Chronic kidney disease (CKD) is marked by a progressive and irreversible decline in kidney function and poses a significant public health issue. Its global prevalence is high, with approximately 10 % of adults worldwide at some stage of the disease, leading to around 1·2 million deaths annually(Reference Bikbov, Purcell and Levey1,Reference Kalantar-Zadeh, Jafar and Nitsch2) . Several hallmarks of CKD are associated with disease progression, including oxidative stress, inflammation, uremia and gut dysbiosis(Reference Mihai, Codrici and Popescu3–Reference Altamura, Pietropaoli and Lombardi5).
In patients with CKD, mitochondrial dysfunction and increased reactive oxygen species production are major contributors to elevated oxidative stress(Reference Daenen, Andries and Mekahli6–Reference Vida, Oliva and Yuste8). Excessive production of reactive oxygen species contributes to inflammation by activating nuclear factor-κB (NF-κB), which promotes the production of cytokines such as IL-1β, IL-6, IL-18 and TNF(Reference Liu, Zhang and Joo9,Reference Kishi, Nagasu and Kidokoro10) . Another signalling complex involved in the induction of renal inflammation and fibrosis in CKD is the NLR family pyrin domain-containing 3 inflammasome, which activates caspase-1 and stimulates the release of IL-1β and IL-18. This complex is linked to a type of programmed inflammatory cell death known as pyroptosis(Reference Huang, Zhang and Zhang11,Reference Henedak, El-Abhar and Soubh12) . Inflammation can alter the regulation of intrarenal microcirculation and perfusion, leading to renal damage, fibrosis and the progression of CKD(Reference Mihai, Codrici and Popescu3,Reference Liu, Lei and Zhang13,Reference Akchurin and Kaskel14) . Previous studies have highlighted the link between inflammation and increased mortality risk in this population(Reference Mihai, Codrici and Popescu3,Reference Akchurin and Kaskel14,Reference Yan and Shao15) .
On the other hand, the cellular antioxidant defense system includes a crucial regulator of redox homeostasis and innate immunity, known as nuclear factor erythroid 2-related factor 2. This factor inhibits the NF-κB pathway, the NLRP3 inflammasome and the genetic transcription of inflammatory cytokines such as IL-1β and IL-6(Reference van der Horst, Carter-Timofte and van Grevenynghe16). Nrf2 positively regulates the expression of genes that encode antioxidant enzymes, including NAD(P)H quinone dehydrogenase 1, heme oxygenase (HO-1), glutathione S-transferases, superoxide dismutase and catalase, thereby orchestrating a detailed protective response(Reference Lin, Hsu and Chang17). However, in patients with CKD, evidence suggests that Nrf2 expression and activity may be impaired or dysregulated, contributing to an imbalance between pro-oxidant and antioxidant pathways(Reference Pedruzzi, Cardozo and Daleprane18). Reduced Nrf2 signalling has been associated with increased oxidative stress and inflammation, which are key drivers of CKD progression(Reference Lim, Ng and Zhang19).
In addition, gut dysbiosis also plays a central role in the pathogenesis of CKD(Reference Rodrigues, Ormanji and Heilberg20) and contributes to inflammation in these patients(Reference Mihai, Codrici and Popescu3,Reference Kadatane, Satariano and Massey21) . Several CKD-related factors have been identified as contributors to dysbiosis, including the uremic environment, low fibre intake and the use of medications such as antibiotics, phosphate binders and oral Fe supplements(Reference Dai, Meijers and Bammens22–Reference Mafra, Borges and Alvarenga24). In CKD, dysbiosis typically involves a loss of short-chain fatty acids-producing bacteria (e.g. Faecali bacterium, Roseburia) and an increase in proteolytic and opportunistic taxa. This imbalance in microbiota composition can lead to bacterial translocation, endotoxin production (e.g. lipopolysaccharides) and favours the generation of uremic toxins such as indoxyl sulfate (IS), p-cresyl sulfate (p-CS), indole-3-acetic acid (IAA) and trimethylamine N-oxide, which accumulate in these individuals due to reduced renal function(Reference Saranya and Viswanathan25,Reference Evenepoel, Stenvinkel and Shanahan26) .
In this context, interventions aimed at restoring and maintaining intestinal microbiota physiology while reducing inflammation and oxidative stress are crucial for slowing the progression of kidney failure and managing complications. Numerous nutritional strategies, including incorporating foods rich in bioactive compounds, can benefit patients with CKD(Reference Cardozo, Stockler-Pinto and Mafra27–Reference Ribeiro, Alvarenga and Coutinho-Wolino32). Eating fruits is widely recognised as reducing the risk of chronic non-communicable diseases. Among these, berries such as blackberries, blueberries, cranberries, raspberries and strawberries have attracted attention for their high levels of bioactive compounds, including anthocyanidins, which are known for their potent antioxidant and anti-inflammatory properties(Reference Mafra, Borges and Lindholm30,Reference Coutinho-Wolino, Melo and Mota33) .
