Background
Chronic kidney disease (CKD) has become a crucial public healthcare issue on account of its progressive, irreversible pathological changes and complications including anemia, dyslipidemia and hyperparathyroidism. Previous studies have demonstrated that the metabolism of protein, which mainly comes from daily diet, is involved in most of these adverse pathological changes [
1]. Thus, dietary intervention with restricted protein intake has been proposed as an extremely important therapeutic strategy to delay the progressive decline of renal function and the development of CKD.
As a simple low protein diet is prone to malnutrition, it is often implemented with other nutritional interventions, such as weight control, sodium diet, keto-analogues [
2‐
4]. Keto-analogues contained appropriate proportioned essential amino acids to meet the needs of human synthesis while reducing the production of metabolic waste. The study of keto-analogues combined with protein restriction started since 1967, Richards etc. suggested that a supply of keto-analogues averted the harmful effect produced by the metabolisms of Sulphur and phosphorus contained in natural foods [
5]. Despite some claims that a conjunctive use of α-ketoacids was superfluous in patients with renal insufficiency, more researchers confirmed that the combined use of keto-analogues with protein restriction reduced the metabolic wastes or toxicities in CKD patients [
6‐
9]. Despite keto-analogues administration playing an important role in clinical CKD adjunctive therapy, research focusing on its basic mechanism involved in improving renal disease were quite few. Furthermore, nephrectomy was the most used kidney disease model in animal studies. In Zhang and Wang’s research, keto-analogues supplemented with low protein diet (LPD) were proved to inhibit the intrarenal renin-angiotensin-system to attenuate proteinuria, and regulate Wnt7a/Akt/p70S6K pathway and apoptotic system to improve muscle mass [
10,
11]. In Dongtao Wang’s study, keto-analogues and LPD were observed to protect 5/6 nephrectomised rats by suppression of oxidative damage and mitochondrial dysfunction [
12]. Another paper mentioned that keto-analogues and LPD increased the expression of Kruppel-like factor-15 (KLF15), a transcription factor proved to reduce cardiac fibrosis, thus reduced the severity of kidney disease in a remnant model [
13]. In a diabetic nephropathy model, it seemed that keto-analogues delayed the progression of diabetic nephropathy via inhibition of oxidative stress [
14]. However, the limitation of animal models caused the mechanical explorations to be incomplete and imperfect. More needs to be done to improve the mechanism of keto-analogues intervention in renal protection.
The renal ischemia-reperfusion (IR) injury model confers the advantage of observing renal repair after acute kidney injury while mimicking transient renal ischemia in clinical cases such as multiple organ failure and pre-transplant kidneys, thus offering insight into allograft survival after kidney transplant [
15,
16]. Keto-analogues therapy with LPD provides sufficient essential amino acid intake without protein waste and excess metabolic toxin production and accumulation. Further understanding of its mechanism of action in CKD would be beneficial for expanding its applications.
In this study, we adopted IR model to explore the molecular mechanism of compound α-ketoacid tablets (KA) in renal disease from the angle of inflammation and apoptosis. KA supplementation inhibited nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, resulting in an alleviation of inflammation and apoptosis, thus staving the progression of CKD. The animal experiment gave further evidence to support the reno-protective effects of KA in CKD as seen in our clinical research.
Methods
Animals
Male C57BL/6 mice aged 2-month-old (weighing 25 g) were purchased from Beijing Huafukang Laboratory Animal Technology Co., Ltd, Beijing, China and were acclimated for 1 week. Mice were anaesthetized with 1% sodium pentobarbital (0.01 mL/g, Sigma, USA) by intraperitoneal injection. IR surgery: bilateral kidneys of mice were clamped in 26 min (Roboz Surgical Instrument Co, Germany). Renal blood flow was restored in a few seconds after murine artery clamp was removed. Whole surgeries were performed at 36.6 °C–37.2 °C using a temperature control machine (FHC, USA). Animals were divided into three groups: Sham, IR + Nacl, IR + KA, n = 4–6/group. Mice in sham group underwent the same surgery excluding clamping renal vessels. Mice in IR + Nacl group underwent with IR surgery and were treated with Nacl. Mice in IR + KA group were operated with IR surgery and treated with KA (FreseniusKabi, Germany). Both kidneys were harvested 28 days after IR surgery. All experiments were approved by the Animal Care and Use Committee of Tongji Hospital (IACUC Number: S851) and performed in accordance with NIH guidelines.
