RNA-seq analysis revealed Bdh1 reduction in DKD mouse model
As a major microvascular complication in diabetic patients, DKD is the leading cause of CKD and ESRD [2-4]. To gain a comprehensive understanding of potential DKD regulators, we performed an RNA-seq analysis comparing gene expression in the kidneys from db/db mice or WT mice after the db/db mice emerged obvious pathological features of DKD (Fig.1A). KEGG analysis showed that the “Synthesis and degradation of ketone bodies” pathway was significantly down-regulated (Fig.1B-C). Given that the KD was reported as an effective treatment for diabetes [33, 34], we supposed that the down regulation of “Synthesis and degradation of ketone bodies” pathway participates the pathogenesis of DKD. Consistently, qRT-PCR analysis confirmed the expression changes of Bdh1, Oxct1, Acat1, and Hmgcs1 (Fig.1D). Notably, among these four pathway members, Bdh1 has been reported to protect heart from heart failure in TCA mouse model. Thus, to identify whether Bdh1 is involved in DKD pathogenesis, we further detected the protein level of Bdh1. As shown in Fig.1E, protein level of Bdh1 in kidneys of db/db mice was significantly lower than that in kidneys of WT mice. Moreover, the decrease in Bdh1 expression was also confirmed by immunohistochemistry (IHC) and immunofluorescence (IF) analysis (Fig.1F). Consistent with the DKD mouse model, we also observed downregulation of Bdh1 in renal tissues of diabetic patients with kidney disease by IHC and IF staining (Fig.1G). These results indicates that the decrease of Bdh1 expression is related to the pathogenesis of DKD.
Bdh1 deficiency mediated high glucose (HG) or palmitic acid (PA)-induced ROS overproduction and inflammation
As it is known, hyperglycemia and hyperlipidemia are the two most obvious characteristics of type 2 diabetes . In view of this, we established HG-induced glucotoxicity and PA-induced lipidtoxicity cell model with HK-2 cells to evaluate the effect of HG or PA on the Bdh1 expression. As expected, either mRNA level or protein level of Bdh1 was obviously reduced by HG or PA treatment in HK2 cells (Fig.2A-C), which was also identified by IF analysis (Fig.2D-E).
To investigate whether the Bdh1 reduction contribute to HG or PA-induced cell injury, we performed Bdh1 knockdown in HK2 cells (Fig.2F). Given that the increased ROS plays a central and prominent role in the pathogenesis of diabetic microvascular complications including DKD  and the overproduction of ROS was related to inflammation , we next detected the ROS level and observed significant increase of ROS in HK2 cells transfected with Bdh1 siRNA (Fig.2G). In addition, the protein level of activated proinflammatory factor, cleaved IL-1β, was also elevated by Bdh1 knockdown (Fig.2H), as well as the secretory IL-1β and IL-18 (Fig.2I-J). Collectively, these results suggest that Bdh1 deficiency might mediated HG or PA-induced cell injury by loss of anti-ROS function.
Either Bdh1 overexpression or βOHB supplementation reversed HG or PA-induced ROS overproduction and inflammation
As the Bdh1 deficiency led to increased ROS and inflammation, we next sought to determine whether HG or PA-induced Bdh1 reduction mediates HG or PA-induced cell injury. To this end, we transfected HK2 cells with flag-Bdh1 overexpression plasmid to block the HG or PA-induced Bdh1 reduction (Fig.3A). Notably, ROS assay showed that the Bdh1 overexpression significantly reduced the HG-induced ROS overproduction (Fig.3B, upper panels). Especially in PA-treated cells, Bdh1 overexpression nearly completely reversed the PA-induced ROS overproduction (Fig.3B, lower panels). As to inflammation, Bdh1 overexpression also reversed the HG or PA-induced activation of IL-1β (Fig.3C) and the increase of secretory IL-1β and IL-18 (Fig.3D-E). These evidences suggests that Bdh1 may play a protective role in DKD pathogenesis and pathological hyperglycemia and hyperlipidemia-induced Bdh1 reduction might mediate cell injury.
Given that Bdh1 is a key enzyme which mainly catalyzes the first step of βOHB metabolism, we next sought to determine whether βOHB supplementation could also exhibit protective effect on HG or PA-treated HK2 cells. In line with Bdh1 overexpression, βOHB supplementation also markedly reversed HG or PA-induced ROS overproduction (Fig.4A). Similarly, βOHB supplementation also reversed the HG or PA-induced activation of IL-1β (Fig.4B) and the increase of secretory IL-1β and IL-18 (Fig.4C-D). Taken together, these findings suggest that Bdh1 mediated βOHB metabolism play important role in protection of HG or PA-induced cell injury.
Bdh1-mediated βOHB metabolism promoted Nrf-2 nuclear translocation through the AcAc-succinate-fumarate metabolic pathway
Given that Nrf2 is a well-known transcription factor that regulates transcriptional induction of ARE-containing genes encoding antioxidant enzymes in response to cellular stresses including ROS [8-10], we next sought to determine whether Bdh1 mediates anti-ROS function through activation of Nrf2. As Nrf2 is a nuclear transcription factor, we detected the protein level of Nrf2 by western blot (WB) in nuclear extracts. Of note, in HK2 cells transfected with Bdh1 siRNA, Nrf2 protein level was significantly lower than that in cells transfected with control siRNA (Fig.5A). Moreover, either HG or PA could induce Nrf2 reduction in nuclear, whereas Bdh1 overexpression could reversed both HG and PA-induced Nrf2 reduction (Fig.5B). Consistent with the observations made in Bdh1 overexpression, βOHB supplementation also reversed both HG and PA-induced Nrf2 reduction in nuclear extracts (Fig.5C), which was further confirmed by Nrf2 nuclear translocation assay with immunostaining (Fig.5D). These data indicate that Bdh1-mediated βOHB metabolism promotes Nrf-2 nuclear translocation.
