Repurposing calcium-sensing receptor activator drug cinacalcet for ADPKD treatment

Abstract

ADPKD is characterized by progressive cyst formation and enlargement leading to renal failure. Tolvaptan is currently the only FDA-approved treatment for ADPKD; however, it can cause serious adverse effects including hepatotoxicity. There remains an unmet clinical need for effective and safe treatments for ADPKD. The extracellular Ca²⁺-sensing receptor (CaSR) is a regulator of epithelial ion transport. FDA-approved CaSR activator cinacalcet can reduce cAMP-induced Cl⁻ and fluid secretion in various epithelial cells by activating phosphodiesterases (PDE) that hydrolyze cAMP. Since elevated cAMP is a key mechanism of ADPKD progression by promoting cell proliferation, cyst formation and enlargement (via Cl⁻ and fluid secretion), here we tested efficacy of cinacalcet in cell and animal models of ADPKD. Cinacalcet treatment reduced cAMP-induced Cl⁻ secretion and CFTR activity in MDCK cells as suggested by ~70% lower short-circuit current (I_sc) changes in response to forskolin and CFTR_inh-172, respectively. Cinacalcet treatment inhibited forskolin-induced cAMP elevation by 60% in MDCK cells, and its effect was completely reversed by IBMX (PDE inhibitor). In MDCK cells treated with forskolin, cinacalcet treatment concentration-dependently reduced cell proliferation, cyst formation and cyst enlargement by up to 50% without affecting cell viability. Cinacalcet treatment (20 mg/kg/day for 7 days, subcutaneous) reduced renal cyst enlargement in a mouse model of ADPKD (Pkd1^flox/flox;Ksp-Cre) by 20%. Lastly, cinacalcet treatment reduced cyst enlargement and cell proliferation in human ADPKD cells by 60%. Considering its efficacy as shown here, and favorable safety profile including extensive post-approval data, cinacalcet can be repurposed as a novel ADPKD treatment.

Introduction

ADPKD is the most common monogenic kidney disease characterized with progressive cyst formation and enlargement leading to renal failure. It accounts for 7–10% of ESKD patients in the world.¹ In ADPKD, mutations affecting the genes encoding the polycystin complex (PKD1 or PKD2) lead to reduced cytoplasmic Ca²⁺ levels in response to tubular flow.² The result of this process is elevated cAMP via reduced phosphodiesterase (PDE) and increased adenylate cyclase activities. This elevated cAMP stimulates cell proliferation and cystogenesis, and drives Cl⁻ and fluid secretion for cyst enlargement via activation of various pathways including PKA, MAPK and ERK.^3–5 Thus, reducing cAMP is considered as a promising treatment strategy in ADPKD.² Although cysts in ADPKD can originate from any part of the nephron, majority of cysts are derived from the collecting duct, where vasopressin is thought to be the major endogenous cAMP agonist.⁶ Tolvaptan is a V2 receptor antagonist that blocks vasopressin effect in the kidney and is the only FDA-approved ADPKD treatment. However, tolvaptan has a black-box warning regarding potential hepatotoxicity and fatal liver failure, which is an important safety concern for a lifelong treatment.⁷ In addition, the levels of various other cAMP agonists, such as forskolin and lysophosphatidic acid were found to be elevated in ADPKD cyst fluid^{2, 8, 9} which can potentially stimulate V2 receptor-independent Cl⁻ and fluid secretion, and limit efficacy of tolvaptan. Treatments reducing cAMP levels independent of V2 receptors can potentially have good clinical efficacy in ADPKD.

Extracellular Ca²⁺-sensing receptor (CaSR) is a major regulator of parathyroid hormone (PTH) secretion in response to serum Ca²⁺. Cinacalcet is the only FDA-approved oral CaSR activator¹⁰ used for treatment of hyperparathyroidism associated with CKD and renal failure.¹¹ Several lines of evidence suggest that CaSR activation inhibits cAMP-induced Cl⁻ secretion in epithelial cells, with a variety of mechanisms proposed including PDE activation.^{12, 13} We recently showed that cinacalcet inhibits Cl⁻ and fluid secretion in mouse and human intestinal cell/organoid models of cholera by promoting cAMP hydrolysis via PDEs.¹⁴ CaSR is also expressed in kidney tubules and ADPKD cyst epithelia.¹⁵ Since elevated cAMP is the key cause of hypersecretion in both cholera and ADPKD, here we investigated the efficacy of the approved-drug cinacalcet in cell and animal models of ADPKD.

