Ketosis Ameliorates Renal Cyst Growth in Polycystic Kidney Disease

Abstract

Mild reduction in food intake was recently shown to slow polycystic kidney disease (PKD) progression in mouse models, but whether the effect was due to solely reduced calories or some other aspect of the diet has been unclear. We now show that the benefit is due to the induction of ketosis. Time-restricted feeding, without caloric reduction, strongly inhibits mTOR signaling, proliferation, and fibrosis in the affected kidneys in a PKD rat model. A ketogenic diet had a similar effect and led to regression of renal cystic burden. Acute fasting in rat, mouse, and feline models of PKD results in rapid reduction of cyst volume, while oral administration of the ketone β-hydroxybutyrate (BHB) in rats strongly inhibits PKD progression. These results suggest that cystic cells in PKD are metabolically inflexible, which could be exploited by dietary interventions or supplementation with BHB, representing a new therapeutic avenue to treat PKD.

Keywords: beta-hydroxybutyrate; ketogenic diet; ketosis; metabolic inflexibility; polycystic kidney disease.

Figure 1:. Serum glucose predicts TKV increase in individuals with ADPKD, and time restricted feeding ameliorates PKD disease progression in a PKD rat model.

A. and B.) Linear regression analysis of the relationship between serum glucose levels (after overnight fasting) and average annual kidney volume change (TKV (A) or height-adjusted TKV (htTKV, B) in normoglycemic, non-diabetic individuals with ADPKD. reveals positive correlation. Line represents best fit and dotted lines represent 95% confidence intervals. C.) Serum β-hydroxybutyrate (BHB) levels from PKD1^cond/cond:Nes^cre mice (PKD; n =6) and wild-type controls (WT; n =6) from our previous research (Kipp et al., 2016) on reduced food intake (RFI; n =8) vs. ad libitum (AL; n =8) controls. D.) Schematic outline of the feeding regimens used in this study. Time-restricted-fed (TRF) rats were allowed access to food from T14 to T22. Food access is indicated by the orange boxes. E.) Average food intake per animal, per gram bodyweight, per week for rats on AL or TRF diet. F.) Average change in body weight for male and female PKD and WT rats on AL or TRF diet. G. and H.) H&E stained kidney sections. Scale bars are 3 mm or 100μm, respectively. I.) Cystic index (percent cystic area) for rats on TRF or AL diet. J.) 2-kidney weight to body weight ratios of TRF and AL treated rats. K.) Serum creatinine levels of AL and TRF animals. L.) Total number of cysts per kidney section in AL and TRF animals. M.) Cyst sizes of cysts counted in L. N.) BHB levels at the start of the experiment (week 3) and the final time point of the experiment (week 8). O.) Blood glucose levels at the start of the experiment (week 3) and the final time point of the experiment (week 8). (See also Fig. S2) Error bars represent SD. Statistical significance was determined using Mann-Whitney analysis. n =8 male and 12 female Cy/+ rats; n =13 male and 8 female wild-type rats for TRF experiments.

Figure 2:. Time restricted feeding reduces mTORC1 signaling, fibrosis, and proliferation in cystic kidneys.

A.) Immunostaining of the mTORC1 marker pS6^S235/236(cyan), in kidneys from WT and PKD rats on AL diet (top and middle) or PKD rats on TRF diet (bottom). B.) Sirius Red stained kidney sections from AL and TRF rats showing collagen deposits (red). C.) Immunostaining of the myofibroblast marker SMA (cyan), in kidneys from WT and PKD rats on AL diet (top and middle) or PKD rats on TRF diet (bottom) D.) Quantification of pS6-positive cells in cyst lining epithelia as a percent of the total number of DAPI stained nuclei. E.) Higher magnification images SMA-stained kidney sections showing pericystic myofibroblasts (cyan). F.) Quantification of SMA-positive cells as the percent of total cells. G.) Ki-67 immunostaining in kidneys from WT and PKD rats on AL diet (top and middle) or PKD rats on TRF diet (bottom). H.) Quantification of Ki-67 positive cells separated by location from the cyst lining, tubular, or interstitial regions and expressed as a percent of the total number of DAPI stained nuclei. I.) Immunostaining of P-STAT3 (cyan), in kidneys from WT and PKD rats on AL diet (top and middle) or PKD rats on TRF diet (bottom). Scale bars=100μm. Error bars represent SD. Statistical significance determined using Mann-Whitney analysis. n =8 male and 12 female Cy/+ rats; n =13 male and 8 female wild-type rats for TRF experiments.

