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Open Access

Peer-reviewed

Research Article

Cafestol ameliorates diabetic nephropathy via Keap1-Nrf2 axis activation: A novel renoprotective mechanism independent of glycemic control

  • Nujud A. Sinan,

    Roles Data curation, Investigation, Methodology, Writing – original draft

    Affiliation Department of Food Science and Human Nutrition, College of Agriculture and Food, Qassim University, Buraydah, Saudi Arabia

    ⨯
  • Mona S. Almujaydil

    Roles Conceptualization, Data curation, Investigation, Methodology, Supervision, Writing – review & editing

    M.almujaydil@qu.edu.sa

    Affiliation Department of Food Science and Human Nutrition, College of Agriculture and Food, Qassim University, Buraydah, Saudi Arabia

    ⨯

Abstract

Diabetic nephropathy is an early complication of diabetes, which triggers lipotoxicity, oxidative stress, inflammation, fibrosis, and apoptosis. This study aimed to investigate the possible protective effects of Cafestol and the underlying molecular mechanisms in type 1 diabetes-induced nephropathy. One dose of streptozotocin (65 mg/kg, i.p.) was used to induce diabetes, then Cafestol was given orally at 5 and 10 mg/kg for eight weeks. In the control rats, Cafestol (10 mg/kg) did not cause any significant changes to any of the measured parameters, including 24-hour urine volume, urinary albumin, creatinine, and renal injury parameters (KIM-1, NGAL, nephrin), suggesting that Cafestol has no adverse renal effect in normal physiology. In T1DM induction rats, renal dysfunction was apparent as marked increases in urine output, enzymatic indicators of injury, inflammatory mediators (IL-6, TNF-α, ICAM-1, nuclear NF-κB), oxidative stress (MDA), and glycation markers (AGEs, sRAGEs), while there were significant decreases in antioxidant defenses (HO-1, SOD, GSH) and nuclear Nrf2 expression. Treatment with Cafestol at doses of 5 mg/kg and 10 mg/kg significantly reduced the pathological parameters when compared to T1DM alone in a dose-dependent manner. Cafestol treatment reduced glomerular and tubular injury, inhibited renal inflammation, restored oxidative balance, and improved antioxidant capacity. Cafestol treatment also reduced renal Keap1 mRNA expression and increased cytoplasmic and nuclear Nrf2 protein levels, indicating that Cafestol activates the Nrf2 pathway without altering Nrf2 mRNA expression. Further, co-administration of Brusatol, an Nrf2 inhibitor, eliminated the renal protection effects of Cafestol for all measured parameters and returned levels to T1DM alone. The results suggest that Cafestol provides substantial renal protective, anti-inflammatory, and antioxidative benefits in a model of diabetes, mainly through the activation of Nrf2. This finding suggests that Cafestol may be a novel agent to activate Nrf2, which may have implications for the treatment of DN.

Citation: Sinan NA, Almujaydil MS (2026) Cafestol ameliorates diabetic nephropathy via Keap1-Nrf2 axis activation: A novel renoprotective mechanism independent of glycemic control. PLoS One 21(5): e0349192. https://doi.org/10.1371/journal.pone.0349192

Editor: Junzheng Yang, Guangdong Nephrotic Drug Engineering Technology Research Center, Institute of Consun Co. for Chinese Medicine in Kidney Diseases, CHINA

Received: December 4, 2025; Accepted: April 26, 2026; Published: May 14, 2026

Copyright: © 2026 Sinan, Almujaydil. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: The Researchers would like to thank the Deanship of Graduate Studies and Scientific Research at Qassim University for financial support (QU-APC-2026).

Competing interests: The authors declare no conflicts of interest.

Introduction

Diabetic nephropathy, or diabetic kidney disease, is a significant microvascular consequence of diabetes mellitus (DM) [1]. Diabetic nephropathy (DN) is a primary contributor to end-stage renal disease (ESRD) worldwide, impacting roughly 30–40% of individuals with diabetes [1,2]. Chronic hyperglycemia and dyslipidemia initiate the development of diabetic nephropathy by inducing oxidative stress, inflammation, fibrosis, and apoptosis, ultimately affecting the glomeruli and tubules [3,4]. Oxidative stress, resulting from elevated production of reactive oxygen species (ROS), diminishes and enhances the antioxidant mechanism, serving as the crucial initiator of all the pathogenic processes [5,6]. Consequently, managing hyperglycemia is the foremost objective in the therapy of diabetic nephropathy. To enhance pathophysiology, oral hypoglycemic agents employed in diabetes management must augment both insulin secretion and sensitivity while diminishing hepatic glucose production through the downregulation of gluconeogenesis [7]. However, there are several risks associated with synthetic oral hypoglycemic agents, and studies have shown that DN-relieving oral hypoglycemic therapies lead to obesity [8].

It has been indicated that nuclear factor erythroid 2-related factor 2 (Nrf2) inhibition has also been reported to be an important mechanism of increased oxidative stress in diabetic nephropathy. Besides, Nrf2 is a potent anti-inflammatory, fibrosis, cell death, and lipogenesis inhibitor through the activation of many transcription factors and signaling proteins [6,9,10]. Activation of Nrf2 has, therefore, been shown to improve kidney function and structure in diabetic kidney lipidosis [11–14].

