Clam Peptides Attenuated Adenine-Induced Chronic Kidney Disease by Improving Inflammation, Oxidative Stress, and Intestinal Flora Composition

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Introduction
Kidney is an important organ responsible for metabolism in the human body, which is responsible for removing endogenous waste and maintaining acid-base and electrolyte balance in the body [1].Numerous adverse factors, such as environmental pollution, family medical history, drug abuse, and metabolic diseases, will lead to chronic kidney disease (CKD) [2], which is defned as a sustained damage of renal parenchyma leading to chronic deterioration of renal function [3].According to statistics, the incidence of CKD is continuously increasing around the world, and if not treated in time, it will develop into chronic renal failure, which will further weaken the detoxifcation ability of the kidney, leading to acid-base imbalance and endocrine disorders in the body [4].
At present, drug treatment is still the main therapeutic approach for CKD in clinic.However, drug treatment is always accompanied with side efects [5,6].For instance, long-term use of angiotensin-converting enzyme inhibitors may induce adverse reactions such as cough and angioedema [7].Te recurrence rate after cyclosporine treatment is more than 50%, and it can cause hypertension and nephrotoxicity [8].Allopurinol and its derivatives may lead to skin allergy and other adverse reactions [9].Tus, it is an inevitable trend to fnd safe and sideefect-free natural food source products to help with the treatment of kidney disease.Among them, increasing evidence suggested that bioactive peptides play an efective role in the maintenance of renal function.For example, soy peptide attenuated renal dysfunction through anti-infammation in adenineinduced CKD in mice [10].Perilla peptides alleviated renal oxidative stress and chronic infammation to improve adenine-induced CKD in mice, thereby improving renal injury [11].
Ruditapes philippinarum (R. philippinarum), commonly known as the clam, is widely distributed and ranks second among the bivalves in aquaculture worldwide [12].It has been shown that peptides obtained after hydrolysis of R. philippinarum are biologically active in many aspects such as antioxidant activity, anticancer [13], antibacterial [14], and antihypertension [15].In our previous study, we prepared peptides from R. philippinarum (RBPs) by Bacillus natto liquid fermentation.Briefy, 5% of total volume (v:v) Bacillus natto and 10% sucrose (compared to dried clam meat powder (w:w)) were added into the substrate of R. philippinarum (1 : 25 ratio of dried clam meat powder-to-water (w:w)) for fermentation at 45 °C for 24 h.Subsequently, the fermentation products were centrifuged to separate the supernatant, which was freeze-dried to obtain RBPs [16].We found RBPs signifcantly reduced blood pressures and restored renal injury in SHRs [16].Inspired by these fndings, we hypothesized that RBPs may improve adenine-induced CKD.
Te adenine-induced CKD rodent model has much in common with the metabolic abnormalities of human CKD and has been frequently utilized to study the pathological basis of CKD [17,18].Terefore, in this study, we established an adenine-induced rat model of CKD to explore the improvement efect of RBPs on kidney damage and to provide a basis for high-value use of shellfsh resources to produce marine drugs or functional foods that improve kidney function.

Experimental Animal.
Male Sprague-Dawley (SD) rats (160-180 g) were purchased from Spafu (Beijing) Biotechnology Co., LTD.(SCXK (Beijing) 2019-0010).Te feeding environment was 23-25 °C, the relative humidity was 55-60%, and the light cycle was 12 h: 12 h.Te experimental animals used in this study were all in accordance with international animal care guidelines and were approved by the Experimental Animal Center of Qingdao University.