In Bdh1-mediated βOHB metabolism pathway, Bdh1 firstly metabolites βOHB into AcAc, which could enter into TCA cycle and then is metabolized into succinate and fumarate in turn (Fig.6A). As the fumarate is a well-known activator of Nrf2 signaling, we next investigated whether Bdh1 activated Nrf2 by increase of fumarate. Interestingly, we found that the concentrations of AcAc, succinate and fumarate were all decreased in HK2 cells transfected with Bdh1 siRNA (Fig.6B). Similarly, to that observed in Bdh1 siRNA transfected HK2 cells, both HG and PA treatment could reduce the levels of AcAc, succinate and fumarate, which was successfully blocked by Bdh1 overexpression (Fig.6C-D). Likewise, βOHB supplementation also reversed HG or PA-induced reduction of AcAc, succinate and fumarate (Fig.6E-F). These findings collectively reveal a metabolic flux composed of βOHB-AcAc-succinate-fumarate, which could be regulated by Bdh1 or βOHB and affected the downstream Nrf2 signaling (Fig.6G).
AAV9-mediated Bdh1 renal expression alleviated the progression of DKD
On the basis of the pronounced capacity of Bdh1 to inhibit ROS overproduction and inflammation, we next explored the therapeutic efficacy of Bdh1 expression in db/db mice. The experimental strategy is shown in Fig.7A. At the time point of 11 weeks after injection of the control or Bdh1-encoding virus, we performed ACR assay, which is the most important function indicator of kidney. Notably, although the Bdh1 renal expression didn’t affect the body weight and fasted blood glucose (Fig.S1A-B), we observed significantly lower ACR in AAV9-Bdh1-injected mice than that in the AAV9-Control-injected mice (Fig.7B). To confirm whether mouse Bdh1 was effectively expressed in the kidney using AAV9 vector, we detected the fluorescence intensity of GFP, which was co-expressed with Bdh1. We found that AAV9 encoding mouse Bdh1 was successfully delivered to the kidneys after 4 weeks of injection (Fig.7C). As expected, we observed increased Bdh1 expression in kidneys from AAV9-Bdh1 injected mice than that in AAV9-control injected mice (Fig.7D). In further histological analysis, AAV9-Bdh1 injected db/db mice showed normal morphology of glomerulus, unlike the glomerular hypertrophy in AAV9-control injected mice (Fig.7E). In addition, the DKD pathology-related fibrosis, inflammation and apoptosis were also substantially reduced by AAV9-Bdh1 injection (Fig.7E-G). These findings collectively provide strong support for the promising application of Bdh1 as a therapeutic target in DKD.
βOHB supplementation alleviated the progression of DKD
As βOHB supplementation showed similar effect to Bdh1 overexpression on HG or PA-induced ROS overproduction and inflammation in HK2 cells, we next sought to determine whether βOHB supplementation could ameliorate DKD. The experimental strategy is shown in Fig.8A. At the time point of 6 weeks after supplementation of βOHB by drinking water, we detected the serum level of βOHB and observed increased serum level of βOHB in db/db mice supplied with βOHB (Fig.8B). After that, we performed ACR assay. Although the βOHB supplementation didn’t affect the body weight and fasted blood glucose (Fig.S1C-D), we found that there was significantly lower ACR in db/db mice supplied with βOHB than that with vehicle (Fig.8C). Moreover, the serum level of βOHB was negatively correlated with the value of ACR, indicating a strong ACR reduction capability of βOHB in DKD (Fig.8D). Interestingly, we also observed increased Bdh1 expression in kidneys from db/db mice supplied with βOHB (Fig.8E). In further histological analysis, db/db mice supplied with βOHB showed normal morphology of glomerulus, unlike the glomerular hypertrophy observed in db/db mice supplied with vehicle (Fig.8F). Consistent with AAV9-mediated Bdh1 renal expression, the DKD pathology-related fibrosis, inflammation and apoptosis were also substantially reduced by βOHB supplementation (Fig.8G-H). These results indicate that βOHB supplementation might ameliorate DKD by increasing renal expression of Bdh1, which finally promotes the βOHB metabolism.
Ketogenic diet alleviated the progression of DKD
The KD has been widely used in clinical studies and reported to have an anti-diabetic effect, while the underlying mechanisms have not been fully demonstrated. Given that the major production of KD is βOHB and Bdh1-mediated βOHB metabolism plays protective role in DKD, we next sought to determine whether KD could ameliorate DKD and whether it functions through Bdh1-mediated βOHB metabolism pathway. As shown in Fig.9A, WT or db/db mice were subjected to a standard diet (SD) or KD for 9 weeks, starting at the age of 8 weeks. Although the KD feeding didn’t affect the body weight (Fig.S1E), the fasted glucose was reversed into normal level in KD-fed db/db mice (Fig.S1F). Compared with SD-fed db/db mice, db/db mice fed with KD showed increased blood level of βOHB (Fig.9B). Notably, KD treatment nearly completely reversed the increase of ACR in SD-fed db/db mice (Fig.9C). Interestingly, we observed increased Bdh1 expression again in kidneys from db/db mice fed with KD (Fig.9D). In further histological analysis, db/db mice fed with KD showed significantly pathological remission in kidneys, including fibrosis, inflammation and apoptosis (Fig.9E-G). These results indicate that feeding KD might ameliorate DKD by increasing blood βOHB and renal expression of Bdh1, which finally promotes the βOHB metabolism.