Jaboticaba, a fruit native to South America, particularly Brazil, is regarded as a Brazilian berry. It belongs to the Myrtaceae family and the Myrciaria genus (also known as Plinia), which includes several species. The most common ones are Plinia cauliflora, Plinia trunciflora, Plinia peruviana, Myrciaria cauliflora and Myrciaria jaboticaba (Reference Fernandes I de, Maciel and Maroldi34). It is characterised by flowers (which are followed by fruits) that emerge directly from the trunks and main branches. Its fruit is a globular-shaped berry, reaching up to 4 cm in size, with a smooth, firm, shiny peel that shifts from green to dark purple or black as it matures. The flesh is white, gelatinous, sweet, slightly acidic and pungent, containing one to four seeds(Reference Inada, Leite and Martins35). Several phenolic compounds, including anthocyanins, flavan-3-ols, proanthocyanidins, flavonols, ellagitannins, phenolic acids, procyanidins and tannins, were identified in jaboticaba(Reference Chang, Alasalvar and Shahidi36).
The consumption of jaboticaba as a fresh fruit is highly appreciated throughout Brazil. However, the epicarp of the fruit (the peel) is typically not used because of its rigidity and astringent taste, leading to significant waste in the production of jaboticaba-derived products. This situation has sparked scientific and commercial interest because the peel contains most of the phenolic compounds, such as anthocyanins and acid derivatives, which are responsible for the remarkable bioactive properties of its extract(Reference Albuquerque, Pereira and Calhelha37).
A pilot, blinded, crossover, placebo-controlled clinical study evaluated the consumption of 27·6 g of jaboticaba peel powder, which contains 1·25 g of total phenolic compounds, and found a significant increase in serum antioxidant capacity in healthy individuals, along with a reduction in glucose and insulin levels compared with the placebo(Reference Plaza, Batista and Cazarin38). Additionally, the authors of this study found that anthocyanins were the main phenolic compounds in the M. jaboticaba peel. However, ellagitannins and gallotannins significantly contributed to the antioxidant capacity(Reference Plaza, Batista and Cazarin38). A randomised, double-blind, placebo-controlled clinical study assessed the effects of a daily intake of 250 ml of jaboticaba juice containing approximately 1300 mg of total polyphenols over 6 d in twenty-four healthy individuals(Reference Ramos Junior, Tavares and Lima39). The study showed that this supplementation prevented the increase in exercise-induced reactive oxygen species and safeguarded both macro- and microvascular functions from damage caused by eccentric exercise in the juice group. Conversely, the group that received a placebo experienced a significant reduction in blood levels of reduced glutathione, brachial artery flow, the oxygen saturation reperfusion slope at 24 h and blood flow at 48 h after eccentric exercise compared with the jaboticaba juice group(Reference Ramos Junior, Tavares and Lima39). Recently, Geraldi et al. (2024) provided patients with metabolic syndrome with either sachets containing 15 g of jaboticaba peel powder (corresponding to approximately 1 g/d of phenolic compounds) or a placebo daily for 5 weeks. They observed a reduction in IL-6 levels and improved glucose control, with no reported adverse effects(Reference Geraldi, de Souza and Norde40).
Evidence from experimental models further supports the biological effects of jaboticaba peel, including anti-inflammatory, antioxidant, metabolic and microbiota-modulating properties(Reference Brito, Silva and Cunha41–Reference Batista, Soares and Mendonça44). A study observed that supplementation with jaboticaba peel extract (50 g/L), rich in polyphenols, in Wistar rats resulted in beneficial modulation of the gut microbiota, increasing Lactobacillus and Bifidobacterium and bacterial metabolic activity, as evidenced by increased levels of microbial metabolites in the colon(Reference da Silva-Maia, Batista and Correa45). Consistent findings have been reported in experimental models of intestinal inflammation, in which an anthocyanin-rich extract from jaboticaba peel also increased the abundance of these beneficial genera and promoted short-chain fatty acid production(Reference da Silva-Maia, Batista and Cazarin43). These effects are attributed to its high polyphenol content, which reaches the colon and interacts with the gut microbiota, influencing its composition and metabolic activity, providing a biological rationale for investigating its effects in patients with CKD who present gut dysbiosis(Reference Li, Chen and Zha46).
