Tolvaptan plus pasireotide shows enhanced efficacy in a PKD1 model

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is a leading cause of ESRD. A central defect associated with ADPKD pathology is elevated levels of 3', 5'-cyclic AMP (cAMP). Compounds such as tolvaptan and pasireotide, which indirectly reduce adenylyl cyclase 6 (AC6) activity, have hence proven effective in slowing cyst progression. Here, we tested the efficacy of these compounds individually and in combination in a hypomorphic PKD1 model, Pkd1(R3277C/R3277C) (Pkd1(RC/RC)), in a 5-month preclinical trial. Initially, the Pkd1(RC/RC) model was inbred into the C57BL/6 background, minimizing disease variability, and the pathogenic effect of elevating cAMP was confirmed by treatment with the AC6 stimulant desmopressin. Treatment with tolvaptan or pasireotide alone markedly reduced cyst progression and in combination showed a clear additive effect. Furthermore, combination treatment significantly reduced cystic and fibrotic volume and decreased cAMP to wild-type levels. We also showed that Pkd1(RC/RC) mice experience hepatic hypertrophy that can be corrected by pasireotide. The observed additive effect reinforces the central role of AC6 and cAMP in ADPKD pathogenesis and highlights the likely benefit of combination therapy for patients with ADPKD.

Keywords: cyclic AMP; polycystic kidney disease; somatostatin; vasopressin.

Figure 1.

C57BL/6 Pkd1^RC/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 μm. (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 12 months, but inbred mice developed similar-size microhamartomas as early as 3 months. Scale bar: 100 μm. (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%±0.11%, outbred, 1.49%±0.20% [P=0.11]; 6 months, inbred, 1.24%±0.05%, outbred, 1.66±0.24 [P=0.15×10⁻³) (Supplemental Figure 1A). (D) The %LW/ERbody wt was elevated in inbred compared with outbred mice at 3 months (inbred, 5.39%±0.34%; outbred, 4.80%±0.30%; P=0.21×10⁻²) and 6 months (inbred, 5.54%±0.40%; outbred, 4.97%±0.48%; P=0.34×10⁻²). Gray dotted lines represent WT (C57BL/6 or outbred) mice. (E) Gross anatomy of representative kidneys from inbred WT mice, inbred Pkd1^RC/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 mean±1×SD %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 μm, 250 μm (inset). (G–J) %KW/body wt (G), renal cystic volume (H), renal fibrotic volume (I), and cAMP levels (J) of saline-treated inbred Pkd1^RC/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 .

Figure 2.

Tolvaptan plus pasireotide treatment showed enhanced efficacy over single drug treatment. (A) Gross anatomy of representative kidneys from inbred WT and inbred Pkd1^RC/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 mean±1×SD %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 μm, 250μm (inset). (C–F) %KW/body wt (C), renal cystic volume (D), renal fibrotic volume (E), and cAMP levels (F) of saline-treated Pkd1^RC/RC 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.

Figure 3.

Hepatic hypertrophy of Pkd1^RC/RC mice can be corrected by pasireotide treatment. (A) %LW/ERbody wt of saline treated, inbred Pkd1^RC/RC control mice (C, green), tolvaptan-treated mice (T, blue), pasireotide-treated mice (P, yellow), and tolvaptan plus pasireotide–treated mice (B, red) (6 months of age) depicted as mean diamonds and SD. Gray dotted line represents C57BL/6 WT value. The %LW/ERbody wt returned to WT level in animals treated with pasireotide. (B) Number of DAPI-positive nuclei per 0.01 mm² in liver cross-sections of Pkd1^RC/RC controls (green), pasireotide-treated mice (yellow), and WT mice (black), depicted as mean diamonds and SD. Per given area, untreated animals had fewer nuclei compared with pasireotide-treated or WT animals, indicative of larger cells (hypertrophy). (C) Representative image of part B. Scale bar: 50 μm. Hepatic hypertrophy has not previously been reported as a characteristic of ADPKD. Because both PC1 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.