Pharmacological Antagonism of HTR2B Reduces ERK and STAT3 Activation and Prevents Renal Fibrosis During the Development of Adenine-Induced CKD in Rats

Abstract

Chronic kidney disease (CKD) is a debilitating condition affecting roughly 1 in 7 people in the United States. CKD is characterized by loss of renal function due to nephron loss and fibrosis. MARY1 is a novel 5-hydroxytryptamine 2B receptor (HTR2B) antagonist that stimulates mitochondrial biogenesis and augments recovery after ischemia/reperfusion-induced acute kidney injury (AKI) in vivo. We evaluated the potential of MARY1 to prevent CKD development in an adenine-induced rat model. Rats were given a normal diet or a diet supplemented with 0.2% adenine for 4 weeks to induce CKD. Adenine-fed rats were simultaneously administered either MARY1 (0.15 mg/kg, IP) or vehicle daily throughout the 4 weeks. Adenine diet resulted in renal cortical fibrosis and upregulation of two kidney damage markers: kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), consistent with CKD development. MARY1 mitigated the induction of KIM-1 and NGAL by 52% and 68%, respectively. Nicotinamide adenine dinucleotide (NAD+) was reduced by 69% in vehicle-treated adenine-exposed rats, which was prevented by MARY1. Adenine increased activation of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3), resulting in increased expression of the pro-inflammatory transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). MARY1 reduced activation of ERK and STAT3, and mitigated NF-κB upregulation by 64%. MARY1 also attenuated adenine-induced fibrosis in the renal cortex by 43%. These findings support HTR2B antagonism as a novel strategy for preventing CKD-associated mitochondrial dysfunction, renal inflammation, and fibrosis during disease development.

Introduction

Chronic kidney disease (CKD) is a debilitating condition that compromises renal function through nephron loss and accumulation of fibrotic burden [1–3]. The Centers for Disease Control and Prevention (CDC) estimates that CKD affects approximately 1 in 7 Americans, with the prevalence expected to rise as the aging population grows. Moreover, common conditions such as diabetes, hypertension, obesity, and heart disease increase the risk of developing CKD [3–5]. Despite this, therapies to treat this disease remain limited, and those that do exist are often expensive and invasive [2, 6, 7]. Dialysis, the most common treatment option for advanced CKD, accounts for 20% of Medicare costs in the United States, while also increasing the risk of cardiovascular complications and imposing an enormous burden on the patient [8, 9]. This underscores the dire need for novel therapeutics to address this increasingly common disease.

CKD is a complex disease potentiated by a broad constellation of factors [1, 2]. Mitochondrial dysfunction is known to play a vital role in the etiology of CKD [10]. Mitochondria are essential to kidney function, supplying energy in the form of adenosine triphosphate (ATP) to tubule cells, fueling the filtration/reabsorption processes necessary for the generation of urine and excretion of waste products [10–13]. In CKD, mitochondria are dysfunctional, leading to reduced ATP and increased reactive oxygen species (ROS) generation, driving a pro-inflammatory environment that ultimately promotes renal fibrosis and cell death [10, 12]. This creates a vicious positive feedback cycle that propagates the onset/progression of CKD [10].

Pharmacological induction of mitochondrial biogenesis (MB), the de novo generation of new and healthy mitochondria, holds promise as a potential therapeutic mechanism for diseases characterized by mitochondrial dysfunction, such as CKD. Our laboratory demonstrated that MB induction restores mitochondrial function and renal recovery in animal models of kidney disease[14–17]. Specifically, we showed that treatment with MARY1 (2,4,6-trifluoro-N-(6-(4-methylpiperazine-1-carbonyl) pyridin-2-yl) benzamide), a novel piperazine-based compound and potent, selective HTR2B antagonist, promotes renal MB and improves mitochondrial and kidney function after bilateral ischemia/reperfusion-induced AKI [18, 19]. MARY1 was also shown to improve ATP generation in the renal cortices of aged mice [20]. Furthermore, antagonism and ablation of HTR2B have been shown to have anti-fibrotic effects in both the liver and the heart [21, 22].

