Tolvaptan Plus Pasireotide Shows Enhanced Efficacy in a PKD1 Model

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Tolvaptan plus Pasireotide Shows Enhanced Efﬁcacy in a PKD1 Model

† Katharina Hopp,* Cynthia J. Hommerding,* Xiaofang Wang,* Hong Ye,* Peter C. Harris,* and Vicente E. Torres*

*Division of Nephrology and Hypertension and †Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota BRIEF COMMUNICATION

ABSTRACT Autosomal dominant polycystic kidney disease (ADPKD) is a leading cause of ESRD. nucleotide–binding (G)-stimulatory pro- A central defect associated with ADPKD pathology is elevated levels of 39,59-cyclic teins,17 and AVPR2 antagonists have alle- AMP (cAMP). Compounds such as tolvaptan and pasireotide, which indirectly re- viated cyst burden in preclinical8,18–21 and duce adenylyl cyclase 6 (AC6) activity, have hence proven effective in slowing cyst clinical22,23 trials. The most prominently progression. Here, we tested the efficacy of these compounds individually and in used AVPR2 antagonist is tolvaptan, combination in a hypomorphic PKD1 model, Pkd1R3277C/R3277C (Pkd1RC/RC), in a which has shown efficacy in multiple ro- 5-month preclinical trial. Initially, the Pkd1RC/RC model was inbred into the C57BL/ dent models, and a long-term clinical trial 6 background, minimizing disease variability, and the pathogenic effect of elevating of 1445 patients that reported a 2.8%/year cAMP was confirmed by treatment with the AC6 stimulant desmopressin. Treatment increase in total kidney volume in the with tolvaptan or pasireotide alone markedly reduced cyst progression and in com- treated population compared with 5.5%/ bination showed a clear additive effect. Furthermore, combination treatment sig- year in the placebo group.23 SSTRs inhibit nificantly reduced cystic and fibrotic volume and decreased cAMP to wild-type AC6 activity through G-inhibitory pro- levels. We also showed that Pkd1RC/RC mice experience hepatic hypertrophy that teins and are activated by the peptide hor- can be corrected by pasireotide. The observed additive effect reinforces the central mone somatostatin.24,25 Because of the role of AC6 and cAMP in ADPKD pathogenesis and highlights the likely benefitof short half-life of somatostatin, more sta- combination therapy for patients with ADPKD. ble synthetic analogues have been tested in preclinical and clinical trials. Octreotide J Am Soc Nephrol 26: 39–47, 2015. doi: 10.1681/ASN.2013121312 or lanreotide, which bind to SSTR2 and -3, have been tested in murine studies26,27 and small clinical trials, in which they Autosomal dominant polycystic kidney have been found in various murine PKD slowed renal and hepatic cyst expan- disease (ADPKD) is the most common models.7–11 cAMP stimulates protein ki- sion.28–31 The recently developed pasireo- monogenic nephropathy and the fourth nase A–mediated signaling and leads to tide binds to SSTR1, -2, -3, and -5 and has leading cause of ESRD. It is characterized increased fluid secretion, proliferation/ shown enhanced efficacy over octreotide by progressive development of bilateral dedifferentiation, and disrupted flow in the Pkd2-/WS25 mouse and PCK rat renal cysts, often accompanied by livercysts sensing/tubulogenesis—key features of models.27 Despite the promising results, and an increased risk for vascular abnor- cystogenesis.12–14 Hence, lowering intra- no treatment for ADPKD has been malities. ADPKD is caused by mutations to cellular cAMP levels has been a major PKD1 or PKD2, encoding polycystin-1 or focus in the development of therapeutic 1,2 14 Received December 17, 2013. Accepted May 14, -2 (PC1/2). Thepolycystinsarethought interventions for ADPKD. 2014. to form a complex that regulates Ca2+ influx Multiple murine and human trials have Published online ahead of print. Publication date in response to extracellular mechanical or indirectly targeted AC6, the AC isoform available at www.jasn.org. chemical stimuli.3 Decreased intracellular predominant in collecting duct principal Ca2+ levels result in downregulation of cells, through the arginine vasopressin Correspondence: Dr. Vicente E. Torres, Division of Nephrology and Hypertension, Mayo Clinic, 200 calcium-dependent phosphodiesterases receptor 2 (AVPR2) or the somatostatin First Street SW, Rochester, MN 55905. Email: and stimulation of calcium-inhibitable ad- receptors (SSTR1–SSTR5).15,16 Binding [email protected] 4–6 enylyl cyclase 6 (AC6). As a consequence, of circulating vasopressin to AVPR2stim- Copyright © 2015 by the American Society of elevated 39,59-cyclic AMP (cAMP) levels ulates AC6 by coupling through guanosine Nephrology

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Figure 1. C57BL/6 Pkd1RC/RC mice have milder PKD than the outbred model and respond to dDAVP treatment. (A) Masson’s trichrome– stained kidney cross-sections of 3-month-old or 6-month-old inbred and outbred animals. Outbred animals developed higher cyst burdens and larger kidneys compared with inbred mice. Scale bar: 500 mm. (B) Masson’s trichrome–stained liver sections of microhamartomas (small, dilated, irregularly shaped bile ducts surrounded by ﬁbrosis). In outbred animals this abnormality was not found before