Therefore, jaboticaba peel extract may serve as a novel adjuvant therapy for common complications in patients with non-communicable diseases, including CKD. Derived from a valued native fruit, it is rich in various bioactive compounds that may modulate cellular pathways and improve the inflammatory profile. Our research group previously conducted a pilot study in patients with CKD undergoing haemodialysis, demonstrating the feasibility and safety of short-term supplementation with jaboticaba peel extract (3·3 g/d for 3 weeks). The intervention attenuated increases in proinflammatory cytokines, such as IL-1β and IL-17E, and complementary in vitro analyses suggested antioxidant effects, providing preliminary evidence of beneficial biological activity(Reference Lima, Brito and Ribeiro-Alves47). However, due to its short duration and study design, a more robust investigation is warranted. Therefore, the present study was designed as a randomised, controlled crossover trial with a longer intervention period and expanded outcome measures to further evaluate these effects.
Consequently, this study is essential, given the significance of understanding the impact of jaboticaba peel extract on patients with CKD. The primary outcome will be the mRNA expression of the inflammatory transcription factor NF-κB. Secondary and exploratory outcomes will include the antioxidant-related transcription factor Nrf2; antioxidant enzymes (superoxide dismutase, glutathione peroxidase and catalase); markers of oxidative stress (malondialdehyde, oxidised LDL and protein carbonylation); gut microbiota composition, assessed by the relative abundance of bacterial genera and diversity indices (alpha and beta diversity); as well as routine biochemical parameters, additional cytokines and uremic toxin levels.
Materials and methods
Objective
This study aims to evaluate the effects of jaboticaba peel extract on inflammation, oxidative stress and intestinal microbiota composition in patients with CKD undergoing haemodialysis.
Hypotheses
We hypothesise that 2 months of supplementation with jaboticaba peel extract may improve patients’ inflammatory profiles by reducing the expression of proinflammatory transcription factors and inflammatory cytokine levels, decreasing oxidative stress through enhanced expression of transcription factors associated with antioxidant pathways and subsequently increasing antioxidant enzyme levels. It may also promote beneficial changes in gut microbiota composition, including shifts in the relative abundance of bacterial genera and improvements in microbial diversity, and reduce uremic toxin production.
Study design
This study is part of a broader research program (called Food as Medicine for patients with CKD) that investigates the role of nutritional interventions and bioactive compounds in modulating inflammation, oxidative stress and the gut microbiota in patients with CKD, integrating clinical and mechanistic approaches. A preliminary pilot study previously demonstrated the feasibility and safety of short-term supplementation and provided initial evidence of potential benefits(Reference Lima, Brito and Ribeiro-Alves47). The present study was therefore designed as a randomised, controlled crossover trial with a longer intervention period and expanded outcome measures to allow a more comprehensive evaluation of these effects.
This is a longitudinal, randomised, double-blind, placebo-controlled crossover clinical trial involving patients with CKD undergoing haemodialysis, including a washout period between interventions. The study has been registered with Clinical Trials and approved by the Research Ethics Committee of the Fluminense Federal University School of Medicine (approval number: 6.953.399). This study’s design follows the Consolidated Standards of Reporting Trials (CONSORT) 2010 statement. This is the protocol version 1.0 (May 2026), consistent with the version registered at ClinicalTrials.gov (Identifier: NCT06394531). The study is expected to begin in July 2026, with the supplementation period estimated to be completed by December 2026, and all subsequent analyses anticipated to be finalised by December 2027. Written informed consent will be obtained from all participants before any study procedures begin, and it will be collected by trained members of the research team, who will provide detailed information about the study and clarify any questions prior to enrollment. Personal information will be stored in secure, password-protected databases accessible only to authorised researchers. Identifiable data will be coded and kept separate from study records to protect confidentiality, and no personal identifiers will be shared outside the research team. All data will be handled in accordance with institutional and regulatory requirements throughout the trial.
Patients will be randomised into either a control group or a jaboticaba group. During the intervention phase, participants will receive four capsules per day, totalling 3·3 g/d of jaboticaba peel, for 2 months. Participants will be instructed to take the capsules in divided doses, twice daily, after lunch and dinner. The capsules containing jaboticaba peel extract were developed, chemically characterised, standardised and will be distributed to participants. The placebo group will receive the same number of placebo capsules (corn starch) for the same period. Patients will undergo a 2-month washout after 2 months of supplementation to eliminate carry-over effects. After this period, patients from the jaboticaba group will switch to the placebo group and vice versa (Figure 1). Clinical crossover trials with polyphenol-rich foods typically use washout periods of 4–8 weeks to minimise carryover effects(Reference Batista, Zanzer and Marostica Junior48–Reference Rivara, Mehrotra and Linke50). Given the high polyphenol content of jaboticaba peel and the assessment of outcomes that may be sustained (e.g. inflammation and gut microbiota), a 2-month washout period was implemented to minimise residual effects.