Methods

Cell culture

MDCK cells (CCL-34, American Type Culture Collection, Rockville, MD) were cultured in a 1:1 mixture of DMEM and Ham’s F12 medium (DMEM/F-12; Gibco, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were grown on snapwell inserts (12 mm diameter, 0.4 μm polyester membrane; Corning Life Sciences, Kennebunk, ME) at 37°C in 5% CO₂ humidified atmosphere. On day 8, apical medium was removed to form an air-liquid interface which promotes the expression of CFTR at the apical membrane of MDCK epithelium.¹⁶ Two days later (Day 10), cells were used for short-circuit current (I_sc) experiments.

Short-circuit current studies

MDCK cells were mounted in Ussing chambers with each hemichamber containing bicarbonate-buffered Ringer’s solution (pH 7.4, in mM: 120 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 10 D-glucose, 5 HEPES, and 25 NaHCO₃). Unless otherwise specified, ion transport modulators were added to both apical and basolateral bathing solutions. CFTR_inh-172 was added when forskolin-induced I_sc reached a stable peak value. The solutions were aerated with 95% O₂/5% CO₂ and maintained at 37°C during experiments. I_sc was measured using an EVC4000 multichannel voltage clamp (World Precision Instruments, Sarasota, FL) via Ag/AgCl electrodes and 3 M KCl agar bridges as described.¹⁷

Intracellular Ca²⁺ and cAMP measurement

For intracellular Ca²⁺ measurement, MDCK cells (2×10⁴ cells/well) were seeded in 96-well plates (Corning Life Sciences) and used 48 hours after plating. Confluent cells were loaded with calcium indicator Fluo-4 NW (Invitrogen, Thermo Fisher Scientific) per manufacturer’s instructions. Fluo-4 fluorescence was measured in each well continuously with a Tecan Infinite M1000 plate reader (Tecan Group, Männedorf, Switzerland) at excitation/emission wavelengths of 494 nm/516 nm after manual addition of 30 μM cinacalcet (or 1% DMSO as vehicle). In some studies, cells were pretreated with PLC inhibitor U73122 (10 μM) for 5 minutes prior to addition of cinacalcet as described.¹⁴

For cAMP assay, MDCK cells (20×10⁴ cells/well) were seeded in 24-well plates until confluence. On day of the experiments, cells were pretreated for 20 minutes with 30 μM cinacalcet ± 500 μM IBMX (PDE inhibitor) or vehicle control (0.2% DMSO). After that, cells were treated with 10 μM forskolin (for 5 minutes) and lysed by repeated freeze/thaw and centrifuged to remove cell debris. The supernatant was assayed for cAMP using the cAMP Parameter immunoassay kit according to the manufacturer’s instructions (R&D Systems, Bio-Techne, Minneapolis, MN).

MDCK cyst model

In vitro cyst assays were performed in MDCK cells as previously described.¹⁸ Briefly, 800 MDCK cells were suspended in 0.4 ml of type I collagen (PureCol; Advanced Biomatrix, Carlsbad, CA) containing 10% Minimum Essential Medium (MEM, 10X), 10 mM HEPES, 27 mM NaHCO₃, 100 U/ml penicillin, and 100 μg/ml streptomycin (pH adjusted to 7.4 with NaOH titration). Cell suspension was seeded into 24-well plates and after gelation, 1.5 ml of MDCK medium containing 5% FBS and 10 μM forskolin was added to each well. Plates were maintained at 37°C in 5% CO₂ humidified atmosphere. To test the effect of cinacalcet on cyst growth, cinacalcet (or DMSO control) was added to medium in the continued presence of forskolin from days 6 to 12. Medium containing compounds was changed every 2 days. Photographs of each well were taken at Days 6, 8, 10 and 12 using Nikon Eclipse Ti-S inverted microscope (Nikon Instruments Inc., Melville, NY). Cyst diameter was measured in each picture using ImageJ software.