40 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 39–47, 2015 www.jasn.org BRIEF COMMUNICATION approved to date. Here we performed a previously reported in the outbred back- 1, H and I) and cAMP levels were signif- preclinical study to test whether tolvap- ground (Supplemental Figure 1, B–E).11 icantly higher (2.8660.49 pmol/mg pro- tan and pasireotide combination ther- Asignificant sex difference was noted tein versus 3.2460.47 pmol/mg protein; 2 apy has enhanced efficacy over single starting at 9 months, with females hav- P=0.29310 1) (Figure 1J). The %LW/ drug treatments. ing more severe disease, an effect likely ERbody wt did not differ between the The preclinical study was performed correlated to the C57BL/6 genetic back- groups, and no sex difference was ob- in the homozygous Pkd1R3277C/R3277C ground. In addition, inbred mice had a served. Together, these results reem- (Pkd1RC/RC) model, which closely mim- higher percentage of liver weight/extra- phasized the central role of cAMP in ics human ADPKD with slowly progres- renal body weight (%LW/ERbody wt) cyst progression/development in PKD1 sive PKD, but to minimize phenotypic and developed microhamartomas as and confirmed the suitability of the in- variability the outbred 129S6; C57BL/6 early as 3 months, compared with 12 bred Pkd1RC/RC model for preclinical Pkd1 RC model11 was fully inbred into months in the outbred background (Fig- trials. the C57BL/6 background. The progres- ure 1, B and D), highlighting that genetic To evaluate the benefitoftolvaptan, sion of PKD of inbred Pkd1RC/RC mice, background effects can vary depending pasireotide, or combination treatment in while more consistent, was milder and on the organ. slowing PKD progression, inbred Pkd1RC/RC slower than in the previously published Totest the pathogenic role of cAMP in mice were treated from 1 to 6 months. outbred model (Figure 1, A and C). the inbred Pkd1RC/RC model, mice were This long-term treatment was chosen to However, cysts continued to originate treated from 1 to 3 months with the syn- better mimic comparable treatment of primarily from collecting ducts (per- thetic vasopressin analogue desmopres- patients with ADPKD, to adequately centage of cysts: proximal tubule, 12%; sin (dDAVP) to increase cAMP levels.20 evaluate the efficacy of the combination collecting duct, 69%; unstained, 19%; Gross anatomy and Masson’s trichrome– therapy, and to assess potential adverse 2 ANOVA: P=9.85310 6) (Supplemental stained kidney sections showed a higher events. At the time of euthanasia, gross Figure 1, F and G). At three months, the cyst burden in dDAVP-treated mice anatomic comparisons between kidneys percentage of kidney weight/body (n=16) than in untreated controls of untreated control (n=22), tolvaptan- weight (%KW/body wt) was 1.74% (n=16) (Figure 1, E and F). This was fur- treated mice (n=21), pasireotide-treated 60.32% in the inbred animals (n=16) ther reflected in %KW/body wt, which mice (n=18), and tolvaptan plus pasireotide– and 2.19%60.24% in the outbred mice was significantly elevated in the treated treated mice (n=20) showed a clear re- 2 (n=6; P=0.54310 2). This difference group (%KW/body wt: control, 1.74% duction in kidney size of mice treated became even more significant at 6 60.32%; dDAVP, 2.47%60.45%; with either drug alone, with an even 2 months (%KW/body wt: inbred P=1.62310 5) (Figure 1G). Correspond- greater reduction (back to the size of [n=22], 1.87%60.23%; outbread ing cystic and fibrotic volumes were also wild-type[WT]kidneys)incombina- 2 [n=6], 3.35%61.10%; P=3.73310 6), significantly increased in the dDAVP- tion-treated animals (Figure 2A). This although there was much less variability treated mice (cystic volume: 47.006 observation correlated with the histo- (Figure 1C). Despite slower progression, 34.86 ml in controls versus 88.646 logic analysis and %KW/body wt (Figure 2 %KW/body wt of inbred mice steadily 46.49 mlindDAVPmice[P=0.92310 2]; 2, B–D, Table 1, Supplemental Figure 2). increased with age and BUN levels were fibrotic volume: 2.1962.00 mlversus Pairwise comparisons showed the 2 significantly elevated at 9 months, as 5.2162.40 ml[P=0.81310 3]) (Figure greatest significance for the combined

