initial hypothesis was that acute CsA-induced hypertension is caused by inhibition of calcineurin in excitatory neural circuits involved in the regulation of central sympathetic outflow.^2,5

On the other hand, a central neural mechanism is difficult to reconcile with additional in vivo rat experiments suggesting that CsA acts on renal and other subdiaphragmatic afferents that project centrally via the vagal nodosal and dorsal root ganglia, thereby reflexively increasing efferent SNA and blood pressure.^6,7 CsA-induced increases in SNA and blood pressure were accompanied by increased discharge of these afferents, and were greatly attenuated when this afferent input was interrupted by subdiaphragmatic vagotomy or dorsal rhizotomy.⁶ Although CsA clearly is a potent stimulus to these afferents, the underlying mechanism and the duration of the response are unknown.

Accordingly, we now have focused our attention on the renal afferent reflex and revised our hypothesis to suggest that CsA stimulates renal afferents by a calcineurin-dependent process. If calcineurin inhibition mediates the increased renal afferent discharge during intravenous CsA, we would predict that these afferents also should be stimulated by FK506 but not rapamycin. If this is the primary mechanism of the prolonged increase in blood pressure, the increase in afferent renal activity should persist for hours. Alternatively, one could postulate a two-step hypothesis in which CsA-induced sympathetic activation is initiated by renal afferent activation via a calcineurin-independent process, and then amplified and maintained by calcineurin-dependent potentiation of neurotransmission at excitatory central synapses in this reflex pathway. Regarding calcineurin-independent effects, the immunophilins (cytosolic receptors for CsA)—FK506, and rapamycin—possess intrinsic rotamase (cis-trans peptidyl prolyl isomerase) activity that is inhibited by all three ligands.^13–15 If rotamase inhibition (leading to changes in protein folding) mediates the increased renal afferent discharge during CsA, we would predict that renal afferent discharge also should be increased by rapamycin as well as FK506 or CsA. If such increases were only transient, this would suggest that additional (ie, central neural) mechanisms must be involved in producing the characteristically long-lasting increases in efferent SNA and blood pressure. To test these alternative predictions, in chloralose-anesthetized rats we compared effects of intravenous CsA with those of FK506 or rapamycin on renal afferent discharge and blood pressure.

Materials and methods

CsA (Sandimmune) was purchased from Novartis (Basel, Switzerland), FK506 (tacrolimus) was kindly provided by Fujisawa (Deerfield, IL), and rapamycin was purchased from Research Biochemicals Institute (Natick, MA). All agents were dissolved in the same vehicle (Cremaphor EL, Sigma, St. Louis, MO), as described before.⁵ Experiments were performed in female Sprague-Dawley rats weighing 220 to 280 g (Charles River, Wilmington, MA). Anesthesia was induced with ketamine HCl (80 mg/kg, intraperitoneal) and methohexital sodium (brevital, 20 mg/kg intraperitoneal) and maintained with α-chloralose (35 mg/kg intravenous, followed by supplemental doses of 10 mg/kg every 30 min). The trachea was cannulated, and the animal was artificially ventilated. The right jugular vein and left carotid artery were cannulated for drug infusion and measurement of intraarterial pressure. Multifiber recordings of afferent renal nerve activity were made from the cut distal end of the left renal nerve that was affixed to bipolar platinum electrodes using dental acrylic as described previously.¹⁶ The nerve action potentials were detected by a high-impedance probe (Grass Model P511K, Quincy, MA), amplified (gain, 5000 to 10,000), and filtered (bandwidth, 100 to 1000 Hz). All data were simultaneously collected and processed by Maclab analog-digit program and stored in a computer for offline analysis. Nerve action potentials were counted using a window discriminator, with the threshold level of detection being set just above the noise level and held constant throughout the experiment.

Experimental protocol

CsA was infused intravenously over 20 min to a total dose of 5 mg/kg (n = 8); FK506 (n = 7) and rapamycin (n = 4) each were infused over 20 min to a total dose of 0.15 mg/kg. These doses have been shown to produce approximately comparable immunosuppression in rats.⁵ Arterial pressure and afferent renal nerve activity were continuously recorded for 2 h after the end of the infusion. At the end of each experiment, euthanasia was performed using methohexital sodium (brevital, 100 mg/kg intravenous). The distal end of the nerve was cut and the remaining electrical activity was measured for 10 min. This activity was considered as noise and subtracted from the total activity recorded during the experiment to obtain an estimate of the true neural activity. The research protocol was approved by the Institutional Animal Care and Research Advisory Committee of the University of Texas Southwestern Medical Center at Dallas.