Study design. Thirty patients undergoing regular haemodialysis (HD) will be recruited for the study and randomised into two groups: a placebo group and an intervention group supplemented with 3·3 g of jaboticaba peel extract. The supplementation will be administered over 2 months, followed by a 2-month washout phase, after which a crossover will occur. Biological samples, including blood and feces, will be collected at the start and end of each study phase.
Figure 1. Long description
The flowchart depicts the study design for a clinical trial involving thirty patients undergoing regular hemodialysis. The patients are randomized into two groups: a placebo group and an intervention group supplemented with 3.3 grams of jaboticaba peel extract. The supplementation phase lasts for two months, followed by a two-month washout period. After the washout, a crossover occurs where the groups switch treatments. Biological samples, including blood and feces, are collected at the start and end of each study phase. The flowchart includes labels for the supplementation phases, washout period, and sample collection points.
Both participants and the research team members involved in the supplementation procedures will remain blinded to group allocation, as the capsules containing jaboticaba peel extract and those containing placebo (corn starch) are identical in appearance, preventing patients and investigators from distinguishing between them. The analysis of potential adverse effects will be conducted using a specific analysis form adapted from the method described by the National Health Surveillance Agency(51). Adherence to the intervention will be monitored by the study researchers through direct supervision at the haemodialysis clinic on dialysis days, periodic telephone contact with participants and the return of capsule bottles at the end of the intervention period.
Inclusion and exclusion criteria
The study will include men and women aged 18–75 years who are on haemodialysis for at least 6 months, have an arteriovenous fistula as their vascular access and adhere to individualised dietary prescriptions. Patients with autoimmune diseases, infectious diseases, diabetes, cancer, AIDS and those using catabolic drugs or antibiotics, as well as antioxidant supplements, prebiotics, probiotics, symbiotics and pregnant women, will not be included.
Criteria for discontinuing or modifying allocated intervention
The allocated intervention may be discontinued or modified under the following circumstances: adverse events or intolerance potentially related to the intervention; initiation of medications or supplements that may interfere with study outcomes (e.g. antibiotics, probiotics, prebiotics or antioxidant supplements); development of exclusion criteria during follow-up (e.g. diagnosis of acute infection, cancer or other relevant clinical conditions); significant clinical deterioration requiring changes in medical management or participant request to withdraw from the study.
To enhance participant retention and ensure complete follow-up, participants will receive regular reminders for scheduled visits via phone calls or text messages, along with flexible scheduling as needed. The research team will maintain close contact with participants throughout the study to reinforce adherence and promptly address any concerns. Participants who discontinue or deviate from the allocated intervention will be encouraged to continue follow-up assessments whenever possible. All efforts will be made to minimise missing data, and reasons for withdrawal or non-adherence will be documented.
Sample calculation, randomisation and blinding
Thirty patients with CKD undergoing haemodialysis will be recruited based on a sample size calculation with a significance level of 0·05 and 80 % statistical power. The estimated effect size corresponds to a minimum 20 % change in NF-κB mRNA expression, based on previous clinical evidence from our research group in CKD patients receiving a dietary intervention(Reference Alvarenga, Salarolli and Cardozo29), supporting the biological plausibility of this magnitude of response. NF-κB was selected as a sensitive mechanistic biomarker of inflammatory activity, given the exploratory nature of the study. For this scenario, twenty-four volunteers would suffice to detect this effect size. We will recruit thirty patients for our study to include a safety margin. Randomisation will be computerised in a 1:1 ratio, with a block size of 15, for the jaboticaba and placebo groups, who will receive either the jaboticaba peel extract or a placebo. An independent statistician will handle randomisation.
Participants, investigators and outcome assessors will remain blinded to group allocation throughout the study. Unblinding will be permitted only in exceptional circumstances, such as a serious adverse event for which knowledge of the allocated intervention is essential for appropriate clinical management. In such cases, unblinding will be performed by a designated member of the research team who is not involved in outcome assessment. The reason for unblinding, the timing and the personnel involved will be fully documented.
Formulation and chemical characterisation
The jaboticaba peel extract (P. cauliflora) will be produced from peels obtained in Natividade da Serra, São Paulo, Brazil. The peels will be dried, milled and subjected to hydroethanolic extraction (ethanol–water solution), followed by concentration, drying and encapsulation of the final product. The proposed daily dosage of 3·3 g of jaboticaba peel extract is equivalent to approximately 45 g of fresh peels. The product contains 666·5 mg of total (poly)phenols per daily dose, expressed as gallic acid equivalents. In addition to its (poly)phenolic profile, the extract also contains naturally occurring polysaccharides derived from the fruit peel matrix.