Here, we examined the therapeutic potential of HTR2B antagonism in preventing sequelae of CKD using an adenine-induced rat model. The aim of this study was to evaluate the efficacy of MARY1 in preventing the onset of CKD and to elucidate the mechanism by which it exerts these effects.

Materials and Methods

MARY1:

The synthetic route and physical characterizations (NMR, LCMS, HPLC) for MARY1 have been previously reported [19]. MARY1 selectively binds to HTR2B with moderate affinity. There is no significant binding to other HTRs, including HTR2A and HTR2C. The characterization of primary and secondary binding affinity of MARY1 across 45 human GPCRs is reported in Santiago et al. (2025) [19]. A daily dose of 0.15 mg/kg IP was chosen based on a naïve dose-response study that indicated this as the lowest effective concentration for induction of MB in the rat renal cortex [19]. Rats receiving vehicle were given a saline injection containing 0.1% DMSO. Treatments were administered daily via intraperitoneal injection.

Adenine Diet:

Dietary adenine exposure was achieved using a 0.2% adenine-supplemented chow for 4 weeks. (Research Diets, Inc; New Brunswick, NJ; Cat.#: D25040401). Control chow was also purchased from Research Diets, Inc. (Research Diets, Inc; New Brunswick, NJ; Cat.#: D16050201)

Animals:

8-week-old male Sprague-Dawley rats were purchased from Charles River Laboratories (Hollister, CA; Cat.#: Crl:CD(SD)-001). All animal experimentation was in compliance with ARRIVE guidelines and performed with rigorous adherence to the recommendations outlined in “The Guide for the Care and Use of Laboratory Animals,” which is curated and distributed by the National Institutes of Health (NIH) [23, 24]. All methodology was reviewed and authorized by the University of Arizona Institutional Animal Care and Use Committee (IACUC). All necessary precautions were followed to reduce animal distress. All animals in this study were housed in groups of 3 or less in temperature-controlled rooms (22° ± 2°C) with a 12-h light-dark cycle and food/water were supplied ad libitum.

Serum creatinine (SCr):

Blood was collected retro-orbitally, kept at room temperature for approximately 30 min, centrifuged at 10,000 rpm for 10 min, and the serum collected. SCr was subsequently measured as previously described [25, 26] with an enzymatic creatinine reagent assay kit (Diazyme; Poway, CA; Cat.#: 7548–120) per manufacturer’s protocol.

Blood Urea Nitrogen (BUN):

BUN was measured from serum using the QuantiChrom Urea Assay Kit (BioAssay Systems; Hayward, CA; Cat.#: DIUR-100) per manufacturer’s protocol.

Albumin to Creatinine Ratio (ACR):

To calculate ACR, urinary albumin was measured using an Exocell albumin ELISA assay (Ethos Biosciences; Logan Township; NJ; Cat.#: NC9182134) per manufacturer’s protocol, and urinary creatinine was determined using the murine creatinine enzymatic reagent assay kit described above.

Protein Isolation and Immunoblot Analyses:

Protein from rat renal cortices was extracted in RIPA buffer composed of 50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 1% Triton X-100 (pH 7.4). Halt Protease and Phosphatase Inhibitor Cocktail (1:100; Thermofisher, Waltham, MA; Cat# 78447) was added to the buffer. Protein concentration was determined using a bicinchoninic acid (BCA) assay. Gel electrophoresis and immunoblot analyses were conducted as previously described [27, 28]. Membranes were blocked in 5% milk or 3% BSA in TBST and then incubated overnight at 4°C with primary antibodies under constant agitation. Following incubation with the appropriate horseradish peroxidase-conjugated secondary antibody, protein bands were detected by chemiluminescence (Thermo Scientific, Waltham, MA) using an Azure 600 imaging system (RRID:SCR_023780; Dublin, CA; Cat# AZI600–01). Band optical density was quantified with ImageStudio Lite software. Antibodies used for immunoblot analyses are listed in Table I.

Table I.