Statistics

Statistical analyses were performed with ANOVA, followed by Newman-Keuls test. A probability (P) level less than .05 was considered statistical significance. All values are expressed as mean ± SE.

Results

Previous studies have shown that CsA increases afferent renal nerve activity.⁷ To determine if the mechanism involves calcineurin inhibition, we compared the effects of CsA with those of two other immunophilin ligands: FK506, which also inhibits calcineurin, and rapamycin, which has no effect on calcineurin. Afferent renal nerve activity increased with CsA (11.6 ± 2.2 to 35.1 ± 6.4 Hz, n = 8, P < .05) or FK506 (13.4 ± 3.6 to 34.5 ± 11.7 Hz, n = 7, P < .05), but was unaffected by rapamycin (11.2 ± 3.6 to 15.0 ± 5.0 Hz, n = 4, P = ns) or vehicle (12.5 ± 3.8 to 14.5 ± 3.9 Hz, n = 6, P = ns) (Fig. 1).

Effects of immunophilin ligands on afferent renal nerve activity. Left panel) segments of the neurograms from four rats showing multifiber recordings of renal afferent nerve activity before and after intravenous CsA, FK506, rapamycin, or vehicle. Right panel) corresponding summary data. *P < .05 v baseline before drug infusion. Afferent renal nerve activity increased with CsA or FK506 but not rapamycin or vehicle.

Fig 1.

Because intravenous CsA characteristically produces increases in SNA and blood pressure that last for hours,^3,6 we extended the observation period for 2 h to determine if the calcineurin inhibitors would produce comparably long-lasting increases in afferent renal nerve activity. The time-course of the afferent renal nerve responses to CsA and the other immunophilin ligands is presented in Table 1. With either CsA or FK506, renal nerve activity increased during the infusion period and then continued to increase progressively over the next 2 hours.

Table 1.

Summary data showing the effects of CsA, FK506, Rapamycin, and vehicle on afferent renal nerve activity and mean arterial pressure

	Baseline	End of Infusion	1 h After Infusion	2 h After Infusion
Afferent renal nerve activity (%)
CsA	100	182 ± 24*†	216 ± 24*†	277 ± 24*†
FK506	100	141 ± 11*†	170 ± 20*†	220 ± 35*†
Rapamycin	100	93 ± 19	101 ± 21	112 ± 23
Vehicle	100	99 ± 11	95 ± 10	133 ± 22
Mean arterial pressure (mm Hg)
CsA	96 ± 7	123 ± 7*†	116 ± 5*†	115 ± 7*†
FK506	86 ± 3	113 ± 7*†	113 ± 5*†	103 ± 6*†
Rapamycin	96 ± 3	96 ± 9	92 ± 4	92 ± 7
Vehicle	93 ± 6	96 ± 5	90 ± 9	87 ± 4

CsA = cyclosporine A; MAP = mean arterial pressure; ARNA = afferent renal nerve activity.

CsA (5 mg/kg, n = 8), FK506 (0.15 mg/kg, n = 7), rapamycin (0.15 mg/kg, n = 4), and vehicle (n = 6) were infused for 20 min. MAP and ARNA were continuously observed for 2 more hours after the infusions. Data are for the following time points: baseline, end of drug infusion, and 1 and 2 h after the infusion.

*

P < .05 v baseline and

†

P < .05 v vehicle.

Discussion

Inhibition of calcineurin-mediated signaling in T lymphocytes is a key mechanism of cyclosporine A (CsA)-induced immunosuppression,^2,8,15 and previous rat studies have suggested that inhibition of calcineurin-mediated signaling in central neuronal pools involved in blood pressure regulation plays an important role in causing acute CsA-induced hypertension.^2,5,11 In contrast, the major new conclusion from this study is that calcineurin inhibitors enhance sympathetic neurotransmission by a novel action localized to renal sensory nerve endings rather than to nerve cell bodies or central synapses. In the rat, calcineurin-dependent activation of renal afferents appears to be the primary mechanism producing the large blood-pressure–raising effect of CsA.