In separate experiments, the effect of cinacalcet on cyst formation was tested. For these studies, cells were prepared as described above and cinacalcet (or DMSO control) was added to medium in the continued presence of forskolin from day 0 onward. On day 6, cysts (diameter > 50 μm) and non-cyst cell colonies in each well were counted as described.¹⁹

Cell proliferation and viability studies

MDCK cells (2×10⁴ cells/well) were plated onto 96-well plates and incubated in DMEM/F-12 medium supplemented with 5% FBS for 24 hours. For the cell proliferation assay, cells were incubated in medium containing 0.2% DMSO or forskolin ± cinacalcet for 48 hours. After that, cell proliferation was determined by MTT assay (CellTiter 96 non-radioactive cell proliferation assay; Promega Corporation, Madison, WI). For acute effects of cinacalcet on cell viability, cells were incubated in medium containing cinacalcet, 0.1% DMSO (vehicle control) or 20% DMSO (positive control) for 24 hours. To measure the effects of long-term cinacalcet treatment on cell viability, MDCK cells were treated with cinacalcet, 0.1% DMSO (vehicle control) or 20% DMSO (positive control) for 6 days with medium change every 2 days. Then cell viability was measured using Alamar Blue (Invitrogen, Thermo Fischer Scientific) according to the manufacturer’s instructions.

Animal studies

The experimental protocols were approved by the UCSF Institutional Animal Care and Use Committee. Animals were bred in UCSF Laboratory Animal Resource Center and experiments were done in adherence with NIH Guide for the Care and Use of Laboratory Animals. The efficacy of cinacalcet was tested in Pkd1^flox/flox;Ksp-Cre mouse model of ADPKD. Breeders of Pkd1^flox/+ mice and Ksp-Cre mice (both in C57BL/6 background) were generously provided by Maryland PKD Research and Translation Core Center (Dr. Patricia Outeda Garcia). Pkd1^flox/flox mice were crossbred with Pkd1^flox/+:Ksp-Cre mice to generate Pkd1^flox/flox;Ksp-Cre mice. The genotype of the mice was confirmed by PCR after birth. Starting postnatal day two, 20 mg/kg cinacalcet hydrochloride or vehicle (saline containing 5% DMSO and 10% Kolliphor HS) were injected subcutaneously on the back of mice once daily for 7 days. Pkd1^flox/+ mice from the same litter were used as littermate controls without PKD. At postnatal day 9, blood samples were collected by cardiac puncture and kidneys were harvested. Serum was isolated by centrifugation and used to quantify BUN and calcium concentrations by QuantiChrom urea assay kit and QuantiChrom calcium assay kit, respectively (BioAssay Systems, Hayward, CA). Kidneys were fixed in 4% paraformaldehyde in PBS and embedded in paraffin. Kidney sections (4 μm thickness) were cut through the hilum, and stained with hematoxylin and eosin (H&E). Imaging was performed on Nikon Eclipse Ci microscope. The cyst and whole kidney area were measured using ImageJ software. Cystic index was calculated as total cyst area divided by total kidney area. To avoid falsely counting normal tubule space as cysts, cystic area was defined as spaces with diameter >50 μm, as described.²⁰