Although there is partial renoprotection by current therapies, such as renin-angiotensin system inhibitors and sodium-glucose cotransporter-2 (SGLT2) inhibitors, of greater importance is the need for novel drugs with multiple disease target actions [15,16]. In this regard, natural products exhibiting anti-inflammatory and antioxidant activities have become more prominent. Recent studies have further emphasised their therapeutic potential in diabetic nephropathy by modulating inflammatory pathways and complex molecular mechanisms, highlighting their potential as therapeutic agents [17,18]. Regular coffee consumption has been consistently associated with a reduced risk for a variety of metabolic and cardiovascular diseases, including diabetes mellitus, obesity, insulin resistance, dyslipidemia, and cardiovascular disease [19–21]. Furthermore, epidemiological evidence shows that consumption of coffee is related to a reduced risk of kidney disease. These health benefits are largely attributed to coffee’s high content of bioactive compounds, particularly chlorogenic acid and the diterpenes cafestol and kahweol, which exhibit potent antioxidants and anti-inflammatory properties [22,23]. Cafestol, a diterpene and lipid-soluble ester found in green coffee beans, has been identified as a bioactive compound with multiple pharmacological properties [22]. Evidence from both in vivo and in vitro studies has demonstrated its capacity to enhance insulin secretion and promote glucose uptake [24,25]. Furthermore, cafestol exhibits significant anti-inflammatory effects, primarily through the inhibition of pro-inflammatory mediators such as NADPH oxidase 2 (Nox2), inducible nitric oxide synthase (iNOS), and the downregulation of the nuclear factor-kappa B (NF-κB) signaling pathway [26,27]. Notably, cafestol has also been found to activate the Nrf2 signalling pathway, which plays a crucial role in cellular defence against oxidative damage in diabetic cardiomyopathy and nephropathy [23,28,29].

Diabetic nephropathy is a progressive renal disorder resulting from persistent hyperglycemia and oxidative stress, which induces inflammation, fibrosis, and renal impairment. Existing therapies are constrained, and cafestol, a bioactive compound found in coffee, is postulated to safeguard the kidneys by diminishing oxidative stress and inflammation, thereby enhancing renal function in diabetic rats. The study aims to assess the preventive effects of cafestol on diabetic nephropathy utilizing a streptozotocin-induced diabetic rat model. The study investigates the effects of cafestol on renal function, histological changes in the kidneys, oxidative stress markers, and inflammatory responses, aiming to clarify its potential as a therapeutic agent for the management of diabetic nephropathy.

Materials and methods

Animal

Adult male Wistar rats (weighing 180 ± 15 g and approximately 8 weeks old) were used and housed at the Department of Food Science and Human Nutrition, College of Agriculture and Food, Qassim University, Saudi Arabia, in standard laboratory cages with appropriate bedding and maintained under controlled conditions, ensuring a stable temperature (20–22°C) and humidity levels (40–60%). To provide a consistent environment and minimise external influences, a 12-hour light/dark cycle was maintained. The rats had unrestricted access to standard food and water throughout the acclimatisation and experimental phases. All animal handling and experimental protocols were conducted in accordance with ethical guidelines. They were approved by the Committee of Research Ethics of Qassim University, Saudi Arabia (approval no. 24-14-01/2024) in compliance with International Animal Ethics, and in accordance with the ARRIVE Essential 10 criteria.

Induction of the T1DM model

T1DM was induced in rats via a single intraperitoneal injection of streptozotocin (STZ, 65 mg/kg; Sigma Aldrich, 142155) freshly dissolved in 0.5 M sodium citrate, as established by others [30,31]. To prevent hypoglycemia, animals received a 0.5% oral glucose solution for three days post-injection. On day 7, blood glucose levels were measured from tail vein samples after anesthesia with a ketamine/xylazine hydrochloride mixture (80/10 mg/kg). Rats with glucose levels >320 mg/dL were classified as diabetic and included in subsequent experiments.

Experimental design

Cafestol acetate (C₂₂H₃₀O₄) powder (purity > 98%) (#81760-48-7) was purchased from Sigma and freshly dissolved in 0.5% carboxymethylcellulose (CMC). This study included five animal groups (n = 12 rats/group). The Control group consisted of non-diabetic rats receiving only 0.5% CMC orally as a vehicle. The Control + Cafestol-treated group included non-diabetic rats administered Cafestol solution (10 mg/kg) orally. The T1DM-model group comprised diabetic rats treated with an oral dose of 0.5% CMC. The T1DM + Cafestol-treated groups included diabetic rats receiving oral Cafestol solution at low and high doses (5 and 10 mg/kg). Lastly, the T1DM + Cafestol + Brusatol-treated group consisted of diabetic rats treated with Cafestol (10 mg/kg) along with an intraperitoneal dose of Brusatol, an Nrf2 inhibitor (2 mg/kg). All treatments were administered thrice per week. Brusatol was always given 1 hour before administering Cafestol. Experiments continued for 8 weeks; a duration established as necessary for developing diabetic nephropathy after such STZ dose administration [32].

Dose selection

Brusatol was used according to established guidelines from earlier studies (to lower the in vivo Nrf2 nuclear translocation and activity across several tissues, including the heart, intestines, and kidneys [30,33]. Furthermore, our early preliminary results showed that oral Cafestol therapy caused a dose-dependent drop in urinary albumin at doses ranging from 1 to 20 mg/kg, with most effects seen at 10, 15, and 20 mg/kg. Previously, a 5 mg/kg dose of Cafestol was demonstrated to protect rats from doxorubicin-induced heart damage [23]. Cafestol treatment at 7 and 14 mg/kg also produced roughly hypoglycemic effects in non-diabetic obesity patients [19].