Figure 3:. A ketogenic diet ameliorates disease progression in juvenile Han:SPRD rats.

A. and B.) H&E stained kidney sections from WT or PKD rats fed ad libitum a Normal Chow (NC) or Ketogenic diet (KD). Scale bars=3mm or 100μm, respectively. C.) 2-kidney weight to body weight ratios of NC and KD treated rats. D.) Cystic index (percent cystic area) from NC and KD cystic rats. E.) Body weight of wild-type and cystic animals on NC and KD. F.) 2-kidney weight to heart weight ratios of NC and KD treated rats. G.) Blood glucose at initial (week 3) and final time point (week 8) of the experiment. H.) Blood β-hydroxybutyrate levels at initial (week 3) and final time point (week 8). I.) Percent change in mass of. J.) Calories consumed per gram of bodyweight per week. K.) Serum creatinine. L.) Total number of cysts per kidney section. M.) Cyst sizes of cysts counted in L. Error bars represent SD. Statistical significance determined from Mann-Whitney analysis. n =8 male and 7 female Cy+ rats; n =13 male and 6 female wild-type rats for ketogenic diet experiments (* P<0.05, **** P<0.0001)

Figure 4:. The ketogenic diet reduces markers of PKD in juvenile Han:SPRD rats.

A.) Immunostaining of the mTORC1 marker pS6^S235/236(cyan), in kidneys from WT and PKD rats on Normal Chow diet (NC, top and middle) or PKD rats on Ketogenic Diet (KD) (bottom). B.) Immunostaining of P-STAT3 (cyan). C.) Immunostaining of the myofibroblast marker SMA (cyan),. Scale bars=100μm D.) Sirius Red/Fast Green stained kidney sections showing collagen deposits (red). Scale bar=300μm E.) Quantification of pS6-positive cells in cyst lining epithelia as a percent of the total number of DAPI stained nuclei in cysts. F.) Quantification of SMA positive cells as a percent of the total number of cells. G.) Quantification of collagen positive area. H.) Ki-67 immunostaining. Scale bar=100μm I.) Quantification of Ki-67 positive cells separated by location from the cyst lining, tubular, or interstitial regions and expressed as a percent of the total number of DAPI stained nuclei. J.) Immunoblot for the indicated markers. K.) Quantification of immunoblots. Error bars represent SD. Statistical significance determined using Mann-Whitney analysis. n =8 male and 7 female Cy+ rats; n =13 male and 6 female wild-type rats for ketogenic diet experiments (* P <0.05, **P <0.01).

Figure 5:. The ketogenic diet reverses kidney disease progression in adult Han:SPRD rats.

A.) Blood glucose levels at initial (week 8) and final time point (week 12) of the experiment. B.) Blood β-hydroxybutyrate levels. C. and D.) Percentage change in NC and KD treated animal’s body, kidney and heart mass over the 4-week treatment period. Bracketed masses reflect the difference between 8 week and 12 week values or differences between NC and KD rats. E. and F.) H&E stained kidney sections. Scale bars=3mm or 100μm, respectively. G.) 2-kidney to body weight ratios. H.) Cystic index (percent cystic area). I.) Serum creatinine. J.) Total number of cysts per kidney section. K.) Cyst sizes of cysts counted in L.) Sirius Red/Fast Green stained kidney sections showing collagen (red). M.) SMA immunostaining (cyan). N.) Immunostaining of the mTORC1 marker pS6^S235/236 (cyan). O.) Ki-67 immunostaining. P.) Quantification of collagen positive area from animals in L and compared to 8 week rats. Q.) Quantification of SMA positive cells as a percent of the total number of cells. R.) Quantification of Ki-67 positive cells separated by location into cyst lining, tubular, or interstitial and expressed as a percent of the total number of DAPI stained nuclei. Scale bars=100μm. Error bars represent SD. Statistical significance was determined using Mann-Whitney analysis. n =12 male and 10 female cystic rats; n =10 male and 14 female wild-type rats for adult ketogenic diet experiments. (* P <0.05, **** P <0.0001).