Primary cultures of human ADPKD cells

Primary human ADPKD cells were generously provided by Dr. Darren Wallace from Kansas PKD Research and Translation Core Center. As previously described,^{21, 22} ADPKD cells were cultured in DMEM/F-12 medium supplemented with 5% FBS; 5 ug/ml insulin, 5 ug/ml transferrin, 5 ng/ml sodium selenite (ITS), 100 U/ml penicillin and 100 ug/ml streptomycin. For these primary cell experiments, cultures were passaged no more than twice before being used in experiments. In vitro cyst assays were performed as previously described.²³ Briefly, 2×10⁴ ADPKD cells were suspended in 0.4 ml of type I collagen. The cell suspension was plated onto 24-well plates and after gelation, 1.5 ml of defined medium (DMEM/F-12 with ITS, 5 × 10⁻⁸ M hydrocortisone, 5 × 10⁻⁵ M triiodothyronine) containing 10 μM forskolin and 25 ng/mL EGF was added to each well. After 24 hours, the concentration of agonists was reduced to 5 μM forskolin and 5 ng/mL EGF. To test the effect of cinacalcet on cyst growth, cinacalcet (or DMSO control) was added to the medium in the continued presence of forskolin from days 4 to 10. Cyst diameter was measured as described above for MDCK cell cyst model. To study cell proliferation in these cells, ADPKD cells (4×10³ cells/well) were plated onto 96-well plates and incubated in DMEM/F-12 supplemented with 1% FBS and ITS for 24 hours. The serum concentration was reduced to 0.002% and ITS was removed for an additional 24 hours before the experiment, as described.²¹ For each experiment, cells were incubated in 0.002% FBS medium containing 0.2% DMSO or forskolin with or without cinacalcet for 48 hours. Then cell proliferation was determined by MTT assay (Promega), as described above. For the cell viability assay, ADPKD cells were plated onto 96-well plates. After 24 hours, cells were incubated in defined medium with cinacalcet or DMSO control. Medium was changed every 2 days and cell viability was quantified by Alamar Blue assay on day 6, as described above.

CaSR immunostaining in human ADPKD cells

ADPKD cells were grown on glass coverslips for 48 hours in DMEM/F-12 medium. After that, cells were fixed with 4% paraformaldehyde in PBS for 20 minutes, and permeabilized and blocked with PBS containing 1% BSA and 0.1% Triton X-100 for 30 minutes. Then cells were incubated with anti-CaSR antibody (#Ab223360; Abcam, Waltham, MA) at a dilution of 1:200 for 60 minutes. After washing with PBS three times, cells were incubated with donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 488 (#A21206; Invitrogen, Thermo Fisher Scientific) at a dilution of 1:1,000 for 60 minutes. Finally, cells were mounted onto glass slides with Prolong Gold antifade reagent with DAPI (#P36931; Invitrogen, Thermo Fisher Scientific) and imaging was performed on Leica DM4000B fluorescence microscope with DFC7000T camera (Leica Microsystems, Wetzlar, Germany).

Statistics

Experiments with 2 groups were analyzed using 2-tailed Student’s t test; for 3 or more groups, analysis was done with 1-way ANOVA and post hoc Newman-Keuls multiple comparisons test. P < 0.05 was considered statistically significant.

Results

Cinacalcet reduces CFTR-mediated Cl⁻ secretion in MDCK cells

Using MDCK cells that natively express CaSR and CFTR,²⁴ we studied the effects of cinacalcet on cAMP-induced Cl⁻ secretion. Cinacalcet (30 μM) treatment inhibited forskolin-induced secretory I_sc by >60% (Fig. 1A and B). As seen in other CFTR-expressing secretory epithelia,^{14, 17} forskolin-induced secretory I_sc was largely reversed by CFTR_inh-172 in MDCK cells. Cinacalcet pretreatment also inhibited CFTR_inh-172-induced reversal of I_sc by 70% (Fig. 1). These results demonstrate that cinacalcet has marked antisecretory effects in MDCK cells.

Figure 1. Cinacalcet reduces CFTR-mediated Cl⁻ secretion in MDCK cells.

A. Short-circuit current (I_sc) traces showing maximal forskolin (10 μM) response and CFTR_inh-172 (10 μM) inhibition following 20 min pretreatment with and without 30 μM cinacalcet. The bottom trace shows that MDCK cells treated with forskolin had stable I_sc response for the duration of the experiments in the absence of CFTR_inh-172. B. Summary of changes in I_sc (Δ I_sc) from experiments as in A. n=6–7 experiments per group. Mean ± S.E.M., Student’s t-test, **P < 0.01, *** P < 0.001.