Previous studies in rats have shown that intravenous CsA stimulates renal and other subdiaphragmatic afferents whose discharge reflexively increases efferent SNA and blood pressure.^6,7 One unanswered question was whether this reflex mechanism alone can account for the long-lasting increases in SNA and blood pressure that persist for hours after a single intravenous dose of CsA. For example, in a study by Moss et al,⁷ afferent renal nerve activity peaked 20 min after CsA but appeared to return toward baseline when the experiment was terminated 20 min later. By using dental acrylic to stabilize the nerve recording,¹⁶ and extending the observation period to 2 h, we were able to demonstrate that afferent renal nerve activity continues to increase for at least 2 h after CsA infusion. The persistence of this neural response enhances the plausibility that, at least in the rat, the large blood-pressure–raising effect of CsA can be explained solely on the basis of CsA acting on renal and other subdiaphragmatic visceral afferents that reflexively increase sympathetic outflow.

Our in vivo experiments provide some important clues about the underlying mechanism by which CsA activates these afferents. FK506 (a macrolide) is structurally unrelated to CsA (a cyclic polypeptide).^17,18 That FK506 closely approximated CsA's effect on renal afferents indicates that renal afferent activation is not a singular side effect of the CsA molecule.

The biologic actions of CsA, FK506, and rapamycin are dependent upon their interaction with two different cytoplasmic receptors, or immunophilins: (1) cyclophilin, which binds CsA, and (2) FK-binding protein (FKBP), which binds both FK506 and rapamycin.^8,15 The CsA-cyclophilin and FK506-FKBP complexes inhibit calcineurin, whereas the rapamycin-FKBP complex has no effect on calcineurin.^8,15 In addition, the unbound immunophilins possess rotamase activity (involved in protein folding), which is inhibited by binding to each of their respective ligands: CsA and FK506, as well as rapamycin.¹⁵ That renal afferent discharge was unaffected by rapamycin indicates that CsA- and FK506-induced renal afferent activation is specifically related to calcineurin inhibition but dissociated from rotamase inhibition.

Previous in vitro studies have demonstrated that calcineurin can modulate neurotransmission by actions localized to nerve cell bodies or central synapses.^9–12 In dorsal root ganglia, FK506 increases the open probability of N-methyl-D-aspartate channels, a subtype of glutamate-receptor–operated channels.¹⁰ In cultured fetal rat cortical neurons, CsA or FK506 increases the frequency of spontaneous glutamate-driven action potentials by enhancing presynaptic release of glutamate.¹¹ In contrast, our in vivo animal data are the first to suggest that calcineurin modulates sympathetic neurotransmission via a novel action localized to visceral sensory nerve endings.

Previous studies have shown that in the rat intravenous CsA evokes a generalized activation of subdiaphragmatic vagal afferents involving not only renal but also genitofemoral and hypogastric afferents.⁷ These afferents mainly exert an excitatory effect on SNA. However, intravenous CsA was found to have no effect on cardiopulmonary vagal afferents,⁶ which typically exert an inhibitory influence on SNA. The mechanism of the preferential activation of excitatory subdiaphragmatic afferents is unknown, but may be related to either differential tissue expression of specific isoforms of immunophilins and calcineurin and/or differential tissue concentrations of the administered immunophilin ligands. CsA and FK506 are highly lipophilic and, during systemic administration, their tissue concentrations parallel the adipose tissue content of various organs, with a comparatively high concentration in kidney and retroperitoneal fat.^19,20 Because the pattern of afferent innervation of the kidney and other subdiaphragmatic viscera may be highly species dependent,^21–23 the present data should not be extrapolated to make general statements regarding the mechanism of CsA-induced hypertension in larger animals or humans.

The precise molecular mechanism by which calcineurin inhibition evokes a prolonged increase in renal afferent discharge is unknown. We might speculate that calcineurin inhibitors interrupt a negative feedback mechanism that normally modulates the excitability of renal afferents. During generator potential formation, entry of calcium into the nerve ending could activate calcineurin, which would dephosphorylate protein substrates (eg, ion channel proteins¹⁰ or those associated with neurosecretory vesicle²⁴) that modulate membrane excitability.

Regardless of the precise underlying mechanisms involved, the present findings strengthen the view that the major side effect of CsA and FK506—hypertension—is inexorably linked to calcineurin inhibition in extralymphoid tissue.^2,5 Development of more selective agents that inhibit calcineurin only in T lymphocytes could eliminate this important secondary form of hypertension.