In contrast to previous studies in which good glucose control prevented, but did not reverse, nephropathy in a model of Type 1 diabetes 
, in the present studies the ketogenic diet reversed nephropathy, as reflected by albumin/creatinine ratios, after it had developed in models of both Type 1 and Type 2 diabetes. The reversal of functional nephropathy was associated with robust normalization of expression of genes induced by oxidative and other forms of stress. In contrast to the complete reversal of nephropathy as reflected by albuminuria and gene expression, histological evidence of nephropathy was only partially reversed in the model for Type 2 diabetes (kidneys from the Akita mice were not available for histological analysis). This suggests, perhaps not surprisingly, that functional and molecular aspects of nephropathy reverse more quickly than morphological aspects of diabetic nephropathy.
The present study also confirmed and greatly extended the number of molecular markers for diabetic nephropathy, especially for genes whose expression in kidney is induced in models of both Type 1 and Type 2 diabetes. The gene most robustly induced in both forms of diabetes was Cdkn1a, also known as p21 (). Cdkn1a was induced over 20-fold in Akita mice though only about 4-fold in db/db mice, consistent with the generally more robust effect of diabetes on gene expression in Akita mice compared to db/db mice, though interestingly albuminuria was greater in db/db mice than in Akita mice. Although induction of p21 has been reported in a rat model of Type 1 diabetic nephropathy 
, the functional significance of this observation is unclear. P21 is induced by a number of cellular stressors and generally appears to play a role in ameliorating stresses, including DNA damage 
, so it seems likely that in the present studies the induction of p21 and its reversal by the ketogenic diet reflects pathological processes rather than mediating the reparative effects of the ketogenic diet. Indeed, this appears to be the case for the vast majority of the genes whose expression was induced in both forms of diabetic nephropathy and whose induction was reversed by the ketogenic diet. Possible exceptions to this pattern are Nox1, Nox4, Txnip, and Duox1, whose induction all plausibly contribute to the development of oxidative stress 
, and thus whose inhibition by the ketogenic diet all plausibly contribute to the restorative effects of the ketogenic diet. Similarly, Casp-8 was inhibited by the ketogenic diet in diabetic mice, and it has been reported that in db/db
mice decreased caspase-8 activity correlates with decreased progression of diabetic nephropathy with moderate exercise 
, and podocyte apoptosis appears to be a feature of advanced diabetic nephropathy 
. More directly reflective of nephropathy are the inhibition of nephrin, podocin, and ZO-1, all of which contribute to normal kidney function and restoration of which by the ketogenic diet plausibly reflects restoration of normal kidney function.
We also note that in contrast to the robust weight loss that the ketogenic diet produces in diet-induced obesity 
, the ketogenic diet increased, rather than decreased, body weight in db/db mice (). Furthermore, weight gain occurred even though caloric intake decreased. It has similarly been reported that the ketogenic diet does not reduce body weight in leptin-deficient ob/ob mice 
, which we have also observed (Mastaitis, unpublished). Thus the weight gain observed in db/db mice, in contrast to the weight loss in wild-type mice, is presumably due to the fact leptin signaling is required for the reduction in body weight by the ketogenic diet, and the weight gain despite reduced caloric intake presumably reflects reduced metabolic rate. It will be of interest to determine which cell types mediate this requirement for leptin in weight loss by the ketogenic diet.
A key issue raised but not resolved in the present study is the mechanism by which the ketogenic diet reverses nephropathy and gene expression profiles associated with nephropathy. One potential mechanism is that the reversal was simply due to reduction in blood glucose. However, since previous studies demonstrated that good glucose control prevented, but did not reverse, diabetic nephropathy 
, and since diabetic complications are thought to be caused by increased cellular metabolism of glucose 
, we hypothesize that at least part of the restorative effect was mediated by reduction of glucose metabolism. This hypothesis is supported by several lines of evidence. First, the ketogenic diet appears to reduce the frequency of epileptic seizures by reducing glucose metabolism 
. Second, molecular responses to the ketogenic diet indicates a re-routing of cellular metabolism away from glucose utilization and toward the use of alternative fuels 
. Finally, we have shown that ketone 3-OHB blocks molecular effects of glucose 
. We therefore hypothesize that the ketogenic diet reverses diabetic nephropathy by raising blood levels of 3-OHB which subsequently reduce glucose metabolism in at least some tissues including kidney. Since ketones and the ketogenic diet are neuroprotective in a wide range of conditions 
, a phenomenon we have corroborated in the present study (), it seems highly likely that the ketogenic diet will be protective in diabetic neuropathy and possibly retinopathy as well.
Although the present studies represent a proof of principle that pathologies produced by diabetes can be reversed by a simple dietary manipulation, many issues remain to be resolved before clinical application can be considered. First, the ketogenic diet is probably too extreme for chronic use in adult patients, and indeed may produce untoward iatrogenic effects. On the other hand, based on hysteretic mechanisms observed with the lac operon 
, it is plausible that only transient exposure to the diet will effectively reverse the gene expression profile and thus in effect “reset” the pathological process. If so it is plausible that only a sustainable transient exposure to the diet may be needed to produce persistent reversal of pathologies associated with diabetes. Furthermore, if the mechanism by which the diet produces its protective effects is in fact the elevation of blood 3-bOHB, it is possible that a pharmacological intervention that mimics these effects might be sufficient to reverse pathology. Although we have not yet been successful in chronically elevating plasma 3-bOHB by chronic mini-pump infusion, dietary supplementation remains a possibility, including supplementation with ketogenic derivatives 
. We have also demonstrated that chronic administration of the structurally similar molecular butyrate (differing only by a single oxygen atom) mimics many of the protective effects of dietary restriction through a mechanism dependent on the transcription factor CBP 
, so assessing the efficacy of this or related molecules in reversing pathologies due to diabetes would also be of great interest.