Cinacalcet effect in MDCK cells is mediated by activation of PLC and PDEs

Under normal conditions, tubular fluid flow leads to elevation of intracellular Ca²⁺ via polycystins, which in turn leads to reduced cAMP by PDE activation. In ADPKD, the reduced intracellular Ca²⁺ response is a key contributor in elevated cAMP, which leads to increased CFTR-mediated Cl⁻ secretion, cell proliferation and cystogenesis.² G_q is the major CaSR signaling pathway in other secretory epithelia and activation of CaSR-G_q pathway leads to elevation of intracellular Ca²⁺ via PLC which in turn promotes cAMP hydrolysis via PDE activation.²⁵ Similarly, we found that CaSR activation in MDCK cells by cinacalcet increased intracellular Ca²⁺, and its effect was abolished by PLC inhibitor U73122 pretreatment (Fig. 2A and B). In addition, 30 μM cinacalcet treatment reduced forskolin-induced cAMP elevation by 60% in MDCK cells, and its effect was completely reversed by PDE inhibitor IBMX pretreatment (Fig. 2C). These results suggest that CaSR activation by cinacalcet elevates intracellular Ca²⁺ and promotes cAMP hydrolysis in kidney cells.

Figure 2. Cinacalcet effect in MDCK cells is mediated by activation of phospholipase C (PLC) and phosphodiesterases (PDEs).

A. Intracellular Ca²⁺ measured by Fluo-4 NW fluorescence in MDCK cells with 30 μM cinacalcet (±PLC inhibitor U73122) or vehicle control (1% DMSO) treatment. B. Summary of data in A. C. cAMP concentration in MDCK cell lysates after maximal forskolin (10 μM) treatment with and without 30 μM cinacalcet (± 500 μM IBMX, PDE inhibitor) pretreatment. n=4–9 per group. Mean ± S.E.M., one-way ANOVA with Newman-Keuls multiple comparisons test, ** P < 0.01, *** P < 0.001, ns: not significant.

Cinacalcet inhibits cyst growth, cyst formation and cell proliferation in MDCK cells

To test the efficacy of cinacalcet, we used forskolin-induced cyst model in MDCK cells. In this model, forskolin treatment starting Day 0 causes progressive cyst formation and enlargement in 12 days. Cinacalcet treatment starting at Day 6 concentration-dependently reduced cyst growth by up to 50% at 10 μM (Fig. 3). In separate experiments, the effect of cinacalcet on forskolin-induced cyst formation was studied by counting cystic and non-cystic cell colonies at Day 6. We found that cinacalcet treatment concentration-dependently reduces cyst formation by up to 80% (Fig. 4A–C). Similarly, cinacalcet treatment inhibited forskolin-induced increased cell proliferation in MDCK cells (Fig. 4D). To rule out any direct toxic effects of cinacalcet in MDCK cells, we assayed cell viability by Alamar blue after acute (24-hour) and chronic (6-day, same as the cyst model) treatment. Cinacalcet did not affect MDCK cell viability up to 30 μM with 24-hour treatment (Fig. S1A). With chronic treatment, cinacalcet was non-toxic in the efficacious concentration range up to 10 μM; however, it slightly reduced cell viability at 30 μM (Fig. S1B). These results collectively suggest that cinacalcet reduces cyst enlargement, cyst formation and cell proliferation in MDCK cells without affecting cell viability at therapeutic concentrations.

Figure 3. Cinacalcet inhibits cyst growth in MDCK cells.

A. Representative pictures showing time course of cyst growth in MDCK cells in the presence forskolin (10 μM, starting Day 0) and effect of cinacalcet (10 μM, starting Day 6). Scale bar is 500 μm. B. The effect of various concentrations of cinacalcet (or 0.1% DMSO control) on cyst diameter change over the study period. C. Summary data for cyst diameter increase from Day 6 to Day 12 in the presence of various concentrations of cinacalcet or vehicle control. n=~100 cysts per group. Mean ± S.E.M., one-way ANOVA with Newman-Keuls multiple comparisons test, * P < 0.05, *** P < 0.001.

Figure 4. Cinacalcet inhibits cyst formation and proliferation in MDCK cells.