Experimental Design.
Fifty-four SD rats were kept in an environment of temperature (22 ± 3 °C), humidity (65 ± 5%), and dark/light cycle for 12 h for one week.After the adaptation period, all rats were divided into two groups: normal group rats (Con, n � 9) gavaged with normal saline and model group rats (Mod, n � 45) gavaged with adenine (250 mg•kg −1 •d −1 ).Both groups had free access to a normal diet.Te systolic blood pressure (SBP) and the diastolic blood pressure (DBP) were measured by BP-2010A (Beijing Soft Invasive Biotechnology Co., Ltd.) at 2-week intervals [17].After 3 weeks of the model building period, 6 model rats and 3 normal rats were randomly selected and sacrifced.Blood samples were collected from the abdominal aorta to detect the content of urea (UR), creatinine (CR), and uric acid (UA) in serum, and the model establishment was evaluated combined with the change in blood pressure.Te rats with successful modeling were divided into fve groups: model group (Con), orally gavaged with 10 mg•kg −1 •d −1 saline each day; SSN-treated positive control group (SSN), orally gavaged with 25 mg•kg −1 •d −1 SSN each day; and low-, medium-, and high-dose clam peptide-treated groups (RBPs-L, RBPs-M, and RBPs-H), orally gavaged with RBPs (50, 100, and 200 mg•kg −1 •d −1 ).In the meanwhile, all groups were given a normal diet and intervened for 8 weeks.Teir body weight was measured at the beginning of the trial and weekly thereafter.

Specimen Collection.
After continuing treatment for 8 weeks, all rats were euthanized after 12 h fasting.Blood was collected from the abdominal aorta and centrifuged at 3500 r/min for 15 min to separate the serum.Te heart, kidney, and liver were separated and weighed.Te excised right kidney and part of the left kidney were wrapped in tinfoil and immersed in liquid nitrogen and frozen at −80 °C until biochemical analysis.A small piece of the left kidney was placed in 4% formaldehyde fxative for histopathological examination.After the last blood pressure measurement, feces were collected from rats in a sterile environment.All serum, tissue, and feces were stored below −80 °C until use.

Body Weight and Organ Index.
During the treatment period of drug administration, the body weight of the rats was weighed weekly, and the body weight of the rats before sacrifce was recorded.After the rats were sacrifced, the kidneys, livers, and hearts of the rats were washed in 0.9% normal saline, wiped with flter paper, and weighed, and the organ index was calculated.Te calculation formula is as follows: organ index � organ weight (g)/body weight (g).

Biochemical Tests in Plasma.
Te levels of NGAL, CR, Ca, UA, IP, blood urea nitrogen (BUN), and cholesterol (CHO) were measured by using an automatic biochemical analyzer.Te levels of TGF-β1, IL-1β, NGAL, L-FABP, and Cystatin C in serum were detected by ELISA.

Histopathological Analysis of Renal Tissue.
After immersion in 10% formalin for 2 days, dehydrated with alcohol, parafn-fxed sections were stained with hematoxylin (HE) for histopathological analysis.Immunohistochemical sections were incubated with dialysate and blocking bufer, Journal of Food Biochemistry with antibodies targeting genes, sequentially treated with peroxisome coupled streptavidin (Nichirei Co.) and 3, 3-diaminobenzidine tetrahydrochloride (DBA reagent), and fnally stained with hematoxylin, and photographs were taken using a microscope.[16].
2.8.Gut Microbial Sequencing.All fecal samples were tested for gut microbial 16S rRNA by Shanghai Personal Biotechnology Co., Ltd., and high-throughput sequencing was performed on the Illumina NovaSeq platform.
2.9.Statistical Analysis.All results were expressed as the mean ± standard deviation, and one-way analysis of variance and Turkey's test were performed by using SPSS 18.0 statistical software.p < 0.05 and p < 0.01 were considered signifcant and had highly signifcant diferences.

Basic Information for the Model Being Successfully Built.
At the end of three weeks, three rats from the Con and Mod groups were randomly selected and sacrifced for determination of UR, UA, and CR contents, and the results were shown in Figure 1.Te results showed that adenine gavage treatment signifcantly increased the levels of UR, UA, and CR in serum (Figures 1(a)-1(c), p < 0.05), indicating that the CKD rat model was successfully established.Besides, their blood pressure was measured twice a week during the model building period, and the results were shown in Figures 1(d) and 1(e).Te blood pressure of Mod rats was higher than that of normal rats, and there was a signifcant time-response relationship (p < 0.05).Terefore, this proved the success of modeling from another aspect.