12 months, but inbred mice developed similar-size microhamartomas as early as 3 months. Scale bar: 100 mm. (C and D) %KW/body wt and %LW/ERbody wt of inbred (green) and outbred (black) mice at 3 and 6 months, depicted as mean diamonds and SDs. (C) The %KW/body wt in inbred mice was less variable among non-littermates but overall was milder and more slowly progressive. Wild-type (WT) C57BL/6 mice also had lower %KW/body wt than WT outbred mice, while body weight remained constant, highlighting a clear background effect in kidney anatomy (%KW/body wt in 10 inbred mice and 4 outbred mice: 3 months, inbred, 1.35%60.11%, outbred, 1.49%60.20% 2 [P=0.11]; 6 months, inbred, 1.24%60.05%, outbred, 1.6660.24 [P=0.15310 3) (Supplemental Figure 1A). (D) The %LW/ERbody wt was 2 elevated in inbred compared with outbred mice at 3 months (inbred, 5.39%60.34%; outbred, 4.80%60.30%; P=0.21310 2) and 6 months 2 (inbred, 5.54%60.40%; outbred, 4.97%60.48%; P=0.34310 2). Gray dotted lines represent WT (C57BL/6 or outbred) mice. (E) Gross anatomy of representative kidneys from inbred WT mice, inbred Pkd1RC/RC control mice, and dDAVP-treated mice (6 months of age) highlights increased kidney size after dDAVP treatment. Scale bar: 0.5 cm. (F) Masson’s trichrome–stained cross-sections of kidneys with mean613SD %KW/body wt. Cross-sections of dDAVP-treated mice showed more severe cystic disease with dilated tubules/ducts (inset, cortex) compared with untreated mice. Scale bar: 500 mm, 250 mm (inset). (G–J) %KW/body wt (G), renal cystic volume (H), renal fibrotic volume (I), and cAMP levels (J) of saline-treated inbred Pkd1RC/RC control mice (C, green) and dDAVP-treated mice (D, purple), depicted as mean diamonds and SDs. Gray dotted lines represent WT values. dDAVP treatment significantly increased %KW/body wt (G), cystic volume (H), and fibrotic volume (I). (J) As predicted, cAMP levels were elevated upon dDAVP treatment. %KW/BW, %KW/body wt. *P,0.05; ** P,0.01; ***P,0.001; ****P,0.0001. Data of outbred animals were obtained from reference 11.

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Figure 2. Tolvaptan plus pasireotide treatment showed enhanced efﬁcacy over single drug treatment. (A) Gross anatomy of representative kidneys from inbred WT and inbred Pkd1RC/RC control mice, C; tolvaptan-treated mice, T; pasireotide-treated mice, P; and tolvaptan plus pasireotide–treated mice, B (6 months of age). Treatment with both drugs showed a clear additive effect, reducing kidney size back to WT range. Scale bar: 0.5 cm. (B) Masson’s trichrome–stained cross-sections of kidneys with mean613SD %KW/body wt. Untreated animals showed multiple cysts and dilated tubules/ducts (inset, cortex) (Supplemental Figure 2). Cyst burden and dilations were reduced upon treatment with either drug alone and were nearly eliminated by combination therapy (Supplemental Figure 2). Scale bar: 500 mm, 250mm (inset). (C–F) %KW/body wt (C), renal cystic volume (D), renal ﬁbrotic volume (E), and cAMP levels (F) of saline-treated Pkd1RC/RC