A. Representative pictures showing the effects of forskolin (10 μM) ± cinacalcet (1–10 μM, starting Day 0) treatments on the number of cysts and non-cyst colonies of MDCK cells on Day 6. Scale bar is 500 μm. B. Number of cystic (diameter > 50 μm) and non-cystic colonies as in A. C. Summary data showing percentage of cyst colonies for the experiments in A. D. Effects of cinacalcet (1–10 μM) treatment on 10 μM forskolin-induced increase in cell proliferation measured by MTT assay in MDCK cells. Control cells were treated with vehicle control (0.2%DMSO) instead of forskolin. n=4–20 wells per group. Mean ± S.E.M., one-way ANOVA with Newman-Keuls multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant

Cinacalcet reduces cyst enlargement in an ADPKD mouse model

To test the efficacy of cinacalcet in an ADPKD animal model, we used Pkd1^flox/flox;Ksp-Cre mice, which develop rapidly progressive disease in the newborn period.^26–28 The mice were treated with cinacalcet (20 mg/kg/day, sc) or vehicle for 7 days starting at P2 (Fig. 5A). At Day 9, mice developed severer cystic phenotype, and cinacalcet treatment reduced renal cyst index by 20% in this model (Fig. 5B). Similarly, cinacalcet treatment resulted in lower kidney/body weight ratio (Fig. 5C) and serum BUN (Fig. 5D) compared to the vehicle group. These results suggest that cinacalcet reduces ADPKD severity in Pkd1^flox/flox;Ksp-Cre mice. Since hypocalcemia is a common therapeutic effect of cinacalcet, we measured serum Ca²⁺ in these mice. We found that cinacalcet treatment (at the dose efficacious for PKD) resulted in mild asymptomatic hypocalcemia in mice (Fig. 5E).

Figure 5. Cinacalcet reduces disease severity in an ADPKD mouse model.

A. Experimental protocol where Pkd1^flox/flox;Ksp-Cre mice were subcutaneously injected with vehicle or cinacalcet (20 mg/kg) for 7 days starting at P2. B. (left) Representative pictures of whole kidney sections stained with H&E. Scale bar is 1 mm. (right) Percent cyst area of vehicle and cinacalcet-treated PKD mice. C. Kidney weight to body weight ratio of vehicle and cinacalcet-treated PKD mice. Non-PKD mice are shown as controls. D and E. Serum urea nitrogen (BUN) and calcium concentrations of mice. n=7–14 mice per group, Mean ± S.E.M., Student’s t-test for panel B, one-way ANOVA with Newman-Keuls multiple comparisons test for panels C-E, * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant

Cinacalcet efficacy in primary human ADPKD cells

To study the efficacy of cinacalcet in a setting with high clinical relevance, we used primary human ADPKD cells which express CaSR (Fig. 6A). Initially, we tested the effect of cinacalcet on cell viability and found that chronic cinacalcet treatment for 6 days does not affect cell viability up to 3 μM. Interestingly, 10 μM cinacalcet was slightly toxic in these primary cells (Fig. S2). Next, we tested the efficacy of cinacalcet on cyst enlargement. We found that 3 μM cinacalcet treatment inhibited forskolin-induced cyst enlargement by 60% in human ADPKD cells (Fig. 6B and C). Similarly, 3 μM cinacalcet inhibited forskolin-induced cell proliferation in these cells (Fig. 6D). These results suggest that cinacalcet is also efficacious in primary human ADPKD cyst epithelial cells.

Figure 6. Cinacalcet reduces cyst growth and cell proliferation in human ADPKD cells.

A. CaSR immunofluorescence staining in human ADPKD cells. Scale bar is 20 μm. B. Representative pictures showing time course of cyst growth in ADPKD cells in the presence forskolin (10 μM, starting Day 0) and effect of cinacalcet (3 μM, starting Day 4). Scale bar is 500 μm. C. (left) The effect of various concentrations of cinacalcet (or 0.1% DMSO control) on cyst diameter change over the study period. (right) Summary data for cyst diameter increase from Day 4 to Day 10 in the presence of 1–3 μM cinacalcet or vehicle control (0.1% DMSO). n=~100 cysts per group. D. Effects of cinacalcet (1–3 μM) treatment on 10 μM forskolin-induced increase in cell proliferation measured by MTT assay. Control cells were treated with vehicle control (0.2%DMSO) instead of forskolin. n=10 wells per group. Mean ± S.E.M., one-way ANOVA with Newman-Keuls multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant

Discussion

Here we showed that approved drug cinacalcet reduces cyst formation and enlargement in cell and mouse models of ADPKD. The mechanism of cinacalcet effect is via targeting elevated cAMP, a key disease driver in ADPKD (Fig. 7). Considering its favorable safety profile, including two decades of clinical use in patients with kidney diseases, cinacalcet is a promising novel treatment candidate for ADPKD.

