A mild reduction of food intake slows disease progression in an orthologous mouse model of polycystic kidney disease

Abstract

Autosomal-dominant polycystic kidney disease (ADPKD) is a common cause of end-stage renal disease, and no approved treatment is available in the United States to slow disease progression. The mammalian target of rapamycin (mTOR) signaling pathway is aberrantly activated in renal cysts, and while mTOR inhibitors are highly effective in rodent models, clinical trials in ADPKD have been disappointing due to dose-limiting extrarenal side effects. Since mTOR is known to be regulated by nutrients and cellular energy status, we hypothesized that dietary restriction may affect renal cyst growth. Here, we show that reduced food intake (RFI) by 23% profoundly affects polycystic kidneys in an orthologous mouse model of ADPKD with a mosaic conditional knockout of PKD1. This mild level of RFI does not affect normal body weight gain, cause malnutrition, or have any other apparent side effects. RFI substantially slows disease progression: relative kidney weight increase was 41 vs. 151% in controls, and proliferation of cyst-lining cells was 7.7 vs. 15.9% in controls. Mice on an RFI diet maintained kidney function and did not progress to end-stage renal disease. The two major branches of mTORC1 signaling, S6 and 4EBP1, are both suppressed in cyst-lining cells by RFI, suggesting that this dietary regimen may be more broadly effective than pharmacological mTOR inhibition with rapalogs, which primarily affects the S6 branch. These results indicate that polycystic kidneys are exquisitely sensitive to minor reductions in nutrient supply or energy status. This study suggests that a mild decrease in food intake represents a potential therapeutic intervention to slow disease progression in ADPKD patients.

Keywords: ADPKD; food restriction; mTOR; polycystic kidney disease.

Fig. 1.

Effects of a moderately reduced food intake (RFI) on control mice. A: disease progression as measured by 2-kidney-to-body weight ratios in untreated PKD1^cond/cond:Nes^Cre mice (solid line) compared with wild-type mice (dashed line; n = 5 animals/time point). B: average food intake per animal per week for mice on ad libitum (AL) diet (solid line) or RFI diet (dashed line). C: weight gain as a percentage of starting weight per week for wild-type mice on AL diet (black) or RFI diet (grey). D: weight gain for polycystic kidney disease (PKD) mice on the AL diet (black) or RFI diet (grey). E: immunoblot analysis of wild-type kidney lysates reveals an increase in overall LKB1 (S307) and AMPK (T172) phosphorylation in mice on the RFI diet compared with AL controls. The RFI diet does not increase LC3-II accumulation as a marker of autophagy.

Fig. 2.

RFI ameliorates disease progression in PKD1^cond/cond:Nes^Cre mice. Shown is a comparison of representative gross kidneys (A) and hematoxylin- and eosin- stained tissue sections (B) from AL wild-type (WT), PKD-AL, and PKD-RFI mice (left to right). C: 2-kidney-to-body weight ratios in untreated mice at the beginning of the treatment regimen (postnatal day 35; p35) and at the end (p84) of the AL or RFI dietary regimen. D: effect of dietary regimen on total body weights at p84. Two-kidney weights (E) and BUN (F) of mice are shown for the start (p35) or end (p84) of the trial. Red circle indicates animals that reached end-stage renal disease (ESRD) by the conclusion of the trial.

Fig. 3.

RFI reduces interstitial fibrosis and proliferation of cyst-lining epithelial in PKD kidneys. A–F: representative images of kidney sections from PKD mice on AL diet (left) or RFI diet (right). A and B: hematoxylin- and eosin-stained tissue. C and D: Masson's trichrome staining to visualize collagen deposition. Intense interstitial collagen staining (blue) is apparent in kidneys of AL-fed PKD mice but is greatly reduced in kidneys of RFI-fed PKD mice. E and F: immunofluorescence microscopy for the proliferation marker Ki-67 (green). Nuclear counterstain is 4,6-diamidino-2-phenylindole (DAPI; red false color). Scale bars = 50 μm. G: quantification of the percentage of Ki67-positive cyst-lining cells among the total number of DAPI-positive cyst-lining epithelial cells. Ki-67-positive cells and nuclei were counted in 5 random, low-magnification fields/kidney (n = 3 mice/treatment condition).

Fig. 4.

RFI inhibits mTORC1 signaling in PKD kidneys. A: immunoblot analysis of total kidney lysates from PKD animals on either AL or RFI dietary regimen reveal a decrease in total phosphorylation of S6 (S240/244) and 4EBP1 (T37/46), and no significant change in the phosphorylation of LKB1 (S307) and AMPK (T132). Actin loading corresponds to the 3 blots loaded above them and each lane represents a single tissue lysate with n = 3 analyzed/treatment group. B–G: localization of mTORC1 activity markers (green) by immunofluorescence microscopy in kidneys from PKD mice on AL diet (left) or RFI diet (right). B and C: pS6 (S240/44). D and E: pS6 (S235/236). F and G: p4E-BP1(T37/46). Nuclear counterstain is DAPI (red false color). Scale bars = 50 μm. All image acquisition and processing were done equivalently.