RBPs Ameliorated Blood Pressures in CKD Rats.
Blood pressure changes in rats during the 8-week RBPs intervention were shown in Figure 2. Te SBP (a) and DBP (b) were dramatically higher in the Mod group than those in the Con group (p < 0.05).Te blood pressure of rats in RBPs and SSN-treated groups showed a decline trend with the increase in administration days and gradually returned to the normal blood pressure range.At the end of treatment, SBP and DBP in the SSN and RBPs groups were lower than those in the Mod group (p < 0.05).Furthermore, a dosedependent response was found in RBP-treated groups.

Efects of RBPs on Growth Parameters in CKD Rats.
After successful modeling, the body weight of the Mod group was signifcantly lower than that of the Con group (p < 0.05), while there was no signifcant diference among the Mod group and the treatment groups (p > 0.05).After 8 weeks of treatment, changes in the body weight of the rats as well as organ indices were shown in Table 1.Te body weight of the rats in the Mod group increased slowly, while that in the other treatment groups dramatically increased compared with the Mod group (p < 0.05).For organ indexes, the kidney, liver, and heart indexes of Mod rats increased by 53.73%, 14.29%, and 16.13%, respectively, compared with the Con group (p < 0.05).Te kidney index, liver index, and heart index of the RBPs-H group were lower than those of the Mod group (p < 0.05).Although the organ indexes of SSN, RBPs-L, and RBPs-M groups showed a decreasing trend, there was no signifcant diference.

Efect of RBPs on the Renal Histopathology of CKD Rats.
As shown in Figure 3(a), the kidneys of the normal group were in the shape of equine bean, with nail color obvious luster.In the model group, the exterior of the kidney was covered with white granules, and the whole kidney showed a frost-like appearance.After RBPs treatment, there was a trend of redness and a dose-dependent relationship.Te therapeutic efect of the positive drug group was similar to that of the RBPs-M group, and the treatment efect of RBPs-H group was the most signifcant.Te efect of RBPs on renal histopathology in rats was shown in Figure 3(b).Compared with the Con group, the renal tissue of the Mod group showed brown crystal deposition in the renal tubulointerstitial, a large number of dilated renal tubules, mesangial proliferation, glomerular atrophy, and infammatory cell infltration in the glomerulus.After administration, the above pathological changes were alleviated in the SSN group and high-, medium-, and low-dose RBPs groups.Te brown crystals disappeared in the RBPs-M group and even completely in the RBPs-H group, and the renal tissue morphology was similar to that in the Con group.So the therapeutic efect of RBPs on renal tissue injury was dosedependent.

Journal of Food Biochemistry
Te results of serum infammatory factors were shown in Figures 5(d)-5(f ), Compared with the Con group, the levels of IL-1β, TGF-β1, and TNF-α in serum of the Mod group were signifcantly increased (p < 0.05).Both RBPs and SSN groups reduced the levels of IL-1β, TGF-β1, and TNF-α compared with the Mod group (p < 0.05).A dose-dependent relationship was noticed among the three RBPs groups.

Efects of RBPs on Gut Microbiota in CKD Rats
3.7.1.Sequence Splicing and Assembly.All stool samples in the six groups were sequenced with the use of Illumina sequencing platforms.A total of 2,218,135 qualifed sequences were detected, which could be clustered into 24,430 OTUs for species classifcation under 97% similarity.Based on the annotation results of OTUs, 299 phyla, 52 classes, 1456 orders, 8040 families, 22097 genera, and 2189 species were obtained from the total samples of the 6 groups, as shown in Table 2.

Venn Analysis.
To identify the number of common and unique OTUs between the experimental groups, Venn analysis was performed.As shown in Figure 6(a), there were 161, 186, 286, 328, and 375 OTUs overlapped with the Con group in Mod, SSN, RBPs-L, RBPs-M, and RBPs-H groups.A dose-dependent increasing trend was also observed in the RBPs groups.Tese results indicated that both RBPs and SSN increased the similarity of OTUs components to normal rats.6

Alpha Diversity
Journal of Food Biochemistry  Journal of Food Biochemistry group, indicating decreased abundance and diversity of microbiota.With the increase of the RBPs dose, species diversity and colony richness in the RBPs group gradually increased, especially in the RBPs-H group, which was higher than that in the Mod group (p < 0.05).Terefore, RBPs can enhance the diversity of gut bacteria to a certain extent.