Figure 7. Mechanisms of cinacalcet effect in ADPKD.

By reducing cAMP, cinacalcet inhibits cyst formation, cell proliferation and cyst growth.

Previous studies investigated potential roles of CaSR in ADPKD and reported variable effects of the investigational CaSR activator R-568 in rodent models of ADPKD. Some studies showed that R-568 could be effective,²⁹ whereas others claimed that R-568 treatment was overall not effective.^{30, 31} However, all these previous studies administered R-568 orally, which can potentially explain its variable efficacy since R-568 has poor and variable oral bioavailability. This bioavailability problem was a major cause for withdrawal of R-568 from clinical development.³² Cinacalcet is the next-generation CaSR activator with good oral bioavailability; however, its effect in ADPKD models has not been studied previously. Since it is already approved by FDA (unlike the investigational drug R-568), cinacalcet can be rapidly repurposed for ADPKD treatment, pending efficacy testing in randomized clinical trials. A recent case series of 12 ADPKD patients on hemodialysis showed that patients on cinacalcet for secondary hyperparathyroidism have reduced annual total kidney volume increase compared with patients not taking cinacalcet.³³ Although this earlier study had several limitations including small sample size and lack of randomization, it implies potential efficacy of cinacalcet in ADPKD patients. Our findings here suggest that cinacalcet can potentially be efficacious at earlier stages of ADPKD to slow down disease progression.

Tolvaptan is the only FDA-approved ADPKD treatment; however, its use is restricted to adult patients at risk for rapid progression, since its efficacy was less prominent in other patient groups.³⁴ Tolvaptan blocks V2R which results in reduced vasopressin-induced cAMP elevation in cyst and tubule epithelia. Although vasopressin is considered as the key physiological cAMP agonist in distal nephron, earlier studies showed that ADPKD cyst fluid contains other cAMP agonists^{2, 8, 9} which can potentially stimulate V2 receptor-independent Cl⁻ and fluid secretion, and limit efficacy of tolvaptan. Other than hepatotoxicity, polyuria is another common side effect of tolvaptan affecting patients’ quality of life and sometimes leading to treatment discontinuation.³⁵ Since polyuria is not a known side effect of cinacalcet, it can potentially be used in ADPKD without any effects on urine output. Since cinacalcet and tolvaptan work through different receptors, they can potentially be used in combination for higher efficacy. This idea is also supported by a recent study that showed synergistic effects of CaSR activator R-568 in mouse and rat models of ADPKD when used together with tolvaptan.³⁶

The roles of CFTR and cAMP-induced Cl⁻ secretion in ADPKD cyst enlargement are well known.^{3, 16, 37} Based on its key role in ADPKD, CFTR inhibitors have been proposed as a treatment approach to reduce cyst enlargement.^{19, 38} However, a major barrier in clinical development of CFTR inhibitors and any other new drug for ADPKD is the need for extensive long-term safety studies, since ADPKD is a chronic and slowly progressing condition.⁶ An FDA-approved drug with favorable safety profile, such as cinacalcet, can potentially be repurposed and rapidly tested in clinical ADPKD trials without extensive safety studies. Another potential limitation for CFTR inhibitors is that they reduce cyst growth, without any effects on cell proliferation or cystogenesis,¹⁹ which might in theory limit their clinical efficacy in ADPKD. By reducing cAMP levels, cinacalcet can reduce cell proliferation, cystogenesis and cyst enlargement as shown here, for potentially greater efficacy than CFTR inhibitors in ADPKD.