Urine and serum collection

Twenty-four hours after the final treatment, rats were placed in metabolic cages for a 2-hour urine collection. Following a 12-hour fasting period, they were anesthetized with a ketamine/xylazine hydrochloride mixture (80/10 mg/kg). Blood samples were then obtained via cardiac puncture and collected into either gel-containing or EDTA-coated tubes. The samples were left at room temperature for 30 minutes before being centrifuged at 500 × g for 15 minutes to separate the supernatant, yielding serum and plasma. All plasma, serum, and filtered urine samples were immediately stored at −20°C for future analysis. All urine, serum, and plasma samples were coded before the biochemical evaluation, and all analyses were performed by investigators blinded to treatment group allocation until the analyses were completed

Preparation of total kidney homogenates

After humanely euthanizing the rats using a two-step method in accordance with the consulting American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals, 2020: first, by inhalation of isoflurane, second, after the animal is unconscious, physical cervical dislocation was done, to confirm death definitively, both kidneys were aseptically removed, cleaned of excess blood, weighed individually, and immediately frozen at −80°C for long-term storage. Detailed records were kept, including kidney weight, animal ID, and collection time. To prepare the total kidney extracts, 30 mg of frozen kidney tissue from each sample was carefully taken and kept on ice to maintain protein stability. The kidney tissue was first washed in cold phosphate-buffered saline (PBS) to remove residual blood. The tissue was then homogenized in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 0.1% Triton X-100, with a protease inhibitor cocktail. The homogenized sample was centrifuged at 10,000 × g for 15 minutes at 4°C to remove cellular debris. The supernatants obtained from all samples, which contained soluble proteins, were collected carefully, labelled, and stored at −80°C for further biochemical analysis.

Preparation of nuclear kidney extract

Nuclear protein was isolated from 30 mg of frozen kidney tissue by homogenization in 500 µL of ice-cold PBS with inhibitors, followed by lysis with hypotonic buffer containing NP-40. After centrifugation at 1,000 × g for 5 minutes at 4°C, the cytoplasmic supernatant was stored, and the nuclear pellet was further processed in a detergent-free buffer. The final nuclear fraction was obtained by centrifugation at 14,000 × g for 10 minutes at 4°C.

Biochemical analysis in plasma and serum

Insulin levels were measured in plasma using the Ann Arbor ELISA Kit (589501, TX, USA), while glucose concentrations in the same samples were assessed with the Cayman Chemicals ELISA Kit (10009582, CA, USA). Plasma Hemoglobin A1c (HbA1c) levels were determined using the BioTrend Chemicals HbA1c ELISA Kit (# CC-80300, BioTrend Chemicals, Köln, Germany), and serum soluble receptor for advanced glycation end products (sRAGE) was evaluated with the MyBioSource Rat sRAGE ELISA Kit (# MBS7606870, MyBioSource, CA, USA). Serum levels of total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) were measured using assay kits provided by AFG Scientific, IL, USA (EK720559, EK720636, EK720660) and # EK720763, respectively. All measurements were conducted in duplicate for 12 serum samples per group, adhering to the manufacturer’s protocols.

Biochemical analysis in the urine

The levels of albumin and creatinine in the urine were measured using assay kits (80662, AFG Scientific, IL, USA, and # 80340, Crystal Chem). Markers of glomerular and tubular function, including kidney injury molecule-1 (KIM-1), Neutrophil gelatinase-associated lipocalin (NGAL), and nephrin, were assessed using various ELISA kits (# EK720751; AFG Scientific, IL, USA; # 80687, Crystal Chem, CA, USA; and CSB-E13957r, CusaBio, MI, USA, respectively). All measurements were performed in duplicate for 12 urine samples per group, following the manufacturer’s protocols.

Biochemical measurement in the kidney total and nuclear homogenate

The levels of TNF-α were quantified using the ThermoFisher ELISA Kit (# BMS622, Frankfurt, Germany). The levels of Intracellular cell-adhesion molecule-1 (ICAM-1) were measured with the Abcam ELISA Kit (Catalog #ab100763, Cambridge, UK). The levels of TNF-α were quantified using the ThermoFisher ELISA Kit (# BMS622, Frankfurt, Germany), Malondialdehyde (MDA), superoxide dismutase (SOD), heme oxygenase-1 (HO-1), and glutathione reductase (GSH) levels were quantified using ELISA kits from AFG Scientific (Catalog #EK720188, #EK720889, #EK720658, and #EK720424, respectively, IL, USA). NF-κB protein levels in the nuclear extract were measured using the MyBioSource ELISA Kit (Catalog #MBS752046, CA, USA). The activity of Nrf2 in the nuclear extracts was assessed using the Abcam rat-specific ELISA Kit (Catalog #ab2072223, Cambridge, UK), and the levels of Kelch-like ECH-associated protein 1 (Keap1) were quantified with an ELISA kit from MyBioSource (Catalog #MBS7218529m, CA, USA). Interleukin-6 (IL-6) levels were quantified with a kit from Lab Science (Catalog #E-EL-R0015, TX, USA). Each experimental group consisted of 12 samples, and all procedures were performed strictly according to the protocols provided by the respective kit manufacturers.

Real-time polymerase chain reaction (qPCR)