Antibodies

Antibody	Dilution	RRID
KIM-1	1:1,000	AB_2116446
NGAL	1:1,000	AB_3665857
PGC-1α	1:1,000	AB_2721267
MFF	1:1,000	AB_2728769
OPA1	1:1,000	AB_2864313
MFN2	1:1,000	AB_10999860
Total OXPHOS antibody cocktail	1:250	AB_2629281
PKC	1:1,000	AB_2799309
PKA	1:1,000	AB_2170170
Phospho-ERK	1:1,000	AB_2315112
ERK	1:1,000	AB_390779
Phospho-STAT3	1:1,000	AB_2491009
STAT3	1:1,000	AB_331757
TNFR2	1:1,000	AB_2809895
NF-κB	1:1,000	AB_776751
VCAM1	1:1,000	AB_2799146
MCP-1	1:1,000	AB_2893318
TGF-β	1:1,000	AB_3670503
α -SMA	1:10,000	AB_11129103
PFKFB3	1:1,000	AB_3095816
MMP9	1:1,000	AB_3714520
PDGFRβ	1:1,000	AB_2162497
Vimentin	1:1,000	AB_2273020
Collagen 1	1:1,000	AB_2922767
Collagen 3	1:1,000	AB_2895112
GAPDH	1:1,000	AB_1642205
β-Actin	1:1,000	AB_2242334
Goat Anti-Rabbit IgG H&L (HRP)	1:10,000	AB_955447
Rabbit Anti-Mouse IgG H&L (HRP)	1:10,000	AB_955440

NAD Assay:

NAD was measured in rat renal cortices using an NAD Assay Kit (Bio-Assay Systems; Hayward, CA; EnzyChrom NAD/NADH Assay Kit; Cat.#: E2ND-100) according to the manufacturer’s directions.

ATP Content:

ATP was measured in rat renal cortices using the ATP Assay kit (Abcam; Cambridge, MA; Colorimetric/Fluorometric Assay; Cat.#: ab83355) as per the manufacturer’s directions.

Mitochondrial DNA (mtDNA) Copy Number:

DNA was isolated from the renal cortex using a Qiagen DNeasy Blood and Tissue Kit (Valencia, CA), followed by quantitative polymerase chain reaction (qPCR) quantification of relative mtDNA copy number. Cytochrome B (MT-CYB), a mitochondrial gene, was measured and then normalized to β-Actin, a nuclear encoded gene. The primer sequences used for qPCR analyses can be found in Table II.

Table II.

Primer Sequences

Gene	Forward Sequence	Reverse Sequence
MT-CYB	5′-CATCAGTCACCCACATCTGC	5′-TGGATGGAATGGGATTTTGT
β-Actin	5′-CCCGCGAGTACAACCTTCT	5′-CGTCATCCATGGCGAACT

Histological Staining:

Kidneys were harvested, fixed overnight in ice-cold 4% paraformaldehyde and then stored in 70% ethanol at 4°C. Samples were taken to the University of Arizona Tissue Acquisition and Cellular/Molecular Analysis (TACMAS) core, where they were paraffin embedded and sliced into 4 μm sections. Periodic Acid Schiff (PAS) histological staining was performed by the TACMAS core facility. 2,8-Dihydroxyadenine (2,8-DHA) crystal deposition was measured by manual counting of deposited crystals in randomly selected tubular regions of interest (ROIs). Additional slices were stained with Masson’s trichrome to assess tubulointerstitial fibrosis using the MasterTech Trichrome Masson’s Stain Kit (StatLab; McKinney, TX; Cat#: STLKTMTRPT) according to manufacturer’s protocol. The slides were scanned using MoticEasyScanOne slide scanner (Motic; Emeryville, CA) at 20x. Positive, blue-stained area was assessed based on isolated cortical region in ImageJ using an open-source macro developed in-house.

Collagen Detection/Analysis:

Collagen deposition was analyzed via the University of Arizona Microscopy Shared Resource (MSR). Nikon NIS-Elements AR (v6.10) software with General Analysis 3 (GA3) was used for image processing and analysis. To detect the entire tissue sample, the following functions were used: Gaussian filter (sigma = 20) and RGB threshold with Smooth (strength = 4μm). To remove false positive detections, Remove Objects Touch Borders and Clean (radius = 100μm) functions were used. To detect collagen within the tissue, the HSI Threshold function was used with clean (length = 2μm) and smooth (strength = 1.3μm) filters applied. The Object Count function was used to quantify the total number of collagen detections, sum collagen area, mean blue intensity in collagen positive area (measured on the unprocessed image), and the total tissue area.