The selected dosage was based on available human clinical trials, previous non-clinical efficacy and safety studies using hydroethanolic extracts and dose-translation approaches in accordance with FDA guidelines, ensuring a physiologically relevant and safe dose for human supplementation. This dose was further supported by our previous pilot study, which demonstrated that it was safe and well-tolerated in patients with CKD(Reference Lima, Brito and Ribeiro-Alves47).
The placebo will consist of capsules containing low-amylose starch, selected to minimise fermentability and avoid potential prebiotic or microbiota-modulating effects.
Food intake and nutritional status
Participants are routinely followed by a dietitian at the haemodialysis clinic and receive general dietary guidance appropriate for haemodialysis care. No additional dietary restrictions will be imposed, except that participants will be instructed to avoid consuming jaboticaba or jaboticaba-derived foods during the intervention period. Food intake will be assessed using the 24-h recall method, conducted at four time points (before and after each intervention phase). The analysis of energy, macronutrient and micronutrient intake will be performed with DietBox® software.
The collected anthropometric data will include body weight, height, arm circumference, triceps and biceps skinfolds, subscapular and suprailiac skinfolds and waist circumference. Nutritional status will be evaluated based on BMI. In addition, body density, fat percentage, corrected arm muscle area and fat-free mass will be calculated. Qualified and experienced nutritionists will carry out both analyses.
Blood collection and biochemical analysis
Blood will be collected from the haemodialysis line using Vacutainer® tubes containing EDTA (1·0 mg/ml) as an anticoagulant for plasma analyses and Vacutainer® SST® II Advance® tubes with clot activator and separator gel for serum collection. Collected blood samples will be transported under refrigerated conditions (approximately 4°C) using insulated containers with ice packs to ensure appropriate temperature control until arrival at the research laboratory. One aliquot from the EDTA tubes will be used for cell separation. The remaining samples will be centrifuged at 3500 rpm for 15 min at 4°C to obtain plasma and serum, which will be stored at –80°C until analysis.
Isolation of peripheral blood mononuclear cells
Whole blood will be transferred to a Falcon tube containing 10 ml of saline solution in a 1:1 ratio. After homogenisation, it will be transferred to another Falcon tube containing 15 ml of Histopaque. Centrifugation will be conducted to form the cell cloud, and 7 ml of the cloud will be transferred to a new tube. A 5 ml saline solution will be added and centrifuged. The supernatant will be discarded, and the pellet will be resuspended in 1 ml of saline and centrifuged again. The supernatant will be discarded, and the nuclear pellet will be resuspended in 1 ml of Recovery™ Cell Culture Freezing Medium and stored at –80°C.
Quantitative real-time PCR analysis
The mRNA expression of Nrf2, Kelch Like ECH Associated Protein 1, BTB domain and CNC homolog 1 (BACH1), NLRP3, NF-κB, HO-1, NAD(P)H quinone dehydrogenase 1, vascular cell adhesion molecule 1, intercellular adhesion molecule-1, E-selectin, toll-like receptor 4, aryl hydrocarbon receptor will be evaluated from peripheral blood mononuclear cell using quantitative real-time PCR. Peripheral blood mononuclear cells will be used for RNA extraction by the SV Total RNA Isolation System (Promega®). The cDNA will be synthesised using the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher®). The Prism 7500 Sequence Detection System (ABI, Applied Biosystems®) and standard cycling conditions will be used for PCR amplification. mRNA expression will be normalised against glyceraldehyde-3-phosphate dehydrogenase, and the expression level will be calculated using the ΔΔCT (delta delta threshold cycle) method.
Biochemical analyses
The activities of the enzymes GSH-Px, catalase and the three types of superoxide dismutase (MnSOD, FeSOD and Cu/ZnSOD) will be determined in plasma using commercial ELISA kits (Cayman®). The Luminex® panel will be used to measure inflammatory cytokines. In this study, serum levels of the following cytokines will be analysed: MCP-1/CCL-2; MIP-1α/CCL-3; MIP-1β/ CCL-4; RANTES/CCL-5; eotaxin-1/CCL-11; IP-10/CXCL-10; GM-CSF/CSF-2; GCSF/CSF-3; FGFβ; IFN-γ; IL-1ra; IL-1β, 2, 4, –5, –6, –7, –8 (CXCL8), –9, –10, –12p70, –13, –15 and –17; PDGF-BB; TNF-α and VEGF. Plasma lipopolysaccharide levels will be determined by ELISA (R&D Systems). Dosages of plasma albumin, creatinine, urea, potassium, phosphorus, Ca, protein C-reactive, ferritin, total cholesterol, HDL and TAG will be determined using commercial kits from BioClin®. LDL values will be calculated using the Friedewald formula (1972): LDL = (TC – HDL) – (TG/5). Erythrocyte sedimentation rate will be measured in whole blood using the Westergren method.