To assess the mRNA expression of Keap1 and Nrf2 in kidney tissues, we utilized a quantitative Real-Time PCR (qPCR) approach. RNA was extracted from frozen kidney samples with the RNeasy Mini Kit (Catalog #74004, Qiagen, Hilden, Germany), and its integrity and concentration were evaluated using a Nanodrop spectrophotometer, which measured absorbance ratios at 260/280 nm. cDNA synthesis was carried out using the Thermo Fisher cDNA Synthesis Kit (Catalog #K1621, Thermo Fisher, Waltham, MA, USA). The following primers were designed for the amplification of the target genes: Keap1 was amplified with the forward primer 5′-CTTCGGGGAGGAGGAGTTCT-3′ and reverse primer 5′-CGTTCAGATCATCGCGGCTG-3′, producing a 132 base pair product (Accession #NM_057152.2). For Nrf2, the forward primer was 5′-AAAATCATTAACCTCCCTGTTGAT-3′, and the reverse primer was 5′-CGGCGACTTTATTCTTACCTCTC-3′, with an amplicon size of 118 base pairs (Accession #NM_031789). β-actin was used as a reference gene with the forward primer 5′-GACCTCTATGCCAACACAGT-3′ and reverse primer 5′-CACCAATCCACACAGAGTAC-3′, generating a 154 base pair product (Accession #NM_031144). The qPCR reactions were carried out on a Bio-Rad system using the Ssofast EvaGreen Supermix (Catalog #172–5200, Bio-Rad, Hercules, CA, USA). Each 20 µL reaction included 10 µL of EvaGreen master mix, 0.2 µL of each primer (final concentration of 500 nM), 2 µL of cDNA template (50 ng final concentration), and 7.6 µL of nuclease-free water. The amplification protocol began with an initial denaturation step at 98°C for 30 seconds, followed by 40 cycles at 98°C for 5 seconds and 60°C for 5 seconds. A melting curve analysis was performed to verify amplification specificity. mRNA expression levels of Keap1 and Nrf2 were normalized to β-actin using the ΔΔCt method. Data were analyzed using the qPCR system software, strictly following the manufacturer’s protocols.

Histological study

Kidney tissues were fixed in formalin, dehydrated through graded ethanol, embedded in paraffin, and sectioned at 5–7 µm. Sections were mounted on slides, deparaffinized with xylene, and rehydrated using a reverse ethanol gradient. Slides were stained with hematoxylin for 5–10 minutes and then eosin for 1–3 minutes to stain the cytoplasm and extracellular matrix, dehydrated through graded ethanol, cleared with xylene, and mounted. Histological analysis was performed using an Olympus CX23 light microscope (Olympus Corporation, Tokyo, Japan). Histological sections were coded before microscopic evaluation, and histological analysis was performed by investigators blinded to treatment group allocation until the analyses were completed.

Statistical analysis

GraphPad Prism software version 8 performed statistical analyses of all data. Before statistical analysis, the normality of the data was assessed using the Kolmogorov-Smirnov test. Analysis was done by two-way ANOVA, and Tukey’s post hoc test was used to determine a significant difference with a p-value less than 0.05. For all datasets, data are expressed as mean ± SD.

Results

Comparative effects of Cafestol and Brusatol on metabolic and lipid parameters

According to Table 1, Control + Cafestol (10 mg/kg) showed no significant changes in body weight, food intake, and plasma glucose, insulin, or HbA1c levels, nor serum levels of total TGs, total CHOL, LDL-c, FFAs, and HDL-c compared to the control group. Conversely, T1DM rats showed significantly lower body weight, higher food intake, and increased plasma glucose, insulin, and HbA1c compared to control rats. Additionally, T1DM rats had significantly elevated serum total TGs, CHOL, LDL-c, and FFAs and showed significantly lower HDL-c than control rats. However, no significant changes in the levels of these parameters were observed when comparing T1DM + Cafestol (5 mg/kg) and T1DM + Cafestol (10 mg/kg) with each other, or with T1DM + Cafestol (10 mg/kg) + Brusatol (2 mg/kg). Moreover, no significant differences were depicted in body weights, food intake, plasma glucose, insulin, HbA1c, and lipid profile when all treated groups were compared with the T1DM model rats.

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Table 1. Changes in food intake and final body weights, as well as alterations in some diabetic indicators and lipid profiles in all experimental groups.

https://doi.org/10.1371/journal.pone.0349192.t001

Cafestol enhances urine output and ameliorates kidney injury markers in T1DM rats in a dose-dependent and Nrf2 Manner

Fig 1 indicates that compared to the control group, administration of Cafestol (10 mg/kg) alone does not induce significant alterations in 24-hour urine volume, urinary albumin levels, or urinary creatinine levels, as well as in urinary markers of glomerular and tubular injury (KIM-1, NGAL, and nephrin), suggesting that Cafestol has no adverse effect on renal function in healthy rats. In contrast, T1DM induction led to a substantial increase in 24-hour urine volume, urinary albumin levels, and urinary creatinine levels, along with a pronounced and significant elevation in urinary levels of KIM-1, NGAL, and nephrin, indicating severe glomerular and tubular damage.

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Fig 1. Effects of Cafestol on renal function and injury markers in control and diabetic rats.

Data are presented as mean ± SD (n = 8 rats per group). Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test. Statistical significance was accepted at p < 0.05. a significantly different from the Control group; b significantly different from the Control + Cafestol (10 mg/kg) group; c significantly different from the T1DM group; d significantly different from the T1DM + Cafestol (5 mg/kg) group; e significantly different from the T1DM + Cafestol (10 mg/kg) group. Brusatol: selective Nrf2 inhibitor.

https://doi.org/10.1371/journal.pone.0349192.g001

Fig 1 also shows that treatment group with Cafestol at both 5 mg/kg and 10 mg/kg doses in T1DM rats significantly mitigated these pathological changes, as evidenced by a dose-dependent reduction in urine output and urinary levels of albumin, creatinine, KIM-1, NGAL, and nephrin levels, suggesting an overall renoprotective effect in diabetic conditions with the highest dose of Cafestol. However, when Brusatol, an Nrf2 inhibitor, was co-administered with Cafestol (10 mg/kg), the beneficial effects of Cafestol were largely reversed. Specifically, a combination of Brusatol with Cafestol led to a significant increase in 24-hour urine volume, urinary albumin, and urinary creatinine, along with an elevation in urinary KIM-1, NGAL, and nephrin levels. Notably, the 24-hour urine output and the urinary levels of all the above markers were not significantly different when T1DM rats were compared with T1DM + Cafestol (10 mg/kg) + Brusatol-treated rats