42 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 39–47, 2015 www.jasn.org BRIEF COMMUNICATION treatment with a marked additive effect trial23; however, more detailed studies are but limiting adverse events. The benefit over single treatments (Table 1). Masson’s required. was seen in terms of %KW/body wt, cystic trichrome–stained kidney sections Treatment with pasireotide reduced and fibrotic volumes, and cAMP levels for showed a reduction in size/number of cysts %LW/ERbody wt back to WT levels (WT, the combined treatment. Renal function and dilated tubules/ducts in tolvaptan- or 4.92%60.32%) (Figure 3A, Table 1). Be- did not significantly differ because these pasireotide-treated mice and only a few cause the observed microhamartomas of inbred animals had relatively mild disease. cysts with mainly normal-appearing cor- untreated animals were small, few in However, correlations between the signif- tex and medulla in animals treated with number, and also found in pasireotide- icant endpoints found here and reduction both drugs (Figure 2B, inset, Supplemen- treated mice (data not shown), it was in BUN in older outbred and inbred tal Figure 2). This was further reflected in unlikely that the reduction of %LW/ER- animals (Supplemental Figure 1E) of this the overall cystic volume, which was sig- body wt correlated to this particular ab- model suggest that renal function differ- nificantly reduced upon single or combi- normality. The effect of a reduction of ences would have been seen if the study nation treatment (Figure 2D, Table 1). In PC1 on hepatocyte morphology is un- had been extended.11 This is analogous to addition, treatment with tolvaptan sig- known. However, it has been reported the correlation between total kidney vol- nificantly reduced fibrotic volume, which that SSTR2 and SSTR4 are expressed in ume in young patients and a subsequent was even further decreased by treatment hepatocyte cell lines and that all SSTRs decline in renal function that has been with pasireotide or both drugs (Figure 2E, mislocalize to hepatocytes in pathologic established in human ADPKD.36 Interest- Table 1). Consistent with the target of the conditions.34,35 Consequently, we evalu- ingly, the effect of pasireotide was gener- treatments, cAMP levels were significantly ated whether hepatocyte hypertrophy or ally greater than for tolvaptan, and this reduced back to WT levels by the combina- hyperplasia may underlie the increase in may be because of the slow, even release tion treatment (WT, 3.2560.88 pmol/mg %LW/ERbody wt. Measuring DNA con- from the minipump compared with the protein)(Figure2F,Table1).Noneof tent per milligram of liver tissue, which nightly ingestion of tolvaptan in food; the treatments resulted in clear adverse was reduced in control animals versus this effect suggests that a split dose or events (see Concise Methods), even though pasireotide-treated and WT mice (data slow-release AVPR2 antagonist may pro- body weight decreased slightly upon not shown), and counting nuclei per vide further benefit. In addition, pasireo- treatment with pasireotide alone or com- 0.01 mm2, highlighted that C57BL/6 tide treatment showed a slight antidiuretic bined with tolvaptan (WT, 26.4663.75 g) Pkd1RC/RC animals experienced hepato- effect, which may benefit patients treated (Table 1). No significant difference was cyte hypertrophy, which was corrected with both drugs because it could reduce noted in BUN, which was in the WTrange bypasireotidetreatment(Figure3,B aquaresis-related symptoms induced by at 6 months of age (Supplemental Figure and C). To date, hepatic hypertrophy tolvaptan. Further, pasireotide corrects 1E), and no significant sex effects were has not been reported as a pathologic the hepatic hypertrophy observed in the observed. feature of ADPKD; elevated liver weights inbred PKD1RC/RC model, an abnormality In addition to the alleviating effect of have been seen in patients but have that may be present but unrecognized in pasireotide on renal abnormalities, the been correlated only to cyst burden and patients, masked by polycystic liver dis- drug also reduced urinary output, coun- not hepatocyte size. Because AVPR2 is ease. teracting the tolvaptan-induced poly- not expressed in the liver, it was not sur- uria in animals treated with both drugs prising that tolvaptan did not decrease (Supplemental Table 1).23 While the %LW/ERbody wt. CONCISE METHODS mechanism for this antidiuretic effect of We have demonstrated in a slowly pasireotide is unclear, similar effects have progressive model of PKD1 the value of Inbreeding of the Pkd1RC/RC Model been reported in patients treated with treatment with tolvaptan or pasireotide into the C57BL/6 Background somatostatin.32,33 This may highlight a fa- alone and the additive effect of combined The animals were inbred into the C57BL/6 vorable effect of the combination treat- therapy, suggesting that using two means background using the IDEXX RADIL speed ment because polyuria is considered an to lower cAMP levels would be beneficial congenic service. First, the sex chromosomes adverse event of tolvaptan treatment and in the human disease. Using the combi- were fixed, followed by microsatellite analysis the reason for treatment discontinuation nation of both drugs may also allow lower of the autosomes. Animals were considered in 8.3% of patients in a recently published single drug doses, achieving similar results inbred when all of the microsatellite markers

controls (green), tolvaptan-treated (blue), pasireotide-treated (yellow), and tolvaptan plus pasireotide–treated mice (red) depicted as mean diamonds and SD. Gray dotted lines represent WT C57BL/6 values. (C) The %KW/body wt was significantly decreased by treatment with either drug alone and even further decreased with use of the combination. (D and E) Similar trends were observed for cystic and fibrotic volume. For these two parameters, pasireotide slightly outperformed tolvaptan. (F) cAMP levels were only significantly different (back to WT levels) in the combination treatment group, highlighting the importance of the additive effect. %KW/BW, %KW/body wt. *P,0.05; **P,0.01; ***P,0.001; ****P,0.0001.

