Triptolide reduces cyst formation in a neonatal to adult transition Pkd1 model of ADPKD

Abstract

Background: Autosomal dominant polycystic kidney disease (ADPKD), a major cause of end-stage renal failure, results from genetic mutation of either polycystin-1 (Pkd1) or polycystin-2 (Pkd2). In order to develop novel therapies to treat the advancement of disease progression, numerous rodent models of different genetic backgrounds are available to study cyst development.

Methods: Here, a Pkd1-floxed inducible mouse model using the interferon responsive Mx1Cre-recombinase was utilized to test the effect of the small molecule triptolide. Relative to other Pkd1 inactivation models, cyst progression in this neonatal to adult transition model is attenuated. Following the characterization of inducible cyst formation in these mice, the development of kidney cysts from triptolide or vehicle-treated animals was analysed.

Results: Although Pkd1 deletion on postnatal Days P10 and P12 resulted in numerous cysts by P35, daily injections with triptolide beginning on Day P16 significantly reduced the total number of cysts per kidney, with a pronounced effect on the number of microcysts and the overall cystic burden. Additionally, renal function as assessed by blood urea nitrogen levels was also improved in triptolide-treated mice at both the P22 and P35 time points. As the Pkd1(flox/flox);Mx1Cre model has not been previously used for drug development studies, the feasibility of a 6-month adult Pkd1 inactivation study was also tested. While kidney cyst formation was minimal and focal in nature, livers of these Pkd1-deficient mice were severely cystic, enlarged and pale.

Conclusions: These results suggest that the Pkd1(flox/flox);Mx1Cre model of ADPKD is amenable to short-term kidney cyst formation drug studies; however, it may be problematic for long-term therapeutic research where widespread liver cysts and fibrosis could compromise drug metabolism.

Fig. 1

Neonatal cyst formation is dependent on pI:pC dosing schedule. (A) Timeline for pI:pC induction experiments. (B) Figures shown are representative H&E-stained kidney sections from P35 Pkd1^flox/flox;Mx1Cre male and female mice induced with 250 μg pI:pC. Variables in the induction schedule are represented as injection at Days P10, P12 or both P10/P12 and the volume of pI:pC delivered in 12.5 or 25 μl.

Fig. 2

Kidney cystic burden increases rapidly in the Pkd1^flox/flox;Mx1Cre neonatal induction model. (A) Representative H&E-stained kidney sections from Pkd1^flox/flox;Mx1Cre mice showing progression of cyst formation following pI:pC induction. (B) Contiguous T2-weighted axial slices from non-cystic (Pkd1^flox/+) and cystic (Pkd1^flox/flox;Mx1Cre) male mice obtained using a multi-slice RARE sequence. Kidneys were harvested at P35 following pI:pC induction at P10/P12; 25 μl. Imaging parameters at 9.4 T were as follows: in-plane resolution = 100 × 100 μm, slice thickness = 500 μm, effective echo time = 34 ms and recycle time = 5 s. (C) Representative H&E-stained liver sections from P35 Pkd1^flox/+ (WT) and Pkd1^flox/flox;Mx1Cre (Cystic) mice (inset shows ×40 magnification of cholangiocyte-derived liver cysts). Alkaline phosphatase (ALP) activity from blood serum was also analysed as an indicator of liver function (WT n = 3, cystic n = 7; P = 0.9776).

Fig. 3

Triptolide reduces cystic burden in the Pkd1^flox/flox;Mx1Cre model of ADPKD. (A) Representative H&E-stained P35 kidney sections from wild-type Pkd1^flox/+ (WT) and cystic Pkd1^flox/flox;Mx1Cre (Cy) mice treated with DMSO or triptolide (×8 magnification). Far right panels are representative H&E-stained outer cortex kidney sections showing areas of small cyst formation (×40 magnification). (B) Analysis of kidney cyst number, % cystic burden and average cyst size (kidneys: n = 42 DMSO and n = 40 triptolide) from cystic P35 Pkd1^flox/flox;Mx1Cre mice. (C) Frequency distribution of the number of cysts present of varying size. Cyst size is represented in pixels as determined from Image J analysis software (kidneys: n = 42 DMSO and n = 40 triptolide). (D) The percent of proliferating cells per cyst as assessed by Ki-67 immunohistochemical staining. Cells per cyst were counted from sagittal cross sections taken from DMSO- or triptolide-treated mice (The number of total cysts analysed for DMSO was 283 and for triptolide was 212). (E) Analysis of kidney weight as a percentage of body weight (%BW) from cystic (n = 21 DMSO, n = 20 triptolide) and non-cystic (WT) (n = 24 DMSO, n = 26 triptolide) P35 mice treated with either DMSO or triptolide. All mice were induced at P10/P12 using a 25 μl pI:pC volume. For all panels: *P < 0.0001, **P < 0.003 and ^#P < 0.05 by Student’s t-test.

Fig. 4

Triptolide preserves renal function. BUN values were measured from cystic mice over time on both P22 and P35 (n = 13 DMSO, n = 11 triptolide; asterisk indicates significance at P < 0.035 by Student’s t-test). Baseline refers to the average BUN values of non-cystic mice treated with DMSO or triptolide taken on P22. Double dagger indicates P < 0.0001 for the increase in BUN from baseline through P35 for DMSO by ANOVA using Bonferroni’s post hoc analysis (α = 0.05). There was no increase in BUN from baseline through P35 (P = 0.10322) with triptolide treatment.

Fig. 5

Widespread liver cysts are present 5 months post-Pkd1 adult deletion. Representative H&E-stained liver sections (×40 magnification) from Pkd1^flox/flox wild-type (WT) or induced Pkd1^flox/flox;Mx1Cre cystic (Cy) mice. Normal hepatic structures such as the central vein and portal triad can be observed in the WT sections. H&E kidney sections are also represented (×8 magnification) from WT and cystic mice. All tissues were obtained at 5 months following 1× 250 μg pI:pC induction at P30.