Cafestol Attenuates renal inflammation in T1DM Rats in a dose-dependent manner through Nrf2 activation

Fig 2 shows no significant alteration in renal total homogenate levels of major inflammatory markers, including IL-6, TNF-α, and ICAM-1, nor in the nuclear NF-κB levels between Control and Cafestol (10 mg/kg)-treated rats. The renal homogenates of T1DM rats demonstrated a significant inflammatory response, marked by a pronounced upregulation of IL-6, TNF-α, and ICAM-1, along with increased nuclear NF-κB activation in comparison to Control rats. This indicates a heightened state of renal inflammation and an intensified activation of pro-inflammatory signaling pathways. Compared with T1DM rats, Cafestol treatment at 5 mg/kg and 10 mg/kg significantly mitigated these inflammatory changes, as evidenced by a dose-dependent reduction in renal levels of IL-6, TNF-α, ICAM-1, and nuclear NF-κB (Fig 2A-D). The data suggests that Cafestol exerts an anti-inflammatory effect, possibly through modulation of NF-κB signaling. However, the co-administration of Brusatol, an Nrf2 inhibitor, with Cafestol (10 mg/kg) in T1DM rats reversed these protective effects as compared to T1DM + Cafestol (5 and 10 mg/kg), where there were significant increases in the renal levels of IL-6, TNF-α, ICAM-1, and nuclear NF-κB levels (Fig 2A-D). In addition, the renal levels of all these inflammatory markers showed no significant changes when T1DM rats were compared with T1DM + Cafestol (10 mg/kg) + Brusatol rats.

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Fig 2. Effects of Cafestol on renal inflammatory markers in control and diabetic rats.

Data are presented as mean ± SD (n = 8 rats per group). Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test. Statistical significance was accepted at p < 0.05. a significantly different from the Control group; b significantly different from the Control + Cafestol (10 mg/kg) group; c significantly different from the T1DM group; d significantly different from the T1DM + Cafestol (5 mg/kg) group; e significantly different from the T1DM + Cafestol (10 mg/kg) group. Brusatol: selective Nrf2 inhibitor.

https://doi.org/10.1371/journal.pone.0349192.g002

Cafestol attenuates T1DM-induced renal oxidative stress and glycation markers via Nrf2 activation in a dose-dependent manner

The administration of Cafestol (10 mg/kg) to control rats significantly lowered levels of advanced glycation end products (AGEs) and soluble receptor for advanced glycation end products (sRAGEs), as well as renal homogenate levels of malondialdehyde (MDA), as compared to Control rats, as shown in Fig 3. It also significantly increased renal homogenate levels of GSH, HO-1, and SOD as compared to control rats (Fig 3A-D). However, in Type 1 diabetes mellitus (T1DM) rats, a profound increase in serum levels of AGEs and sRAGEs, alongside renal levels of MDA, was observed, accompanied by a marked reduction in HO-1, SOD, and GSH levels as compared to Control rats, reinforcing the notion of severe oxidative imbalance and compromised antioxidant defenses in diabetic conditions. When compared to T1DM rats, the administration of Cafestol at either 5 mg/kg or 10 mg/kg to T1DM rats significantly ameliorated these pathological alterations, as evidenced by the significant decline in serum levels of AGEs and sRAGEs, reduction in renal MDA levels, and the substantial restoration of HO-1, SOD, and GSH levels (Fig 3). However, these effects were dose-dependent and were more profound on all parameters with the 10 mg/kg dose of Cafestol.

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Fig 3. Effects of Cafestol on renal oxidative stress/antioxidant markers in control and diabetic rats.

Data are presented as mean ± SD (n = 8 rats per group). Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test. Statistical significance was accepted at p < 0.05. a significantly different from the Control group; b significantly different from the Control + Cafestol (10 mg/kg) group; c significantly different from the T1DM group; d significantly different from the T1DM + Cafestol (5 mg/kg) group; e significantly different from the T1DM + Cafestol (10 mg/kg) group. Brusatol: selective Nrf2 inhibitor.

https://doi.org/10.1371/journal.pone.0349192.g003

These findings underscore potent antioxidative and glycation-inhibitory properties of Cafestol in a dose-dependent manner, highlighting its therapeutic potential in mitigating diabetes-induced oxidative stress and related complications. Importantly, co-administration of Brusatol along with Cafestol in T1DM rats completely abrogated the protective effects of this drug, as reflected by the re-elevation of serum levels of AGEs, sRAGEs, and renal MDA levels that were coupled with the significant depletion of HO-1, SOD, and GSH as compared to T1DM rats that were treated with the 5 or 10 mg/kg Cafestol.

Fig 3 also shows that no significant changes were observed in the serum and renal levels of all these markers when T1DM + Cafestol (10 mg/kg) + Brusatol rats were compared to T1DM rats. This reversal strongly indicates that the beneficial effects of Cafestol on oxidative stress and glycation markers are mechanistically dependent on Nrf2 activation.