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Table 1. Summary of measurements for the combination treatment preclinical trial with pasireotide received the same diet P Valuesb without the drug. Pasireotide (Novartis) a Group Measurements6SD was administered by osmotic minipump C(n=22) T (n=21) P (n=18) B (n=20) at a dosage of 10 mg/1kgbodyweightper %KW/body wt hour. The minipump of control animals and C1.8760.23 – 0.01017b 0.00023b 5.51E-10b those receiving only tolvaptan contained T1.6960.20 0.01017b – 0.58514 0.00019b P1.6160.14 0.00023b 0.58514 – 0.01832b saline. All minipumps were replaced every B1.4360.16 5.51E-10b 0.00019b 0.01832b – 3 weeks. %LW/ERbody wt C5.5460.40 – 0.86211 1.23E-06b 4.91E-07b Urine Collection/Analysis T5.6060.34 0.86211 – 7.48E-08b 2.75E-08b One week before euthanasia, all animals in P4.9660.43 1.23E-06b 7.48E-08b – 0.99986 each treatment group and the control group b b B4.9460.29 4.91E-07 2.75E-08 0.99986 – were placed in a metabolic cage from 6 pm to m Cystic volume ( l) 6 am (in each group, two to four mice were 6 – b b C 91.31 36.73 0.14538 0.00018 5.16E-08 combined in one metabolic cage). Urine T 71.85634.19 0.14538 – 0.09899 0.00027b volumes were recorded the next morning P 50.37620.58 0.00018b 0.09899 – 0.25587 and osmolality was measured using pHOx B 32.48616.17 5.16E-08b 0.00027b 0.25587 – Fibrotic volume (ml) Ultra (Nova Biomedical). C5.1861.98 – 0.02634b 4.33E-07b 7.26E-08b T3.7261.85 0.02634b – 0.01107b 0.00381b Tissue and Blood Harvest/Analysis b b P2.1061.31 4.33E-07 0.01107 – 0.99367 The animals were euthanized by CO2 expo- B1.9061.35 7.26E-08b 0.00381b 0.99367 – sure, and the body weight of each animal was cAMP (pmol/mg protein) recorded. Blood was then collected via car- b C4.4561.53 – 0.68829 0.54057 0.01671 diac puncture, and kidneys, liver, spleen, and 6 – T4.061.32 0.68829 0.99251 0.21733 heart were harvested and weighed. The left 6 – P3.921.37 0.54057 0.99251 0.38158 kidney and small pieces of each liver lobe B3.2860.59 0.01671b 0.21733 0.38158 – were flash frozen, and the right kidney plus Body wt (g) all other organs were fixed in 4% parafor- C 26.3263.17 – 0.92433 6.71E-06b 4.32E-10b T 25.7263.90 0.92433 – 0.00018b 4.79E-10b maldehyde. For the tolvaptan/pasireotide P 23.3962.27 6.71E-06b 0.00018b – 0.01202b trial, total blood was used for a complete B 21.3762.23 4.32E-10b 4.79E-10b 0.01202b – chemistry and electrolyte analysis measuring C, control (Pkd1RC/RC, untreated); T, tolvaptan; P, pasireotide; B, both (tolvaptan plus pasireotide). 14 variables (Abaxis, VetScanVS2, Com- aSix-month-old inbred animals. prehensive Diagnostic Panel). This panel b fi , Signi cant at P 0.05. included BUN and a liver function test quan- tifying aspartate aminotransferase, alanine matched the C57BL/6 strain. Thereafter, ani- weight per hour) (V1005, Sigma-Aldrich) via aminotransferase, and alkaline phosphatase. mals were maintained as homozygotes. osmotic minipump (Alzet model 1004) as All BUN values were in the range of WT described previously.37 The minipumps values. The aspartate aminotransferase and Experimental Animals and Study were replaced every 3 weeks, and all animals alanine aminotransferase values did not sig- fi Design survived the trial period. ni cantly differ from WT values, and alka- The Institutional Animal Care and Utilization line phosphatase values in the control and Committee approved the use of the C57BL/6 Tolvaptan and Pasireotide Treatment tolvaptan-treated mice were slightly lower Pkd1RC/RC model, maintained at the animal Inbred Pkd1RC/RC mice were divided into than WT values but within normal range facilities of the Mayo Clinic, Rochester, MN, four groups (control, tolvaptan, pasireotide, (normal ranges provided by VetScanVS2 and all experimental protocols described in tolvaptan plus pasireotide) at 3 weeks of age profiles). BUN levels of animals beyond 6 this report. (n=88; 11 animals per treatment per sex). months of age were measured using pHOx Littermates of the same sex were sorted into Ultra. Desmopressin Treatment different treatment groups. Treatment with RC/RC Inbred Pkd1 mice were divided into a tolvaptan and/or pasireotide was started at Histomorphometric Analysis control group and a treatment group at 3 1 month of age, and all animals were eutha- Cystic volumes and fibrotic volumes were weeks of age (n=32; eight animals per treat- nized at 6 months of age. Tolvaptan-treated calculated as previously described20 using ment group and sex). Littermates of the same animals received 0.1% tolvaptan (Otsuka three cross-sections per kidney. In short, sex were sorted into different treatment Pharmaceuticals) in their ground rodent the cystic volume was calculated by measur- groups. From 1 to 3 months of age, animals chow, by homogenous mixing, whereas ing the percentage cystic area of the three received saline or dDAVP (30 ng/100 g body control animals and those treated only cross-sections, adjusted to kidney weight

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kidney (one in the pasireotide group and one in the combination treatment group) and hence were excluded from the analysis. No other gross anomalous abnormalities were observed, and no abnormalities were noted from the histologic analysis. The blood panel (Abaxis, VetScanVS2, Compre- hensive Diagnostic Panel) was used to evaluate potential ion imbalances as well as liver toxicity. No signiﬁcant differences were noted upon treatment with either drug alone or the combination. Consequently, no deﬁ- nite adverse events were noted within our study.