Although CFTR is considered as the major cAMP-gated Cl⁻ channel in the kidney, here we found that 10 μM CFTR_inh-172 partially inhibits forskolin-induced secretory currents in MDCK cells, consistent with earlier studies in this model.¹⁶ It has been previously shown that 10 μM CFTR_inh-172 fully inhibits CFTR in vitro.^{14, 17, 39} Thus, the partial effect of 10 μM CFTRinh-172 on forskolin-induced secretory currents here may suggest the presence of other non-CFTR Cl⁻ channels such as Clc-2 in MDCK cells, similar to what has been reported in other CFTR-expressing epithelial cells.^{17, 40} Future studies investigating the roles of these non-CFTR channels in MDCK and other relevant models might be informative for better understanding of the mechanisms of ion transport in ADPKD.

After 200 mg single dose treatment in humans, the range of maximum plasma cinacalcet concentration (Cmax) was found to be 21.2–170 ng/mL (0.06–0.5 μM).⁴¹ Although these concentrations may seem slightly lower than the in vitro efficacious concentrations in our study, we would like to note that cinacalcet has very high volume of distribution (~1000 L) in humans, which demonstrates that majority of the drug is distributed to the tissues (such as kidney) with only a small fraction in the plasma.⁴² For this reason, the plasma concentration may not be an ideal surrogate for in vivo efficacy of cinacalcet in ADPKD. A recent study investigated the relationship between in vitro potency and the clinically efficacious plasma concentrations of 164 approved drugs. They found that 70% of the compounds have lower plasma concentrations than in vitro efficacious concentrations.⁴³ Thus it is recommended that predicting the in vivo efficacious concentrations in humans using only in vitro potency data should be avoided.⁴⁴ Perhaps a more relevant translational measure is the in vivo efficacious dose of cinacalcet in animal models. For parathyroid-related indications, the maximum FDA-approved daily cinacalcet dose is 180–360 mg (~2.5–5 mg/kg for 70 kg adult).⁴⁵ In this study, cinacalcet treatment had good efficacy at 20 mg/kg in mouse ADPKD model, a dose previously used in rodent studies⁴⁶ and equivalent to 1.6 mg/kg in humans when body surface area conversion factor is considered.⁴⁷ These suggest that cinacalcet can potentially have clinical efficacy in ADPKD patients at regular doses.

Since CaSR is also expressed in the parathyroid gland, cinacalcet use can cause mild hypocalcemia, as found in mice in our study. KDIGO CKD-MBD guidelines state that mild and asymptomatic hypocalcemia in the context of calcimimetic treatment can be tolerated.⁴⁸ In addition, a meta-analysis showed that calcimimetics do not increase serious adverse event frequency in patients with secondary hyperparathyroidism.⁴⁹ Thus cinacalcet can potentially be used safely in ADPKD patients with hyperparathyroidism. However, its safety in ADPKD patients without hyperparathyroidism may need to be formally tested in clinical studies.

In conclusion, we showed efficacy of cinacalcet in cell and mouse models of ADPKD including human primary ADPKD cyst epithelia. Our results can enable formal testing of cinacalcet in ADPKD clinical trials in the near future.

Background

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic kidney disease and a leading cause of kidney failure. Elevated cyclic adenosine monophosphate (cAMP) in kidney epithelia is a key driver of ADPKD progression. Extracellular Ca²⁺-sensing receptor (CaSR) activity regulates cAMP levels in epithelial cells and cinacalcet is an approved CaSR activator used for treatment of hyperparathyroidism in chronic kidney disease patients.

Translational Significance

We found here that cinacalcet reduces disease burden in cell and mouse models of ADPKD including primary human ADPKD cells by targeting elevated cAMP. Considering its safety profile and two decades of clinical use in patients with chronic kidney disease, cinacalcet can be repurposed as a novel ADPKD treatment.

Abbreviations:

ADPKD

autosomal dominant polycystic kidney disease

cAMP

cyclic adenosine monophosphate

CaSR

Ca²⁺-sensing receptor

PLC

phospholipase C

PDEs

phosphodiesterases

CFTR

cystic fibrosis transmembrane conductance regulator

I_sc

short-circuit current

IBMX

3-Isobutyl-1-methylxanthine

ESKD

end-stage kidney disease

PKA

protein kinase A

MAPK

mitogen-activated protein kinase

ERK

extracellular signal-regulated kinase

V2 receptor

vasopressin receptor 2

FDA

food and drug administration

PTH

parathyroid hormone

MDCK

Madin-Darby canine kidney