Cafestol activates renal Nrf2 cytoplasmic and nuclear translocation by downregulating Keap1 in both control and T1DM rats

Fig 4 displays Cafestol Activates Renal Nrf2 Cytoplasmic and Nuclear Translocation by downregulating Keap1 in Both Control and T1DM Rats. In control rats, administration of Cafestol (10 mg/kg) did not significantly alter renal mRNA levels of Nrf2 but significantly reduced Keap1 mRNA expression, which was accompanied by marked increases in both cytoplasmic and nuclear Nrf2 protein levels, indicating that Cafestol activates basal Nrf2 signaling under physiological conditions. In contrast, T1DM rats exhibited a substantial upregulation of Keap1 mRNA expression compared to control rats, accompanied by a significant reduction in renal cytoplasmic and nuclear Nrf2 protein levels, despite no apparent changes in Nrf2 mRNA expression. The administration of Cafestol at either 5 mg/kg or 10 mg/kg to T1DM rats significantly mitigated these alterations, as evidenced by a marked reduction in Keap1 mRNA expression and a concomitant increase in nuclear Nrf2 protein levels, despite no significant changes in Nrf2 mRNA expression (Fig 4). This restoration of Nrf2 signaling by Cafestol was dose-dependent, with the 10 mg/kg dose exerting a more pronounced effect. However, co-administration of Brusatol, an Nrf2 inhibitor, with Cafestol (10 mg/kg) in T1DM rats abolished the Cafestol-induced reduction in Keap1 mRNA expression and the increments in the cytoplasmic and nuclear Nrf2 protein levels (Fig 4A-D). This reversal strongly indicates that the protective effects of Cafestol on Nrf2 signalling are mediated through an Nrf2-dependent mechanism, further supporting its role in enhancing Nrf2 nuclear translocation and activity in the diabetic kidney.

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Fig 4. Effects of Cafestol on renal Keap1/Nrf2 pathways in control and diabetic rats.

Data are presented as mean ± SD (n = 8 rats per group). Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test. Statistical significance was accepted at p < 0.05. a significantly different from the Control group; b significantly different from the Control + Cafestol (10 mg/kg) group; c significantly different from the T1DM group; d significantly different from the T1DM + Cafestol (5 mg/kg) group; e significantly different from the T1DM + Cafestol (10 mg/kg) group. Brusatol: selective Nrf2 inhibitor.

https://doi.org/10.1371/journal.pone.0349192.g004

Discussion

The findings from the streptozotocin-induced T1DM rat model elucidate the characteristic pathophysiological features of untreated T1DM, including cachexia, increased food consumption, hyperglycemia, hyperinsulinemia, elevated HbA1c levels, and dyslipidemia. Cafestol supplementation at 10 mg/kg did not significantly alter these parameters in non-diabetic controls, indicating its neutral effect in healthy states. In T1DM models, cafestol (5 or 10 mg/kg) and its combination with brusatol failed to improve body weight, food intake, glycemia, or lipid profiles, suggesting limitations related to dosage, duration, or model-specific factors affecting beta-cell function [34,35]. While cafestol shows efficacy in type 2 diabetes models by lowering glucose and improving insulin sensitivity, its effects in T1DM appear limited, primarily benefiting insulin-resistant states rather than addressing severe hyperglycemia or oxidative stress pathways [36].

Diabetes mellitus induces hyperglycemia, which can progress to micro- and macrovascular problems and ultimately become a primary cause of death. Since increased ATP dephosphorylation following streptozotocin administration provides a substrate for xanthine oxidase, which in turn produces superoxide radicals, hydrogen peroxide, and hydroxyl radicals, the mechanism of STZ as a diabetogenic agent is mediated by reactive oxygen species [37]. Hyperglycemia-driven oxidative stress underlies much of the diabetic pathology observed here. STZ’s diabetogenic effect involves ATP depletion and excessive ROS production via xanthine oxidase activation [38,39]. Our T1DM group exhibited hallmark metabolic derangements and dyslipidemia consistent with prior reports, including elevated triglycerides, cholesterol, LDL, and free fatty acids alongside reduced HDL [40–42].

Results revealed that the control group’s metabolic parameters were not significantly altered by cafestol (10 mg/kg), indicating that cafestol at this dosage does not significantly disrupt normal physiological or metabolic homeostasis [23]. A comparison of T1DM + Cafestol (10 mg/kg) with T1DM + Cafestol (5 mg/kg) did not show any significant difference in the metabolic and lipid parameters. Similarly, in groups of T1DM + Cafestol (10 mg/kg) + Brusatol (2 mg/kg). All the cafestol-treated groups, when compared with untreated T1DM models, did not display significant differences in terms of body weight, food intake, plasma glucose, insulin, HbA1c, and lipid parameters. The findings suggest that neither cafestol alone nor cafestol together with brusatol significantly ameliorated the metabolic and lipid abnormalities associated with T1DM at the dosages tested in the study. It has been observed that the beneficial impact of certain compounds on metabolic parameters using the STZ model [43,44]. This requires a further investigation of the specific mechanism and dose of cafestol in the STZ-induced diabetes model.

The results show that Cafestol, at 10 mg/kg, does not adversely affect renal function in healthy rats, as indicated by stable 24-hour urine volume, urinary albumin, creatinine, and markers of glomerular and tubular injury (KIM-1, NGAL, nephrin). However, in T1DM rats, Cafestol treatment at 5 and 10 mg/kg reduced the pathological increases in these markers and urine output, demonstrating a dose-dependent renoprotective effect. This protective action is likely mediated through activation of the Nrf2 pathway, a key regulator of antioxidant defense that mitigates oxidative stress and inflammation in diabetic nephropathy [45]. Co-administration of Brusatol, an Nrf2 inhibitor, reversed the protective effects of Cafestol, leading to kidney injury markers and urine output like untreated diabetic rats, illustrating the importance of Nrf2 activation in Cafestol’s renoprotection. Modern studies support that Cafestol activates Nrf2, which in diabetic nephropathy reduces oxidative damage and fibrosis, consistent with the observed functional and biochemical improvements [46,47]. On the other hand, blocking Nrf2 makes kidney damage worse in diabetes by letting oxidative stress and fibrotic signaling go unchecked, as shown by Brusatol co-treatment [48].