Immunofluorescence Labeling/ Analysis Tissue were prepared for immunofluorescence labeling as previously described.11 Bio- tinylated-LTA (1:500; Vector Laboratories) Figure 3. Hepatic hypertrophy of Pkd1RC/RC mice can be corrected by pasireotide treat- and Aqp2 (1:100; Santa Cruz Biotechnology) ment. (A) %LW/ERbody wt of saline treated, inbred Pkd1RC/RC control mice (C, green), tolvaptan-treated mice (T, blue), pasireotide-treated mice (P, yellow), and tolvaptan plus were used as proximal tubule and collecting pasireotide–treated mice (B, red) (6 months of age) depicted as mean diamonds and SD. duct markers, respectively. Staining against Gray dotted line represents C57BL/6 WT value. The %LW/ERbody wt returned to WT level Tamm-Horsfall protein (1:200; Santa Cruz in animals treated with pasireotide. (B) Number of DAPI-positive nuclei per 0.01 mm2 in Biotechnology) was also performed but not liver cross-sections of Pkd1RC/RC controls (green), pasireotide-treated mice (yellow), and quantified because ,2 dilations/cyst per WT mice (black), depicted as mean diamonds and SD. Per given area, untreated animals animal stained positive for the maker. Quan- had fewer nuclei compared with pasireotide-treated or WT animals, indicative of larger tification was performed using ImageJ soft- m cells (hypertrophy). (C) Representative image of part B. Scale bar: 50 m. Hepatic hyper- ware, and cysts/tubular dilations were counted trophy has not previously been reported as a characteristic of ADPKD. Because both PC1 if their diameter exceeded 50 mm. and PC2 are expressed in hepatocytes (Supplemental Figure 3), reduced expression or function of PC1 may be the cause for the observed higher %LW/ERbody wt. %LW/ERBW, %LW/ERbody wt. *P,0.05; ****P,0.0001. cAMP Analysis Pieces from the flash-frozen left kidney were ground to finepowderandusedforthecAMP assay as previously described following the (approximate kidney volume). Fibrotic vol- Evaluation of Adverse Events of manufacture’sprotocol(EnzoLifeSciences).11 ume was calculated from the percentage fi- Tolvaptan/Pasireotide-Treated brotic area of 10 cortical images and adjusted Animals Crude Membrane Preparation and to kidney weight (approximate kidney vol- Because the trial required numerous pump Western Blotting ume). Hepatic hypertrophy was evaluated replacements, five animals died immediately Huh7 and renal cortical tubular epithelial cells using three females and three males with av- after surgery or within the following 2 days were cultured in DMEM media containing erage %LW/ERbody wt. DNA was isolated (one in the tolvaptan group, three in the 10% FBS, penicillin/streptomycin, and glucose. from frozen tissue originating from two dif- pasireotide group, and one in the combina- Cells grown to confluence were washed three ferent lobes using the DNeasy Blood & Tissue tion treatment group). These deaths were times with cold PBS, scraped and eluted in low- kit from Qiagen. Before isolation, the tissue probably not drug-related, so death was not ionic-strength buffer (10 mM Tris HCL [pH, was weighed and the isolated DNAwas quan- considered an adverse event. At the time of 7.4], 2.5 ml MgCl2, 1 mM EDTA) containing tified in duplicates using the Qubit 2.0 fluo- euthanasia, every animal was inspected for protease inhibitor (Roche), and incubated for rometer. For the nuclear counts, tissue was gross health issues. Body weight had decreased 30 minutes. Cells were then homogenized stained with DAPI as previously de- slightly in animals treated with pasireotide using a 26.5-gauge needle and nuclei were scribed.11 Fifteen 340 images from three dif- and the drug combination (Table 1). However, spun out at 2500 g for 5 minutes (4°C). The ferent liver lobes (five images per lobe) were no notable differences in feeding/drinking supernatant was collected and membrane frag- taken using the Zeiss AxioObserver and habits were observed; thus, the underlying ments were pelleted at 20,000 g for 30 minutes quantified using the Zen software (Carl cause and significance of this observation re- (4°C). Pelleted membranes were eluted in low- Zeiss). All histologic images were taken as mainsunknown.Inaddition,twoanimals ionic-strength buffer, and protein content was previously described.11 presented with one severely dysplastic quantified using a bicinchoninic acid assay