Beta Diversity Analysis-NMDS Analysis.
Beta diversity analysis was used to determine diferences in the structure of intestinal microbiota among experimental groups via NMDS analysis.Te closer the face-space distance of each point, the smaller the diference in gut microbiota between groups.As shown in Figure 6(d), there was no overlap and a long distance between the Con and Mod groups, indicating that there was a signifcant diference in the composition of intestinal fora between the groups.Tis was followed by the SSN group, while the RBPs group was closer to the Con group, indicating that both SSN and RBPs signifcantly improved the gut microbiota composition in CKD rats. Figure 7(e) showed the most abundant intestinal fora at genus levels in each group.Compared with the Con group, Muribaculaceae and Lactobacillus decreased and Romboutsia, Ruminococcaceae_UCG-005, and Christensenella ceae_R-7_group increased in the Mod group (p < 0.05).Intervention of RBPs decreased the relative abundance of Romboutsia and Ruminococcaceae_UCG-005 compared to the Mod group (p < 0.05).Compared with the Mod group, the RBPs-M group displayed higher levels of Lactobacillus (p < 0.05).Te relative abundance of Muribaculaceae was higher, and that of Christensenellaceae_R-7_group was lower in the RBPs-H group than in the Mod group.Compared with the Mod group, the SSN group displayed lower levels of Romboutsia, Ruminococcaceae_UCG-005, and Christensenellaceae_R-7_group and higher levels of Lactobacillus (p < 0.05) (Figures 7(f )-7(j)).