Findings showed that in the control group, cafestol (10 mg/kg) alone could not induce significant changes in 24-hour urine volume, urinary albumin, and urinary concentrations of creatinine. Similarly, urinary glomerular and tubular injury markers include KIM-1, NGAL, and nephrin. The evidence proved that cafestol at this level does not harm the renal function of healthy rats [23]. T1DM, on the other hand, caused significant renal impairment, resulting in an increase in 24-hour urine volume, urinary albumin, and urinary creatinine. Urinary KIM-1, NGAL, and nephrin levels were also markedly increased, showing generalised glomerular and tubular damage and conforming to diabetic nephropathy models [49,50]. There has been considerable use of the biomarkers for early detection of diabetic kidney injury [46]. These pathological changes were reduced in T1DM treated with cafestol at doses of 5 mg/kg and 10 mg/kg in a dose-dependent manner. This nephroprotection was demonstrated by decreasing urine output and urinary excretion of albumin, creatinine, KIM-1, NGAL, and nephrin; the 10 mg/kg dose produced the greatest benefit, which implies that cafestol has therapeutic potential for preventing DN.

The findings indicate that Cafestol markedly alleviates renal inflammation in T1DM rats by dose-dependently decreasing critical pro-inflammatory markers IL-6, TNF-α, ICAM-1, and nuclear NF-κB levels, underscoring its anti-inflammatory function in diabetic nephropathy. These findings correspond with studies indicating that Cafestol suppresses pro-inflammatory cytokines and endothelial adhesion molecules by modulating NF-κB signaling, a pivotal mechanism in the inflammation and progression of diabetic kidney disease [34,51]. The simultaneous administration of Brusatol, an Nrf2 inhibitor, negates the protective benefits of Cafestol, highlighting the role of Nrf2 signaling in this anti-inflammatory process [51]. Nrf2 activation is recognized for its ability to inhibit NF-κB-mediated inflammatory responses by augmenting antioxidant defenses and diminishing oxidative stress, both of which are pivotal in diabetes-induced kidney inflammation [52,53]. Cafestol’s renal protective effects in diabetes seem to function through a crosstalk mechanism that involves Nrf2-mediated suppression of NF-κB signaling pathways, aligning with the current comprehension of diabetic nephropathy pathophysiology and the anti-inflammatory capabilities of Nrf2 activators.

Cafestol’s renoprotective effects rely on the Nrf2 pathway; brusatol inhibits Nrf2, blocking cafestol’s benefits and increasing kidney injury markers in diabetic rats. This finding has profound implications that cafestol does work to exert its renoprotective action, at least partially, by inducing the activation of the Nrf2 signaling pathway that is of vital importance in antioxidant defense and against oxidative stress and inflammation of DN [22,49,54]. Our findings elucidate the vast anti-inflammatory potential of Cafestol in T1DM-induced kidney inflammation, in addition to Nrf2 signaling causality in regulating such protective effects. To begin with, we observed that cafestol (10 mg/kg) administered to healthy rats did not initiate any measurable changes in the renal total homogenate level of significant inflammatory markers. This means that the tested dose of cafestol does not induce an inflammatory response in healthy renal tissue, and this also agrees with previous experiments demonstrating its safety and lack of pro-inflammatory effects in non-diseased states [29]. However, in renal homogenates, T1DM rats displayed an inflammatory response in contrast to controls, with markedly elevated nuclear NF-κB activation and levels of IL-6, TNF-α, and ICAM-1 expression. This is in line with the established pathophysiology of DN and implies increased renal inflammation and activation of the pro-inflammatory signalling cascade [55,56]. By encouraging cellular dysfunction and fibrosis, elevated levels of these inflammatory markers, specifically TNF-α and IL-6, are known to result in diabetic kidney injury [57,58].

Interestingly, both 5 mg/kg and 10 mg/kg of cafestol were effective in halting these inflammatory changes in the T1DM group. The potent anti-inflammatory effects of cafestol were demonstrated by the dose-dependent reduction of renal IL-6, TNF-α, ICAM-1, and nuclear NF-κB. This suggests that cafestol affects the inflammatory environment, potentially through blocking NF-κB signalling, a crucial modulator of the expression of inflammatory genes [59]. This is consistent with studies that demonstrate that cafestol inhibits inflammatory mediators in various disease models, typically due to its antioxidant properties and alteration of inflammatory processes [21,60]. Further mechanistic analysis revealed that Nrf2 signalling, when Brusatol and Cafestol (10 mg/kg) were given together, the protective effects were removed in the T1DM group. Reversal was indicated by the sharp rise in renal levels of IL-6, TNF-α, ICAM-1, and nuclear NF-κB compared to the levels of untreated T1DM.

This clearly shows that the anti-inflammatory activity of Cafestol occurs due to its activation of the Nrf2 pathway. Nrf2 is a key transcription factor that helps the body fight against oxidative stress. Cafestol can activate it, and this leads to its signalling pathways that can help to induce protection from diabetic nephropathy [61,62]. Studies have shown the complex crosstalk between Nrf2 and NF-κB pathways. Further, Nrf2 activation can inhibit NF-κB activity and downregulate the biosynthesis of pro-inflammatory cytokines [63]. In line with the present findings, evidence for such an interaction pertaining to the therapeutic beneficial action of Cafestol is strong.