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(Thermo Fisher Scientific). For SDS PAGE, 3. Nauli SM, Alenghat FJ, Luo Y, Williams E, kidney disease. J Am Soc Nephrol 25: 18–32, 25 mg (input ratio of 1) of membrane prepara- Vassilev P, Li X, Elia AE, Lu W, Brown EM, 2014 Quinn SJ, Ingber DE, Zhou J: Polycystins 1 15. Chabardès D, Firsov D, Aarab L, Clabecq A, tion was loaded onto a 3%–8% Tris-acetate gel and 2 mediate mechanosensation in the pri- Bellanger AC, Siaume-Perez S, Elalouf JM: Lo- (150 V for 2.5 hours). The gel was transferred mary cilium of kidney cells. Nat Genet 33: calization of mRNAs encoding Ca2+-inhibitable to a polyvinylidene fluoride membrane and 129–137, 2003 adenylyl cyclases along the renal tubule. probed for PC1 (7e12, 1:1000) or PC2 4. Kusano E, Murayama N, Werness JL, Functional consequences for regulation of fi – (Yce2, 1:2000; Santa Cruz). Christensen S, Homma S, Yusu AN, Dousa the cAMP content. 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Chabardès D, Imbert-Teboul M, Elalouf JM: tion and aquaporin-2 phosphorylation and were performed by 2-way ANOVA and least- Functional properties of Ca2+-inhibitable trafficking in inner medulla. JAmSoc – square means t post hoc test (analyses of Figure type 5 and type 6 adenylyl cyclases and role Nephrol 21: 2059 2068, 2010 of Ca2+ increase in the inhibition of in- 18. Gattone VH 2nd, Maser RL, Tian C, Rosenberg 1). Linear regression was used to test for potential tracellular cAMP content. Cell Signal 11: JM, Branden MG: Developmental expression synergism of tolvaptan and pasireotide, but the 651–663, 1999 of urine concentration-associated genes and analysis was not significant (P=0.98). A P value 7. Yamaguchi T, Nagao S, Kasahara M, their altered expression in murine infantile- below the a level of 0.05 was considered to Takahashi H, Grantham JJ: Renal accumula- type polycystic kidney disease. Dev Genet 24: – represent a statistically significant difference. All tion and excretion of cyclic adenosine 309 318, 1999 monophosphate in a murine model of slowly 19. Torres VE, Wang X, Qian Q, Somlo S, Harris data are represented as mean6SD. progressive polycystic kidney disease. Am J PC, Gattone VH 2nd: Effective treatment of Kidney Dis 30: 703–709, 1997 an orthologous model of autosomal domi- 8. Gattone VH 2nd, Wang X, Harris PC, Torres nant polycystic kidney disease. Nat Med 10: VE: Inhibition of renal cystic disease de- 363–364, 2004 ACKNOWLEDGMENTS velopment and progression by a vasopressin 20. Wang X, Gattone V 2nd, Harris PC, Torres VE: V2 receptor antagonist. Nat Med 9: 1323– Effectiveness of vasopressin V2 receptor an- Wewould like to thank Drs. Scott L. Nyberg and 1326, 2003 tagonists OPC-31260 and OPC-41061 on 9. Smith LA, Bukanov NO, Husson H, Russo RJ, polycystic kidney disease development in Raymond Hickey (Department of Molecular Barry TC, Taylor AL, Beier DR, Ibraghimov- the PCK rat. JAmSocNephrol16: 846–851, Medicine, Mayo Clinic, Rochester, MN) for Beskrovnaya O: Development of polycystic 2005 providing the Huh7 cell line and Otsuka kidney disease in juvenile cystic kidney mice: 21. Meijer E, Gansevoort RT, de Jong PE, van der PharmaceuticalsandNovartisforprovidingthe Insights into pathogenesis, ciliary abnormal- Wal AM, Leonhard WN, de Krey SR, van den tolvaptan and pasireotide used in this study. ities, and common features with human dis- Born J, Mulder GM, van Goor H, Struck J, de ease. JAmSocNephrol17: 2821–2831, Heer E, Peters DJ: Therapeutic potential of This work was supported by National In- 2006 vasopressin V2 receptor antagonist in a stitutes of Health grants DK44863 (V.E.T.), 10. Starremans PG, Li X, Finnerty PE, Guo L, mouse model for autosomal dominant poly- DK058816 (P.C.H.), and DK090728 (Mayo Takakura A, Neilson EG, Zhou J: A mouse cystic kidney disease: optimal timing and Translational PKD Center); the Zell Founda- model for polycystic kidney disease dosing of the drug. Nephrol Dial Transplant 9 – tion; and the American Society of Nephrology through a somatic in-frame deletion in the 5 26: 2445 2453, 2011 end of Pkd1. Kidney Int 73: 1394–1405, 2008 22. Higashihara E, Torres VE, Chapman AB, Ben J. Lipps Research Fellowship (K.H.). 11. Hopp K, Ward CJ, Hommerding CJ, Nasr SH, Grantham JJ, Bae K, Watnick TJ, Horie S, Tuan HF, Gainullin VG, Rossetti S, Torres VE, Nutahara K, Ouyang J, Krasa HB, Czerwiec Harris PC: Functional polycystin-1 dosage FS; TEMPOFormula and 156-05-002 Study DISCLOSURES governs autosomal dominant polycystic kid- Investigators: Tolvaptan in autosomal domi- ney disease severity. J Clin Invest 122: 4257– nant polycystic kidney disease: Three years’ None. 4273, 2012 experience. Clin J Am Soc Nephrol 6: 2499– 12. Yamaguchi T, Pelling JC, Ramaswamy NT, 2507, 2011 REFERENCES Eppler JW, Wallace DP, Nagao S, Rome LA, 23. Torres VE, Chapman AB, Devuyst O, Sullivan LP, Grantham JJ: cAMP stimulates Gansevoort RT, Grantham JJ, Higashihara E, the in vitro proliferation of renal cyst epithe- Perrone RD, Krasa HB, Ouyang J, Czerwiec 1. Harris PC, Torres VE: Polycystic kidney dis- lial cells by activating the extracellular signal- FS; TEMPO 3:4 Trial Investigators: Tolvaptan ease, autosomal dominant. In: GeneReviews, regulated kinase pathway. Kidney Int 57: in patients with autosomal dominant poly- edited by Pagon RA, Adam MP, Ardinger HH, 1460–1471, 2000 cystic kidney disease. NEnglJMed367: Bird TD, Dolan CR, Fong CT, Smith RJH, 13. 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J Am Soc Nephrol 26: 39–47, 2015 Combination Therapy in ADPKD 47 SUPPLEMENTAL MATERIALS