Discussion
A novel peptide named RBPs was prepared in our previous research by fermentation of R. philippinarum with Bacillus natto [19].In this study, we proved for the frst time that RBPs had signifcant therapeutic efects on adenine-induced CKD.Mechanism studies have shown that RBPs may repair renal injury by regulating the balance of oxidative status and infammation and improving intestinal fora in CKD rats.
In previous studies, induction of CKD with adenine has been recognized as one of the most successful methods [15].Te efect of adenine-induced CKD is signifcant and conducive to observe.Long-term gavage of adenine can lead to chronic kidney injury similar to human, which is characterized by signifcant elevation in the serum levels of renal injury markers like CR, BUN, and UA [20].In vivo, adenine is reduced to 2, 8-dihydroxyadenine, which enhances pressure on the capillaries around them by blocking the renal tubules, leaving them in a state of hypoxia, and then attaches to the renal tubules in the form of deposits, causing damage of renal tubular epithelial cells and leading to kidney diseases such as infammation and fbrosis [21].In this study, adenine was used to successfully induce the occurrence of CKD in rats.Subsequently, we found that RBPs protected against adenine-induced CKD in rats.In the body, UA can cause oxidative stress and infammation, and the kidney is mainly responsible for the regulation of UA levels.Serum UR and CR are the most common indicators to assess CKD status [22].In general, the kidney will exclude excess UA, CR, and UR in the body [11].When the kidney is damaged, the blood levels of UA, CR, and UR will increase due to the inability to excrete them in time.NGAL is frst found in the granules of human neutrophils, and its content is signifcantly increased in the blood during infammation in many organs, like the kidney.L-FABP, as a lipid-binding protein, can bind fatty acids and transport them to mitochondria to provide energy for renal tubular cells.Both NGAL and L-FABP are markers used to predict the occurrence of early kidney injury [23,24].In addition, Cystatin C, as an endogenous cysteine peptidase inhibitor, has been intensively investigated as a biomarker of kidney function for many years [25].When renal function is impaired, Cystatin C is freely fltered in the glomeruli and increased in blood.After RBP treatment, the elevated blood levels of UA, CR, UR, L-FABP, NGAL, and Cystatin C in the CKD rats were all signifcantly decreased, indicating the efectively protective efect of RBPs on kidney injury.Numerous previous reports indicated that peptides played CKD alleviative roles.For instance, Perilla peptides ameliorated kidney diseases by improving renal cell apoptosis and oxidative stress and maintaining intestinal barrier [11].Soy peptide alleviates renal injury in hypertensive rats by activating the SIRT1-PGC1α/Nrf2 pathway [26].Our study was the frst to prove the CKD protective efect of peptides from R. Philippinarum.
Te exact mechanisms of CKD are complex and largely unknown.However, it has been shown that CKD is closely related to oxidative stress and infammatory response [27].Oxidative stress and infammation are linked with CKD by oxygen-derived free radicals, and the main pathological mechanism of which is characterized by the activity of intracellular and extracellular oxygen-derived free radicals causing kidney damage and then triggering an infammatory response.First, free radicals usually interact with the molecular components of the nephron [28], causing a series of kidney damage reactions, such as lipid peroxidation of the cell membrane leading to membrane deactivation [29], kidney DNA breaking down and cross-links causing harmful mutations [30,31], and oxidation of amino acids preventing some important functions [29].Second, since free radicals interact with the nephron to produce secondary free radicals with the same harmful efects, the nephron will continue to be damaged [32], and the infammatory response will stimulate the formation of additional free radicals.Te damaged neutrophils were transferred across the membrane through the nicotinamide adenine dinucleotide phosphate oxidase system and coupled with molecular oxygen to produce superoxide [33].Superoxide and free and their targets continue to promote persistent renal infammation [34].Infammatory signals promote the generation of reactive oxygen species (ROS) and cause nephron damage [31].Finally, with long-term efects, the nephron interacting with free radicals starts to degrade gradually, and renal damage becomes more and more pronounced [35].
After adenine-induced CKD being built in this study, the activity of CAT, an antioxidant enzyme in renal tissue, thus caused peroxidative damage in renal tissue [36].MDA is a product of peroxidation.Te Serum T-AOC level is a commonly used indicator to refect the overall antioxidant capacity in an organism [37].After RBPs treatment, the serum level of MDA was increased and that of CAT and T-AOC in the CKD rats was enriched, indicating the antioxidant capacity was enhanced and renal oxidative damage was improved.TGF-β1 plays a regulatory role in renal fbrosis, and its level will increase during the development of CKD [38].IL-1β and TNF-α as proinfammatory factors will also show an upward trend [39].After RBPs treatment, the contents of IL-1β, TGF-β1 and TNF-α were decreased in the serum of CKD rats, indicating the notable antiinfammatory efects of RBPs.Our results suggested that RBPs alleviated CKD by inhibition oxidative stress and infammatory response.Te gut is rich in microbes that play critical roles in maintaining host health [40].When renal function is impaired, it is often accompanied by changes in the composition of intestinal fora [41], and in turn, the changes in intestinal fora promote the development of CKD [42].It has been shown that Firmicutes are positively correlated with plasma CR and BUN levels, while Bacteroidetes are negatively correlated with them [43].Te F/B ratio is usually used as a marker refecting the condition of gut microbiota [44].In our research, Firmicutes showed an upward trend in feces of CKD rats, while Bacteroidetes showed a downward trend, and the ratio of Firmicutes to Bacteroidetes (F/B) increased, which was consistent with previous reports [45].After RBP treatment, the proportion of Firmicutes at the phylum level was reduced and the proportion of Bacteroidetes and F/B was increased, suggesting that RBPs could act by regulating the proportion of the microbiota.At the genus level, higher abundance of the genus Muribaculaceae was associated with lower levels of serum proinfammatory factors [46].Romboutsia, Christensenellaceae_R-7_group, and Ruminococca-ceae_UCG-005 were reported to be enriched in the feces of CKD animals and were positively correlated with infammation and oxidative stress [47][48][49].In our study, RBPs treatment increased the fecal levels of Muribaculaceae and decreased Romboutsia, Christensenellaceae_R-7_group, and Ruminococcaceae_UCG-005, which are in line with the protective efect on CKD.Lactobacillus is benefcial bacteria that produce short-chain fatty acids (SCFAs).SCFAs can protect the kidney by preventing oxidative stress and infammation in the kidney [50].Te proportion of Lactobacillus in the feces was increased after RBPs treatment, supporting the benefcial efect of RBPs on intestinal health.In addition, the correlation analysis also indicated that the modulated bacteria were signifcantly correlated with CKDrelated indices.Benefcial bacteria (Bacteroidetes, Muribaculaceae, and Lactobacillus) were negatively correlated, while destructive bacteria (Firmicutes, Romboutsia, Christe nsenellaceae_R-7_group, and Ruminococcaceae_UCG-005) were positively correlated with kidney injury, oxidative stress, and infammation.Overall, our fnding suggested that the improvement efect of RBPs on adenine-induced CKD 12 Journal of Food Biochemistry might be at least partially attributed to the modulation of intestinal fora.