KA and LPD preparation and administration
Compound α-ketoacid tablets were confected by mixture of 0.375 g α-ketoacid to 6 mL 0.9% Nacl, to a final concentration of 62.5 mg/mL and were administered by gastric gavage. Administration concentration was 1000 mg per kg mouse weight per day. LPD was supplied by WQJX BIO-Technology (Wuhan, China). The diet was composed of casein (6.5%), starch (66.45%), glucose (10%), soybean oil (7%), fibrin (5%), mineral salt (3.5%), vitamin (1%), l-cystine (0.3%) and choline chloride (0.25%) in accordance with rodent diet of American Dietetic Association, with a calorie of 3.5 kcal/g to meet energy need. In IR + KA group, mice were treated with KA and LPD. In comparison, mice in the IR + Nacl group were treated with 0.9% Nacl and LPD.
BUN, TC and TG detection
Blood urea nitrogen (BUN), total cholesterol (TC) and triglyceride (TG) were tested using kits (Changchunhuili, China) according to the manufacturer’s instructions.
Histological, immunocytochemical and immunofluorescent staining
Bilateral kidneys of mice were fixed in 4% paraformaldehyde and embedded in paraffin. The paraffin-embedded kidneys were sectioned at 3 μm. Pathological staining for Periodic Acid-Schiff staining (PAS), and Sirius red were performed to evaluate renal tubular injury grade and interstitial fibrosis. For immunofluorescent (IF) staining, sections were stained with primary antibody Kim-1 (1:800; R&D), lotus tetragonolobus lectin (LTL, 1:100, Vector Lab), alpha-smooth muscle actin (α-SMA, 1:100, Abcam), Collagen I (1:100, Abcam), CD45 (1:50, Biolegend), CD3 (1:50, Guge, Wuhan) and Ly6G (1:50, Biolegend) at 4 °C overnight and fluorescent labeled secondary antibodies. Nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI). Data was analyzed by Image Pro Plus software (Media Cybemetics, Rockville, MD, USA) in a blinded manner.
Western blot
Renal tissues were lysed in RIPA lysis buffer (Promoter, Wuhan, China) containing protease inhibitor (Promoter, Wuhan, China). Cell proteins were collected in the same way. Total protein concentration was determined using a BCA protein assay kit (Promoter, Wuhan, China). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (PVDF, Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk in TBS with 0.1% Tween-20 for 1 h at 37 °C and then probed with antibodies against α-SMA (1:5000, Abcam), PDGFR-β (1:3000, Abcam), Fibronectin (1:1000, Abcam), Bcl-2 (1:1000, Abclonal), BAX (1:1000, Abclonal), Caspase 3 (1:1000, Abclonal), NFATc-1 (1:1000, Abcam), total and phosphorylated-65, -38 and -ERK (1:1000, cell signaling technology, USA) and GAPDH (1:4000, Abbkine) at 4 °C overnight. The blots were washed next day and incubated with HRP-conjugated secondary antibodies for 1 h at 37 °C. The signal intensities of targeted band were quantified using Image J (NIH, USA).
Quantitative real-time PCR
Total RNA was extracted using Trizol reagent according to the manufacturer’s instructions (Invitrogen, USA). One microgram RNA was reverse transcribed into first strand cDNA using the GoScript reverse transcription system (Promega, USA) in a 20 μL reaction system. The cycling parameters were used as follow: 40 cycle pf denaturation at 95 °C for 15 s and annealing at 60 °C for 60 s. Triplicated experiments for each sample were processed. Quantitative PCR was conducted using SYBR master mix (Qiagen, Germany) on the Roche light 480II. The mRNA expression levels of several markers, including TGF-β, fibronectin and Collagen I were conducted via the comparative cycle threshold (Ct) method and normalized to the expression levels of GAPDH. Primer sequences were listed in Table
1.
Table 1
Primer sequences for qRT-PCR [
52]
GAPDH | Forward primer: 5′-TTGATGGCAACAATCTCCAC-3′ Reverse primer: 3′-CGTCCCGTAGACAAAATGGT-5′ |
TGF-β | Forward primer: 5′-CTTCAATACGTCAGACATTCGGG-3′ Reverse primer: 3′-GTAACGCCAGGAATTGTTGCTA-5′ |
Fibronectin | Forward primer: 5′-GCTCAGCAAATCGTGCAGC-3′ Reverse primer: 3′-CTAGGTAGGTCCGTTCCCACT-5′ |
Collagen I | Forward primer: 5′-ATGGATTCCCGTTCGAGTACG-3′ Reverse primer: 3′-TCAGCTGGATAGCGACATCG-5′ |
A total number of 894 CKD outpatients and inpatients were recruited from April 2015 to September 2018 in Tongji hospital, Tongji medical college, Huazhong university of science and technology for cross-sectional and retrospective study. A total number of 452 patients were excluded, (1) hemodialysis, peritoneal dialysis or renal transplantation at the beginning of enrolled time; (2) the follow-up time was less than 2 times; (3) basic data substantially missing. Among the remaining enrolled 442 patients, 119 were at stage 1 and 2, only 9 patients were prescribed KA, which greatly influenced the result of analysis. For the remaining 323 patients at stage 3 to 5, 148 patients took KA were divided into KA group, the other 175 patients were divided into No-KA group. The clinical study was approved by Medical Ethics Committee of Tongji Hospital, Tongji medical college, Huazhong University of Science and Technology (TJ-IRB20180501). We registered in Chinese Clinical Trial Registry (ChiCTR1800016536).