Tolvaptan plus Pasireotide Shows Enhanced Efficacy in a PKD1 Model

Katharina Hopp*, Cynthia J Hommerding*, Xiaofang Wang*, Hong Ye*, Peter C Harris*+ and Vicente E

Torres*

*Division of Nephrology and Hypertension, +Department of Biochemistry and Molecular Biology, Mayo

Clinic, Rochester, Minnesota

Corresponding Author:

Vicente E. Torres

Division of Nephrology and Hypertension

Mayo Clinic

200 First Street SW

Rochester, MN 55905

Phone: 507-284-5009

Fax: 507-266-9315 email: [email protected]

Supplemental Figure 1 | Analysis of the inbred Pkd1RC/RC phenotype up to one year (A) Masson Trichrome stained cross-sections of WT inbred (I) and outbred (O) animals highlight the larger kidney size of outbred animals compared to inbred mice. This background effect may in part drive the larger %KW/body wt of Pkd1RC/RC outbred animals compared to inbred mice. Scale bar: 500µm. (B) Masson Trichrome stained cross-sections of 9m and 12m old inbred Pkd1RC/RC mice show continuous cyst progression with age. A marked difference of disease severity was noted between these older inbred females and males. Scale bar: 500µm (C) Close up of (B) reinforcing the gender difference and showing that cyst development and growth, rather than just dilated tubules, is the major disease manifestation in this model. Scale bar: 100µm. (D/E) %KW/body wt and BUN depicted as mean diamonds and SD. (D) %KW/body wt of inbred Pkd1RC/RC separated by males (M, dark green) and females (F, light green) illustrates the significance of the gender difference at 9m and 12m. This is likely a background effect as it was not noted in the outbred strain1 (E) BUN analysis, which becomes significant at 9m in females, highlights a decline in renal function with advanced disease.

2 (F) Immunofluorescence staining of inbred Pkd1RC/RC animals shows that the majority of cysts are of collecting duct (Aqp2) origin. A few dilated tubules/cysts stain positive for LTA (proximal tubule) especially at 9m and 12m of age. Scale bar: 100µm. (G) Quantification of the number of cysts per cross-section (avrg. in parentheses) positive for LTA, Aqp2 or neither of 6m old mice (n=6). Collecting duct cysts account for the majority (69%) of cysts and are significantly more frequent then unstained or LTA positive cysts (p=9.85x10-6). Only 12% of LTA positive cysts (avrg. 3/21) have a diameter greater 100µm, highlighting that the majority are dilated tubules. Scale bar: 100µm. p-values: **<0.01, ****<0.0001. %KW/BW, %KW/body wt.

Supplemental Figure 2 | High magnification histology images of tolvaptan/pasireotide preclinical trial mice. Cortical and outer medullary images were taken of Masson Trichrome stained kidney cross-sections from two animals per treatment group with the average %KW/body wt. Untreated control group images show significant cyst burden in the cortex with some dilated tubules in the outer medulla (Animal #1). Cyst burden and size decreased and tubular dilation were less common in animals treated with either drug. The tubular structure and parenchyma of animals treated with both drugs was comparable to WT in many aspects. Scale bar: 100µm. %KW/BW, %KW/body wt.

Supplemental Figure 3 | Hepatocytes express PC1 and PC2. Membrane preparations of the hepatocellular carcinoma cell-line Huh7, an accepted hepatocyte culture model, show that both PC1 (7e12) N-terminal glycoforms (NTP), and the full length (FL) protein are expressed, as characterized previously1. This has been shown at the mRNA level2. The cells also express PC2 (Yce2). Both proteins are present at a much lower level when compared to a renal cortical tubular epithelial cell-line (RCTE).

5 Supplemental Table 1 | Urine volume and osmolality analysis

12h Urine Volume1 (ml) Urine Osmolality (mOsm/kg) Control 0.84±0.51 1124.85±147.25 Tolvaptan 1.38±0.47A 902.64±61.49B

Pasireotide 0.63±0.39A 1328.88±417.15B

Both 0.87±0.28 1082.50±176.27

1 Urine was collected overnight for 12h in metabolic cages housing 2-4 animals, provided measurements are per animal A Tukey-Kramer HSD: T vs. P p=0.0197, all other comparisons were not significant B Tukey-Kramer HSD: T vs. P p=0.0185, all other comparisons were not significant

6 References

1. Hopp, K, Ward, CJ, Hommerding, CJ, Nasr, SH, Tuan, HF, Gainullin, VG, Rossetti, S, Torres, VE, Harris, PC: Functional polycystin-1 dosage governs autosomal dominant polycystic kidney disease severity. J Clin Invest, 122: 4257-4273, 2012. 2. The European Polycystic Kidney Disease Consortium: The polycystic kidney disease 1 gene encodes a 14 kb transcript and lies within a duplicated region on chromosome 16. Cell, 77: 881- 894, 1994.