Conclusions
In conclusion, RBPs could improve CKD by protecting the structure of the kidney and reducing the serum levels of NGAL, CR, Ca, UA, TG, IP, L-FABP, BUN, CHO, and Cystatin C. Potential mechanisms might be lying in the enhancing of antioxidant capacity and anti-infammation properties and restoring the composition of intestinal fora (as shown in Figure 9).Our results led novel insights for exploring RBPs as functional foods for CKD treatment.

Figure 1 :
Figure 1: Changes of CKD-related indices in rats after adenine administration.(a) Serum levels of urea nitrogen (UR).(b) Serum levels of urea nitrogen (UA).(c) Serum levels of urea nitrogen (CR).(d) Changes in systolic blood pressure within three weeks of modeling.(e) Changes in diastolic blood pressure within three weeks of modeling (all data were presented as the mean ± SD; * p < 0.05 vs. the Con group).

Figure 2 :
Figure 2: Efects of RBPs on blood pressure in rats during the eight-week treatment period.(a) Changes in systolic blood pressure in rats during the 8-week treatment period.(b) Changes in diastolic blood pressure in rats during the 8-week treatment period.All data were presented as the mean ± SD.Compared with the model group, * p < 0.05; * * p < 0.01.Con: normal group; Mod: model group; SSN: Shenshuaining capsule-treated group; RBPs-L group: low-dose clam peptide-treated groups; RBPs-M group: medium-dose clam peptidetreated groups; RBPs-H group: high-dose clam peptide-treated groups.

Figure 3 :Figure 4 :
Figure 3: Efect of RBPs on the renal morphology and pathology of CKD rats.(a) Morphological change of rat kidneys from six experimental groups.(b) RBP treatment restored glomerular morphological changes (black arrows) and infammatory infltrates caused by renal injury (red arrows) in CKD rats.Te scale bar was 100 µm.
Analysis.Alpha diversity analysis indices were used to assess the species diversity of the microbial communities in experimental groups.Te Chao1 index refects the community richness, and the Shannon index shows the community diversity.As shown in Figures6(b) and 6(c), compared with the Con group, the Chao1 index and Shannon index were lower in the Mod

3. 7 . 5 .
Gut Microbiota Composition.Te top 10 phyla were shown in Figures7(a)-7(d).Firmicutes and Bacteroidetes were the two most important phyla.Te decreased proportion of Bacteroidetes and the increased relative abundance of Firmicutes were found in the Mod group compared with the Con group.Compared with CKD rats, the proportion of Bacteroidetes at the phylum level was signifcantly increased after RBPs-M and RBPs-H treatments (p < 0.05), while Firmicutes signifcantly decreased (p < 0.05).No signifcant change was observed among RBPs-L and SSN on these two phyla.Furthermore, the ratio of Firmicutes and Bacteroidetes (F/B) was calculated.Both medium-and high-dose RBPs treatments reduced the elevated ratio of F/B in the Mon group (p < 0.05).Tere were no signifcant diferences in other phyla among the groups.

Figure 9 :
Figure 9: Potential mechanisms of the RBPs improving adenine-induced chronic kidney disease.

Table 1 :
Efects of RBPs on body weight and organ indices in CKD rats.

Table 2 :
Sequencing information of intestinal bacteria in each group.