Determination of toxins plasma levels p-cresyl sulfate, trimethylamine N-oxide, indoxyl sulfate and indole 3-acetic acid
The concentrations of p-CS, IS and IAA will be determined after heat-induced deproteinisation and analysed by reverse-phase HPLC (RP-HPLC) with fluorescence detection. Plasma concentrations of trimethylamine N-oxide will be analysed using LC-MS/MS.
Determination of oxidative stress markers
Lipid peroxidation will be estimated by measuring thiobarbituric acid-reactive substances, including malondialdehyde, using the modified Ohkawa method(Reference Ohkawa, Ohishi and Yagi52). The samples will be diluted with thiobarbituric acid (0·6 % w/v), SDS (8·1 % w/v) and phosphoric acid (1 % w/v), then heated at 95°C for 60 min. The microtubules will be centrifuged at 4000 rpm for 20 min at 20℃, the supernatant will be separated and the absorbance will be measured in a Synergy H1M microplate reader (Biotek) at 532 nm. The plasma thiobarbituric acid-reactive substances levels will be expressed in nanomoles per milliliter.
Plasma levels of oxidised low-density lipoprotein (ELABSCIENCE) will be determined using a commercial ELISA kit (Elabscience®, Catalog No: E-EL-H0124). Using the Sandwich-ELISA principle, the plate provided in this kit is pre-coated with a specific antibody for human oxLDL. 100 μl of each sample will be added to the wells of the plate and incubated for 90 min at 37°C with a specific antibody. Then, a detection antibody specific for human oxLDL and an HRP conjugate will be added to each well of the microplate, and the plate will be incubated for 1 h at 37℃. The wells containing human oxLDL, biotinylated detection antibody and avidin–HRP conjugate will react, producing a blue colour. The enzyme–substrate reaction will be stopped by adding a stop solution, which changes the colour to yellow. The optical density will be measured spectrophotometrically at 450 ± 2 nm. The optical density value will be proportional to the concentration of human oxLDL. The concentration of human oxLDL in the samples will be calculated by comparing the samples’ optical density with the standard curve.
Protein carbonylation will be analysed in the plasma using an adapted technique described by Levine et al. (1990)(Reference Levine, Garland and Oliver53). Briefly, 50 μl of the samples will be mixed with 500 μl of 2,4-dinitrophenylhydrazine and incubated for 15 min at room temperature. Subsequently, the proteins will be precipitated with 10 % trichloroacetic acid, resuspended in an ethanol/ethyl acetate solution and then solubilised in guanidine. The carbonyl content will be measured at 370 nm using a PowerWave XS microplate reader (Biotek®). The results will be expressed as nmol of carbonyls per mg of protein. The Bradford method will be used for protein quantification.
The oxygen radical absorption capacity test will measure the inhibition of peroxyl radical oxidation, reflecting the ability of antioxidants to interrupt the radical chain reaction. Peroxyl radicals are free radicals that, under physiological conditions, will prevail in lipid oxidation in biological systems and food products. The producer of the peroxyl radical will be 2,2′-Azobis(2-methylpropionamidine) dihydrochloride. In this method, the peroxyl radical emitted by a generator interacts with a fluorescent sample, resulting in a loss of fluorescence that is recorded by a fluorimeter. Trolox will serve as a reference standard antioxidant, and disodium fluorescein will be used in the reaction. The samples will be prepared with 100 mcL of plasma, 100 mcL of dimethyl sulfoxide and 800 mcL of phosphate-buffered saline. Samples at different concentrations will be pipetted into the plate in triplicate. After adding the reagents and incubating at 37°C for 10 min, the plate will be read on a fluorimeter.
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay will be used to determine antioxidant activity, using the stable free radical DPPH. This test will analyse the scavenging capacity of antioxidants. The reaction will be monitored by measuring changes in DPPH colour at 520 nm; the loss of its intense violet colour will indicate antioxidant activity(Reference Munteanu and Apetrei54).
The ferric reducing antioxidant power assay will measure, in an acidic medium, the reduction of the ferric ion (Fe3+)-ligand complex to the ferrous complex (Fe2+)(Reference Munteanu and Apetrei54). Antioxidant activity will be indicated by a rise in absorbance at 595 nm. The linking ligand used in this test will be tripyridyltriazine.