Figure 6:. Acute fasting leads to rapid cystic cell death, and reduced cyst and kidney size in polycystic kidneys.

A.) Blood glucose and B.) β-hydroxybutyrate levels of untreated and 48-hour fasted Han:SPRD and WT rats. C.) Animal mass. D.) Cystic index (percent cystic area). E.) Single kidney masses. F.) 2-kidney to heart mass ratios. G.) Heart masses. H.) TUNEL stain. Kidneys from WT rats did not demonstrate significant levels of apoptosis. Scale bar=100μm I.) Percentage of TUNEL positive cells as the percent of the total number of DAPI stained nuclei in cystic epithelia, tubules, interstitial or cells within lumen. J.) Sirius red/fast green stain of ad libitum and fasted Han:SPRD rats indicating collagen and basement membranes in red. Arrowheads denote fragmented epithelial cells. Arrows denote denuded epithelium. Asterisks denote red blood cells in cysts. K.) Oil O red stain. Scale Bar=50μm. L.) Blood glucose values and M.) BHB levels from 24-hour fasted Pkd1^cond/cond:Nes^cre mice. N.) Single kidney mass of AL and fasted Pkd1^cond/cond:Nes^cre mice. O.) 2-kidney to bodyweight ratio of AL and fasted Pkd1^cond/cond:Nes^cre mice. P.) TUNEL stain of kidney sections from Pkd1^cond/cond:Nes^cre mice after a 24-hour fast. Scale bars=100μm. Q.) Percentage of TUNEL positive epithelial cells or cells within cyst lumens as a percent of the total number of DAPI stained nuclei in AL vs. fasted Pkd1^cond/cond:Nes^cre mice in cystic. R.) CT scans of PKD cats before and after 72 hours fast. * Denotes the same cyst in each image. S.) Quantification of total kidney volumes of PKD cats determined ~1.5 years prior to the study, immediately before fasting or immediately after fasting. Error bars represent SD. Statistical significance was determined using Mann-Whitney analysis. n =8 male and 5 female cystic rats; n =7 male and 5 female wild-type rats, n =4 polycystic cats, and n =4 female cystic Pkd1^cond/cond:Nes^cre mice; n =3 male and 5 female wild-type mice were used for fasting experiments. (* P <0.05, ** P <0.01, **** P <0.0001)

Figure 7:. BHB administration ameliorates disease progression in the Han:SPRD rat model of PKD.

A.) Gross kidney images of untreated cystic (left), BHB-treated cystic (middle) and BHB-treated wild-type (right) rats. Scale bar=1cm B.) Sirius Red/Fast Green stained kidney sections. Scale bar=2mm and 50μm respectively. C.) 2-kidney to bodyweight ratios, D.) animal masses, and E.) cystic areas of water, salt and BHB treated rats. F.) Serum creatinine. G.) Percent collagen area from Sirius Red/Fast Green stained sections. H.) Ki-67 immunofluorescence stain. I.) Ki-67 quantification. J.) SMA immunofluorescence stain for myofibroblasts (cyan). Scale bar =50μm. Error bars represent SD. Statistical significance was determined using Mann-Whitney analysis. n =7 male and 5 female cystic rats; n =3 male and n =5 female wild-type rats treated with BHB and n =4 male and 4 female cystic rats; n =2 male and 5 female wild-type rats treated with Na/K salt. (* P <0.05, ** P <0.01, *** P <0.001).