Statistical analysis
Data was expressed as count value (%) for categorical variables and mean ± SD for continuous variables. Comparisons between the two groups were made by nonparametric T test for continuous variables while χ2 test for categorical variables. Kaplan–Meier analysis and COX proportional hazard regression model were adopted to calculate the cumulative probability to reach the end-point and hazard ratio of renal function deterioration. The statistical analyses were performed by SAS v9.4 (SAS, Inc., 200 Cary, NC, USA), and all P-values calculated as two-sided. The association was considered significant with p-values less than 0.05.
Discussion
In this study, we adopted IR-induced renal injury model to observe chronic progression of renal tubular injury and interstitial fibrosis. Firstly, we found that KA reduced IR-induced abnormal serum BUN concentration as well as total triglycerides with statistically significant difference, improved renal function and hyperlipidemia in mice. Kidneys play an important role in the protein reabsorption and excretion of protein metabolites. Renal injury causes abnormal protein metabolism, which can affect enzyme-related protein synthesis and metabolism, resulting in hyperlipidemia [
25,
26]. KA acted as an essential protein supplement and alleviated hypoproteinemia and protein synthesis in liver, we inferred from our results that KA might increase the expression of lipoprotein lipase, hepatic lipase and very low-density lipoprotein receptor, and as a result increase triglycerides clearance and decrease their formation in liver, thereby modifying the hemostasis of triglyceride. KA also caused a slight but statistically insignificant improvement on total cholesterol compared to the IR group. This might be due to the different metabolic mechanisms involved for cholesterol and triglycerides. Cholesterol biosynthesis is dependent on the enzyme activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) in liver. In chronic kidney disease both the expression and activity of HMG-CoA showed a marked increase [
27]. The reduction of hypercholesterolemia induced by renal injury depends on the inhibition of HMG-CoA, such as statins.
Secondly, we observed an anti-inflammatory and -apoptotic effect of KA in mice. As reported, at the early stage of renal injury, inflammatory response is a protective measure to attenuate injury and promote repair. When inflammatory infiltration is uncontrolled, abundant inflammatory cells recruited from circulation or resident cells accumulate in the tubular interstitium, producing massive amounts of proinflammatory molecules, such as IL-1β, IL-6, tumor necrosis factor-α, and dysregulated renal immunoreaction further deteriorates renal injury [
21,
28]. Apoptosis is another cause accounting for renal injury. Proximal tubular epithelial cells are highly susceptible to IR injury, the necrotic or apoptotic tubular cells release harmful molecules, form cell casts, resulting in renal obstruction which further exacerbates renal injury [
29]. NF-κB pathway was widely reported as transcription factors participating in renal inflammation and apoptosis by regulating the expression of some pro-inflammatory cytokines and chemokines genes [
30‐
35]. It was shown that the MAPK signaling pathway, which includes ERK and p38 kinase, is activated by inflammatory stimuli and involved in regulating p65 NF-κB activation to promote inflammation [
36]. In the present study we observed increased inflammatory T cells and neutrophils infiltration and tubular apoptosis in IR group as well as activation of NF-kB and MAPK signaling pathways. KA administration alleviated inflammatory infiltration of mature T cells and neutrophils in mice. Also, tubular apoptosis caused by IR was attenuated in KA group too, exhibiting the anti-apoptotic effect of KA treatment. Furthermore, NF-kB and MAPK signaling pathways were significantly inhibited in the IR + KA group too, as well as downstream molecule NFATc-1. We inferred that the decrease of inflammation and apoptosis brought by KA administration might have some links with an inhibited activation of NF-κB and MAPK pathways. As MAPK are catalytically inactive in base condition and their activation requires specialized enzyme to phosphorylate their activation loops. KA might inhibit MAPK pathway by the reduction of associated phosphatases. NF-κB is distributed in the cytoplasm and translocates into nucleus when stimulated, KA might inhibit the NF-κB pathway activation by reduction of stimulating factors or the receptor activator of NF-κB, or by the inhibition of MAPK pathway. Further studies to explore the detailed mechanisms of KA need to be done.