Determination of intestinal microbiota composition
Participants will receive standardised stool collection kits and detailed instructions for home collection. Samples should be collected within 48 h prior to the scheduled visit, stored immediately at –20°C and transported to the laboratory under cold conditions. Upon arrival, samples will be aliquoted and stored at –80°C until analysis. Microbial DNA will be extracted using the Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo Research), following the manufacturer’s instructions. DNA concentration and purity will be assessed using spectrophotometry (NanoDrop 2000, Thermo Fisher Scientific). The V4 region of the 16S rRNA gene will be amplified by PCR using universal primers such as 515F and 909R, and sequencing will be performed using a high-throughput platform (Illumina MiSeq). Raw sequencing data will be processed using a standardised bioinformatic pipeline (DADA2) to generate amplicon sequence variants. Microbial composition will be evaluated based on relative abundance and diversity metrics, including α diversity (e.g. Shannon index, observed amplicon sequence variants) and beta diversity (e.g. Bray–Curtis dissimilarity with principal coordinates analysis). Longitudinal changes in microbial diversity and taxonomic profiles will be assessed over the course of the intervention. Further statistical and bioinformatic analyses will follow approaches appropriate for compositional microbiome data, as described in the statistical analysis section.
Statistical analysis
The results will be expressed as mean (sd) or median (min–max), depending on the data distribution. Normality and homoscedasticity will be assessed using the Shapiro–Wilk and Levene tests, respectively. To appropriately account for repeated measures and the non-independence of observations, linear mixed-effects models will be used to evaluate the effects of supplementation, including subject as a random effect and treatment, period and sequence as fixed effects, thereby allowing assessment of potential carryover effects. Analyses will be conducted primarily according to the intention-to-treat principle, including all randomised participants. Per-protocol analyses will also be performed as sensitivity analyses, including only participants who complete the study with adequate adherence to the intervention. When appropriate, models will be adjusted for pre-specified covariates, including age, sex, dialysis vintage, BMI, diabetes status and baseline values of the respective outcomes, selected a priori based on their clinical relevance and potential influence on the study endpoints. Model assumptions will be verified through residual diagnostics. Post hoc comparisons will be performed using Bonferroni correction. When model assumptions are not met, non-parametric methods will be applied, including the Wilcoxon signed-rank test for paired comparisons, the Mann–Whitney U test for independent samples and the Friedman test for repeated measures. Correlations between variables will be assessed using repeated-measures correlation or mixed-model-based approaches, as appropriate, to account for within-subject dependence. Additionally, the net effect of supplementation (Δ) will be calculated as the within-subject difference between intervention and placebo conditions. These effects will be analysed using linear mixed-effects models or, when appropriate, corresponding non-parametric tests. Statistical significance will be set at P < 0·05, with 95 % CI reported. All analyses will be conducted using R software and GraphPad Prism.
Bioinformatics analysis
Sequencing will be performed using the Illumina MiSeq platform (Illumina) according to standard protocols. Raw FASTQ reads will be imported into the QIIME2 platform and processed using the DADA2 pipeline to perform quality filtering, denoising, chimera removal and inference of amplicon sequence variants. Taxonomic assignment will be conducted using a reference database such as SILVA(Reference Yilmaz, Parfrey and Yarza55). Potential contaminants introduced during DNA extraction and library preparation will be identified and removed using the decontam package in R, based on sequence prevalence in negative controls relative to biological samples. Microbiome data will be analysed in R using appropriate packages (e.g. phyloseq, vegan). Alpha diversity will be assessed using indices such as Shannon diversity and Pielou’s evenness, while beta diversity will be evaluated using Bray–Curtis dissimilarity and visualised through principal coordinates analysis. Differences in overall microbial community structure will be tested using PERMANOVA, with appropriate stratification to account for repeated measures. To address the compositional and sparse nature of microbiome data, count data will be normalised using the centered log-ratio transformation after appropriate zero handling. Differential abundance analysis will be performed using methods specifically designed for compositional microbiome data with longitudinal structure, such as ANCOM-BC2 (or MaAsLin3), incorporating mixed-effects models to account for repeated measurements within individuals over time. False discovery rate will be controlled using the Benjamini–Hochberg procedure. Co-occurrence networks will be constructed at the genus level to reduce sparsity and noise. Correlations will be estimated using the SparCC algorithm, and network inference will be performed using the SpiecEasi package. Networks will be generated separately for each time point (e.g. baseline and post-intervention), and changes over time will be explored using differential network approaches. The longitudinal structure of the data will be addressed by restricting correlations within time points or by aggregating at the subject level. Key taxa (network hubs) will be identified using the PageRank algorithm.
Discussion
Jaboticaba is a native Brazilian fruit of great ecological and economic importance, widely recognised for its high levels of bioactive compounds, including anthocyanins, tannins and other phenolic compounds. These compounds are mainly concentrated in the peel and have been associated with antioxidant, anti-inflammatory and metabolic regulatory effects(Reference Inada, Leite and Martins35). Such properties highlight the potential of jaboticaba peel in mitigating complications commonly observed in chronic non-communicable diseases, including chronic kidney disease.
