Reversal of diabetic nephropathy by a ketogenic diet

Abstract

Intensive insulin therapy and protein restriction delay the development of nephropathy in a variety of conditions, but few interventions are known to reverse nephropathy. Having recently observed that the ketone 3-beta-hydroxybutyric acid (3-OHB) reduces molecular responses to glucose, we hypothesized that a ketogenic diet, which produces prolonged elevation of 3-OHB, may reverse pathological processes caused by diabetes. To address this hypothesis, we assessed if prolonged maintenance on a ketogenic diet would reverse nephropathy produced by diabetes. In mouse models for both Type 1 (Akita) and Type 2 (db/db) diabetes, diabetic nephropathy (as indicated by albuminuria) was allowed to develop, then half the mice were switched to a ketogenic diet. After 8 weeks on the diet, mice were sacrificed to assess gene expression and histology. Diabetic nephropathy, as indicated by albumin/creatinine ratios as well as expression of stress-induced genes, was completely reversed by 2 months maintenance on a ketogenic diet. However, histological evidence of nephropathy was only partly reversed. These studies demonstrate that diabetic nephropathy can be reversed by a relatively simple dietary intervention. Whether reduced glucose metabolism mediates the protective effects of the ketogenic diet remains to be determined.

Figure 1. Development of albuminuria in mouse models of type 1 and type 2 diabetes and its reversal by the ketogenic diet.

ACR in diabetic Akita mice (A) and in diabetic db/db mice (B) with respective age-matched control mice, fed either the ketogenic diet or chow diet for 8 weeks. Urine was collected and ACR measured 8 weeks after starting the diet regimens. Data are means ± SE (n = 10–14 for all groups). *p<0.05 (ANOVA) vs. WT-Chow group (or vs. diabetic-Chow groups as indicated by the horizontal brackets).

Figure 2. Diabetic nephropathy is associated with a similar profile of gene expression for oxidative stress and toxicity related transcripts in models of Type 1 (Akita) and Type 2 (db/db) diabetes – reversal with ketogenic diet.

Real-time PCR gene expression was assessed by RT² Profiler PCR Arrays from SABiosciences. The ketogenic diet reverses the kidney gene expression profile associated with diabetic nephropathy in both the Akita and db/db mice. Data for each gene was normalized to a panel of housekeeping transcripts and expressed as fold change compared to the WT-Chow group. Data are means ± SE (n = 4–5 for all groups). *p<0.05 (ANOVA) vs. WT-Chow group (or vs. diabetic-Chow groups as indicated by the horizontal brackets).

Figure 3. Nephropathy-related gene expression in the diabetic Akita mice is reversed by the ketogenic diet.

(A) Kidney gene expression changes associated with the Akita mutation and not in the db/db diabetics that are reversed by the ketogenic diet. The pattern of reversal is particularly evident in the clusterograms form the entire (B) Oxidative Stress and Antioxidant Defense PCR Array and (C) Stress & Toxicity PCR array from SABiosciences. The clusterograms (heat maps) represent relative expression levels for all samples and all genes included on the real-time PCR arrays. Data for each gene in (A) was normalized to a panel of housekeeping transcripts and expressed as fold change compared to the WT-Chow group (except for nephrin, podocin, and zo-1 – see Research Design and Methods ). Data are means ± SE (n = 4–5 for all groups). *p<0.05 (ANOVA) vs. WT-Chow group (or vs. diabetic-Chow groups as indicated by the horizontal brackets).

Figure 4. Kidney expression of duox1 and sod1 is affected by the ketogenic diet in db/db diabetic mice.

(A) Ketogenic diet induces Sod1 expression and normalizes Duox1 expression in the db/db mice. The transcripts that show consistent patterns across db/db data sets are depicted in the clusterograms from the (B) Oxidative Stress and Antioxidant Defense PCR Array and (C) Stress & Toxicity PCR array from SABiosciences. The clusterograms (heat maps) represent relative expression levels for all samples and selected genes included on the real-time PCR arrays. Data for each gene in (A) was normalized to a panel of housekeeping transcripts and expressed as fold change compared to the WT-Chow group. Data are means ± SE (n = 4 for all groups). *p<0.05 (ANOVA) vs. WT-Chow group (or vs. diabetic-Chow groups as indicated by the horizontal bracket).

Figure 5. Improvement in glomerular histopathology in diabetic db/db mice fed the ketogenic diet.

Morphometric study demonstrating the mean values of glomerular deposition of PAS positive material(A) (% of total glomerular area) in db/db using light microscopy in fixed kidney stained with periodic-acid-Schiff stain as described in Research Design and Methods . Images of representative glomerular histology from WT-Chow (B), WT-Keto (C), db/db-Chow (D), db/db-Keto (E). *p<0.05 (ANOVA) vs. Control groups (or vs. db/db-Chow group - horizontal bracket).

Figure 6. The ketone 3-beta-hyroxybutyrate (3-OHB) is cytoprotective in cells exposed to 100 uM hydrogen peroxide, at 0 or 15 mM glucose.

p<0.001, ANOVA, significant effect of 3-OHB at both glucose concentrations. * p<0.05 vs. 0 3-OHB. Viability determined by a colorimetric (CCK-8; Dojin Laboratories, Kumamoto, Japan).