In addition, we explored the effect of KA intervention on CKD patients. Although numerous clinical trials on the effect of KA and LPD on CKD were conducted in past years, the results were controversial [
37‐
40]. Some studies found that the potential benefits of KA and LPD were mainly focused on reducing waste products of protein metabolism, such as modification of creatine concentration. However, their influence on renal function protection or alleviating CKD progression required more careful and adequate attention [
37,
41‐
44]. The uncertain effect of protein restriction on CKD protection might be related to the progressive and irreversible characteristic of CKD, especially CKD patients at stages 4 and 5. Most patients recruited in KA and LPD studies were with eGFR less than 30 mL/min, or in a pre-dialysis state [
8,
45,
46]. Their worse physical conditions made KA and LPD intervention only an adjunctive therapy to main therapy such as glucocorticoids and immuno-biologicals. In the meantime, different pathological types showed inconsistent progression rates, for example CKD development in patients with membranous nephropathy exacerbated more rapidly than in patients with mesangial proliferative nephritis. Different degrees or types of pathological nephropathies could also influence the results of clinical study. Furthermore, as the study on KA and LPD was conducted over a long time period, often more than 6 months, the compliance of patients recruited was especially important. An unstrict administration of LPD could influence the final result of the clinical study.
Our clinical research collected data from CKD patients from 2015 to 2018 in Tongji hospital, which partially represent the population in the area of central China along the Yangtse River. Among all the CKD patients enrolled, 119 were at CKD stages 1 and 2, only 9 patients were prescribed with KA, compared to 323 at stages 3 to 5, with 148 prescribed with KA. Thus, only CKD patients at stages 3 to 5 were taken into consideration. Our next step was to analyze the renal function between KA and No-KA group. There was a great difference in baseline renal function indexes between KA and No-KA groups in patients at CKD stage 3, patients on KA appeared to be in worse renal conditions. We speculated this may be due to the deep relationship between eGFR and the accumulation of metabolic protein waste. The lower the eGFR was, the less efficiently protein wastes were excreted from our body and the greater the accumulation of waste and toxicities, the more urgent the need for a protein intake intervention. Thus, patients at stages 3 to 5 were more often prescribed KA treatment to reduce renal load than patients at earlier stages. Using early research as reference, we defined a reduction of > 50% eGFR as an end-point event. The patients with dialysis or renal transplantation at the time of enrolment were excluded. According to Kaplan–Meier analysis, the cumulative probability to reach the end-point was lower in the KA group than the No-KA group among patients at stages 4 and 5. Otherwise, according to our data the age of patients at CKD stages 3 to 5 peaked at middle age, mainly between 34 and 60 years old (Table
2). It presented a probable protective effect of KA on CKD middle aged patients at stages 4 and 5, which meant KA was a protective factor in staving CKD progression. Our study implied that a supplement of KA could defer the progression of CKD in patients at stages 4 and 5, which was consistent to the animal experiment results and previous research [
2,
47‐
49].
CKD is a chronic progressive disease which could affect the whole-body system and finally irreversibly enter end-stage kidney stage [
50,
51]. Finding a way to slow down the development of chronic renal injury was important for both family and social burdens. This study demonstrated the effect of KA on IR-induced murine CKD, by inhibiting the activation of NF-κB and MAPK pathways, thereby alleviating inflammatory infiltration and apoptosis, finally attenuating renal tubular injury and interstitial fibrosis. Meanwhile, a clinical trial was conducted to demonstrate its effect on delaying CKD progression. There were still some limitations to which we are still determining the best approach: (1) In animal experiments, there was no evidence to prove that the inhibition of NF-κB and MAPK directly attenuated inflammation and apoptosis. As NF-κB and MAPK pathways participate in several physical and pathological processes, inhibitors of these two pathways might be needed to demonstrate the direct relationship between the pathways and inflammation and apoptosis caused by KA. (2) In clinical trials, apart from KA, other medications were not strictly consistent. (3) Human inflammatory and apoptotic factors need to be measured for a further demonstration of anti-inflammatory effect from KA. More longitudinal studies are required to perfectly support the anti-inflammatory and -apoptotic effects of KA and LPD on CKD progression.
Authors’ contributions
RZ and YY designed the study; HZ, MW, HX and HZ carried out experiments. Data analysis was carried out by MW, Li Li and FZ. Reagents were contributed by OLCLS and HZ, and the mice were managed by HX, and ZZ. Clinical patient information was collected by WL, KQ and CZ and the paper was written by MW, OLCLS and HG. All authors read and approved the final manuscript.