Ketogenic Diet And β-hydroxybutyrate Alleviates Renal Fibrosis By Enhancing Fatty Acid Oxidation Through The Free Fatty Acid Receptor 3 Pathway

Objective: Allograft fibrosis leads to chronic renal allograft dysfunction,the main causes of graft loss.Because the intricate mechanisms of renal fibrosis remain unclear,there is still a lack of feasible targeted therapies that can alleviate or reverse fibrosis progression.Recent studies report that fatty acid oxidation plays a key role in renal fibrosis.With respect to what is known about the catabolic pathway,carnitine palmitoyltransferase 1a(Cpt1a)and acyl-coenzyme A oxidase 1(Acox1)are its rate-limiting enzymes in fatty acid oxidation.Cluster of differentiation 36(CD36)promotes the absorption of long-chain fatty acids.Peroxisome proliferator-activated receptor-α(PPARα)and PPAR-γ coactivator-1a(PPARGC1a)are the key transcription factors that regulate the expression of target genes(Cpt1a and Acox1)involved in fatty acid oxidation.The ketogenic diet has gained importance in the treatment of solid organ structural remodeling and enhances myocardial fatty acid oxidation to prevent cardiac dysfunction and fibrosis in mice,but its role in renal fibrosis has not been explored.β-hydroxybutyrate(β-OHB),as a ketone body metabolized by ketogenic diet,accounts for approximately 70% of the total ketone bodies.β-OHB is not only a simple energy intermediate metabolite but also plays an important role as a signaling molecule.β-OHB binds to two G protein-coupled receptors,hydroxycarboxylic acid receptor 2(HCAR2)and free fatty acid receptor 3(FFAR3).The mechanism of renal fibrosis improvement by β-OHB and ketogenic diet was further investigated in renal tubular epithelial cells.Methods: Male C57BL/6 mice were fed a normal diet or a ketogenic diet for 6 weeks prior to unilateral ureteral obstruction(UUO).Seven days after UUO,serum and kidney samples were collected.Serum β-OHB concentrations were measured and renal fibrosis was assessed through the histological analysis.Using immunohistochemistry 、immunofluorescence、western blotting and real-time q PCR analysis,the fibrosis-related markers(α-SMA,Col1a1,Col3a1 and fibronectin-1(Fn-1)),the fatty acid oxidation-related proteins(Cpt1a 、 Acox1 、 PPARα 、 PPARGC1 a 、 CD36)and pro-inflammatory cytokines(IL-1β、IL-6、TNF-α and F4/80)were quantitatively analyzed.Etomoxir(a Cpt1 a inhibitor,for 6 d)was injected intraperitoneally 1d before UUO.NRK52 E cells(rat renal tubular epithelial cells)were treated with TGFβ1,a fibrosis-inducing cytokine,and with or without β-OHB for 48 h.The expression of fibrosis-related markers、the fatty acid oxidation-related proteins and β-OHB binding receptors was measured by western blotting and real-time q PCR analysis.To further demonstrate the role of FFAR3,NRK52 E cells were co-stimulated with AR420626(a FFAR3 agonist)and TGFβ1 for 48 h.Cells were transfected with small interfering RNA(si RNA)targeting FFAR3 24 h before TGFβ1 stimulation.After 24 h of incubation,cells were treated with TGFβ1 and with or without β-OHB.Then the expression of the fibrosis-related markers and the fatty acid oxidation-related signaling molecules was analyzed using western blotting and real-time q PCR.Results: Ketogenic diet significantly enhanced serum β-OHB levels in mice(from 1.068±0.063 mmol/L to 4.689±0.377 mmol/L,P < 0.001).H&E staining revealed that structural destruction caused by ureteral obstruction was significantly alleviated by ketogenic diet.Masson’s trichrome and picrosirius red staining demonstrated that ketogenic diet effectively attenuated excessive collagen deposition in the obstructed kidneys.Immunohistochemistry、westerm blotting and real-time q PCR analysis revealed that ketogenic diet significantly reduced the expression of the fibrosis-related markers(α-SMA,Col1a1,Col3a1 and Fn-1)in the obstructed kidneys,compared to those fed with the normal diet.The expression levels of Cpt1 a,Acox1,PPARα,PPARGC1 a,and CD36 were dramatically reduced in obstructed kidneys and partially restored by ketogenic diet.However,the protective effect of ketogenic diet was abolished by etomoxir.Besides,ketogenic diet significantly suppressed UUO-induced macrophage infiltration and the expression of IL-6 in the obstructive kidneys. In NRK52 E cells,the fibrosis-related signaling molecules were increased by TGFβ1 and reduced by β-OHB.β-OHB treatment restored the impaired expression of Cpt1 a and PPARα.β-OHB significantly reverted the reduced expression of FFAR3 induced by TGFβ1.Although the m RNA level of HCAR2 was markedly elevated by TGFβ1,β-OHB did not alter its expression in NRK52 E cells.AR420626 similarly mitigated fibrosis induced by TGFβ1 and greatly strengthened fatty acid oxidation.The expression of FFAR3 was inhibited immensely by si RNA.The protective effect of β-OHB was blocked by si RNA targeting FFAR3.Conclusions: Ketogenic diet and β-OHB attenuated renal fibrosis by enhancing fatty acid oxidation and reduction in macrophage infiltration via the FFAR3-dependent pathway,which provides a promising dietary therapy for renal fibrosis.