The administration of Cafestol (10 mg/kg) in control rats reduced serum AGEs and sRAGEs, as well as kidney MDA, while increasing antioxidant indicators such as GSH, HO-1, and SOD, thus illustrating its antioxidative and anti-glycation properties. In T1DM rats, elevated serum AGEs, sRAGEs, and renal MDA, together with decreased antioxidant indicators, signified substantial oxidative stress and lower antioxidant capability typical of diabetes. Cafestol administration dose-dependently ameliorated these anomalies in T1DM mice, reducing oxidative damage and reinstating antioxidant enzyme levels, aligning with its function in alleviating diabetes-induced oxidative stress and glycation [21,64]. The elimination of these protective benefits with the co-administration of Brusatol, an HO-1 inhibitor, substantiates the essential function of HO-1 induction in Cafestol’s mode of action [64]. These results correspond with recent research emphasizing Cafestol’s capacity to augment antioxidant defenses, diminish glycation end products, and facilitate glucose metabolism regulation in diabetic models, thereby reinforcing its therapeutic potential for managing oxidative and glycation stress associated with diabetes [45,47,65].

Cafestol diminishes Keap1, thereby augmenting Nrf2 signaling and antioxidant defense in both normal and diabetic rats; Brusatol inhibits this effect, validating Cafestol’s Nrf2-dependent protective mechanism. This effect aligns with recent studies demonstrating Cafestol’s potential to promote Nrf2 nuclear translocation and subsequent antioxidant gene expression, therefore mitigating oxidative stress and inflammation in renal and cardiac tissues [23,64]. The regulation of the Keap1/Nrf2 pathway is an established therapeutic target for diabetic complications, as Nrf2 activation alleviates oxidative damage associated with diabetic nephropathy [66,67].

When compared to the T1DM group, T1DM + Cafestol (10 mg/kg) + Brusatol rats showed no significant change in these markers in the kidney. Comparing these two models reinforces the idea that the Nrf2 pathway is a major mediator of Cafestol’s anti-inflammatory effects since blocking the Nrf2 pathway abolishes the protective effects and results in an inflammatory pattern comparable to that seen under diabetic conditions, where Nrf2 activation is lost or impaired. As per the above results, the Nrf2-dependent anti-inflammatory mechanism of cafestol has potential in combating DN. The current study has some limitations. First, mechanistic conclusions have been drawn mainly from a streptozotocin-induced T1DM rat model and the use of the pharmacological inhibitor, Brusatol; thus, while these data support the hypothesized role of the renal Keap1-Nrf2 axis in mediating the effects of cafestol on renal Nrf2 activation, experimental validation at the cellular level via gene-specific methods (i.e., siRNA or CRISPR/Cas9) to confirm Nrf2 activation induced by cafestol in renal cells subjected to high-glucose conditions was not performed. Second, although changes in Nrf2 levels within both the cytoplasmic and nuclear compartments of the kidney were measured in fractionated extracts from the kidney (and shown to correlate with corresponding changes in expression of Keap1 and antioxidant markers), the level of nuclear Nrf2 was not validated by either Western blot or immunofluorescent analysis, both of which would have provided stronger, more semi-quantitative support for Nrf2 redistribution at the subcellular level. Third, a limited panel of antioxidant mediators downstream of Nrf2 (HO-1, SOD, and GSH) were evaluated, as opposed to performing a more extensive analysis of the classical Nrf2 targets at either the mRNA or protein level. Lastly, while histological evaluations were performed, the majority of these analyses were qualitative in nature and morphometric determinations of several renal brunches (e.g., glomerular volume, mesangial expansion, and tubular injury scores) were not completed. Fifthly, even though the initial doses selected for cafestol (5 mg/kg and 10 mg/kg) were based on their preliminary dose-response characteristics as well as previous empirical support, there were no plasma or renal tissue concentrations of cafestol assessed; therefore, we cannot definitively prove that these dosing levels have direct physiological relevance or translational equivalence. Also, the design of this study was for an endpoint design over an 8 weeks period, which was relevant to the development of diabetic nephropathy in the present model system; however, there was no time-point analysis so it is unknown what temporal relationship Nrf2 activation has to later changes in renal function, oxidative stress, and inflammation. Future studies should include renal cell-type specific genetic validation, broader profiling of Nrf2 regulated genes, quantitative analysis of renal morphology, pharmacokinetic evaluation and time course analysis of how long it takes for cafestol to exert its renoprotective effects (at intervals of 2 weeks up until 2 weeks and 4 weeks) to properly establish the molecular and temporal dynamics of cafestol mediated renoprotection

Conclusions

The present findings reveal that streptozotocin-induced T1DM is associated with severe metabolic, lipid, renal, and inflammatory disorders, which are consistent with previous diabetic models. Although cafestol did not improve hyperglycemia, dyslipidemia, or other systemic metabolic disorders related to T1DM at the doses tested, it had significant renoprotective and anti-inflammatory actions. Cafestol significantly decreased the parameters of diabetic nephropathy, such as albuminuria, urinary injury biomarkers (KIM-1, NGAL, nephrin), and pro-inflammatory mediators (IL-6, TNF-α, ICAM-1, and NF-κB activation) in a dose-dependent fashion. These protective actions were completely lost by the concomitant administration of brusatol, an Nrf2 inhibitor, indicating that the beneficial renal and anti-inflammatory effects of cafestol are largely exerted through the activation of the Nrf2 signaling pathway. Collectively, the data indicates that while cafestol does not restore systemic metabolic dysfunction in T1DM, it nonetheless exerts significant protection against renal oxidative and inflammatory insult. The outcome underlines the therapeutic potential of cafestol as a Nrf2-dependent renoprotective agent in diabetic nephropathy and justifies further study on optimal dosing, molecular mechanisms, and applicability in broader diabetic contexts.

Supporting information

S1 File. Orginal data- cafestol.

https://doi.org/10.1371/journal.pone.0349192.s001

(PDF)

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