Fruit peels are recognised as particularly rich sources of polyphenols, often exceeding the concentrations found in the edible pulp, and there is growing evidence supporting their biological activity. Clinical studies using peel-derived interventions have demonstrated beneficial effects on inflammatory and metabolic outcomes. For instance, supplementation with pomegranate peel extract has been shown to reduce inflammatory markers, such as hs-CRP, and to improve liver enzymes and the lipid profile in individuals with non-alcoholic fatty liver disease(Reference Baghdadi, Shidfar and Mokhtare56). Similarly, interventions using jaboticaba peel in both healthy individuals and those with metabolic syndrome have been reported to reduce IL-6 and oxidative stress, alongside improvements in glycaemic responses(Reference Geraldi, de Souza and Norde40,Reference Batista, Zanzer and Marostica Junior48) .
The present study was designed to test the hypothesis that supplementation with jaboticaba peel extract may influence key pathways involved in chronic kidney disease. Specifically, this study will evaluate whether supplementation affects inflammatory status, including NF-κB activation and plasma levels of proinflammatory cytokines (e.g. IL-6 and TNF-α), as well as oxidative stress markers, such as lipid peroxidation (malondialdehyde), oxLDL and protein carbonylation, and antioxidant capacity (oxygen radical absorption capacity, DPPH and FRAP). Additionally, the study will assess potential effects on Nrf2 expression and antioxidant enzyme activity (GPx, CAT and superoxide dismutase), clinical routine parameters and gut microbiota composition, including plasma levels of uremic toxins (IS, p-CS, IAA and trimethylamine N-oxideTMAO).
To our knowledge, few studies have investigated the effects of jaboticaba peel supplementation on human health and disease conditions. However, clinical evidence for jaboticaba supplementation in humans suggests potential benefits. Previous studies have reported improvements in antioxidant capacity, glycaemic control and inflammatory markers following the consumption of jaboticaba-derived products in healthy individuals and patients with metabolic disorders(Reference Plaza, Batista and Cazarin38–Reference Geraldi, de Souza and Norde40).
In this context, a pilot study conducted by our research group in patients undergoing haemodialysis demonstrated that short-term supplementation (3 weeks) with jaboticaba peel extract (3·3 g/d) attenuated increases in proinflammatory cytokines, such as IL-1β and IL-17E. In addition, in vitro experiments using a jaboticaba peel formulation (1·5 %) demonstrated antioxidant effects, including reduced reactive oxygen species and a trend towards decreased protein oxidation. Mechanistically, these findings were associated with modulation of the TLR–MYD88–NF-κB signalling pathway, supporting the role of jaboticaba peel in regulating key inflammatory and oxidative stress pathways(Reference Lima, Brito and Ribeiro-Alves47). However, evidence regarding the bioavailability and dose–response relationship of jaboticaba peel extract in humans remains limited and should be considered when interpreting the potential effects of supplementation. Further dose–response studies are needed to better understand its effects. Additionally, no significant changes were observed in other outcomes following 3 weeks of jaboticaba peel supplementation, including C-reactive protein, oxidative stress markers (malondialdehyde and protein carbonylation) and uremic toxins (IS, p-CS and IAA)(Reference Lima, Brito and Ribeiro-Alves47). These outcomes will be reassessed in the present study, whose longer duration and crossover design may enhance the detection of delayed or subtle biological responses that were not evident in the pilot study.
Building on findings from our previous pilot study in patients with CKD, the present study will be a randomised, controlled, crossover trial to further evaluate the safety and tolerability of a predefined dose of jaboticaba peel extract. By integrating clinical, biochemical, inflammatory and microbiota-related outcomes, this study will provide a more comprehensive understanding of its potential effects on common disease complications. These findings are expected to advance knowledge in this field and support future research and evidence-based approaches to the clinical use of bioactive compounds.
The trial results will be disseminated through peer-reviewed publications, conference presentations and updates in the trial registry. A plain-language summary will be made available to participants and other relevant stakeholders. No individual participant data will be disclosed.
Acknowledgements
We would like to thank the Clinical Research Unit (UPC) of the Antônio Pedro University Hospital, where the laboratory analyses will be performed.
This work is supported by the Fundação Carlos Chagas Filho de Apoio à Pesquisa do Estado do Rio de Janeiro (FAPERJ) (E-26/210.615/2023) and by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (150936/2023-0).
J. S. B.: Conceptualisation, Methodology, Investigation, Data curation, Writing – original draft preparation. L. S. L.: Conceptualisation, Investigation, Data curation, Writing – original draft preparation. A. B.: Methodology, Writing – review and editing. E. B. C.: Writing – review and editing. D. M.: Conceptualisation, Methodology, Formal analysis, Writing – review and editing, Supervision, Project administration, Funding acquisition.
A. B. works at the Research, Development, and Innovation Department of Apis Flora Indl. Coml. Ltda. All authors declare no conflicts of interest.