Glomeruli Detection/Analysis:

Glomerular number and size were analyzed via the University of Arizona MSR. Nikon NIS-Elements AR (v6.10) software with General Analysis 3 (GA3) and NIS.ai was used for image processing and analysis. To achieve automated glomeruli detection, the SegmentObjects.ai module was utilized to generate a trained classifier for glomeruli identification. For AI training, all glomeruli from 3 kidney slides were manually annotated using the Binary Layers and Binary Toolbar modules. These 3 annotated images were used to create a trained SegmentObjects.ai classifier for 1000 iterations. To refine the glomeruli detections made by the trained SegmentObjects.ai classifier, the following functions were used: Clean filter (Radius = 10μm), Connect Objects (Maximal distance = 2μm), and Fill Holes to fill the entire glomeruli annotation. The Object Count function was used to quantify the mean area and total count of glomeruli in each image files. The Object Measure function was used to quantify the area of each detected glomeruli.

Immunohistochemistry (IHC):

Kidney sections (4 μm) were deparaffinized and IHC staining performed using the VECTASTAIN Elite ABC-HRP Kit (Vector Labs; Scottsdale, AZ; Cat.#: PK6200). Primary antibodies used for IHC staining were: PDGFRβ (1:200; RRID:AB_2809341) and AQP1 (1:400; RRID: AB_2810992). Substrate binding was visualized using Pierce^™ DAB substrate kit (Thermo Fisher Scientific; Waltham, MA; Cat.#: 34002). The slides were scanned using MoticEasyScanOne slide scanner (Motic; Emeryville, CA) at 20x. Slides were analyzed by selecting randomized cortical ROIs and scoring using the Colour Deconvolution plugin for ImageJ [29].

Data and Statistical Analysis:

Tissue/blood/urine isolated from a single animal represent n=1. For all experiments, n=8 rats/group. Prior to analyses, statistical outliers were confirmed via the ROUT outlier test. All data subsequently underwent a Shapiro-Wilk test to determine the normality of distribution. All data that were determined to be normally distributed with no significant differences between the standard deviations underwent an Ordinary one-way ANOVA test followed by a Tukey’s post-hoc test. Alternatively, data that were normally distributed but had significant differences between their standard deviations underwent a Brown-Forsythe one-way ANOVA that was then followed by a Dunnett T3 post-hoc test. Data that did not conform to a normal distribution underwent a nonparametric ANOVA (Kruskal-Wallis H-test followed by a Dunn’s post-hoc test). Data were subsequently analyzed with GraphPad Prism software V10.4.2 (RRID:SCR_002798; La Jolla, CA), and p<0.05 was regarded as statistically significant. Statistical comparisons between groups are represented using Compact Letter Display (CLD), whereby different letters are used to indicate statistically significant differences between means. All data in this study are presented as mean ± SEM (standard error of the mean).

Results

MARY1 administration attenuates renal injury in a rat model of adenine-induced CKD

Male Sprague-Dawley rats were fed either control diet or a 0.2% adenine-supplemented diet for 4 weeks [30–32]. During this time, adenine-fed rats were administered vehicle or MARY1 (0.15 mg/kg; IP) daily. Body weight, blood, and urine were collected at the beginning and end of the study. There was no difference in body weight across groups throughout the 4-week period (Fig. 1A). After 4 weeks, kidney mass was elevated ~1.7-fold in adenine-fed rats compared to controls, regardless of MARY1 treatment (Fig. 1B). Serum BUN, a clinical measurement for assessing renal function [33], increased 3-fold to 42 mg/dL in vehicle-treated adenine-supplemented rats compared to controls. MARY1 administration mitigated this increase by 30% (32 mg/dL) (Fig. 1C). Serum creatinine and urinary ACR, measures of renal function and clearance capacity [1, 2, 34], were elevated ~4- and 4.5-fold, respectively, in adenine-fed rats, with or without MARY1 treatment (Fig. 1D–E).

Figure 1. MARY1 administration attenuates renal injury in a rat model of adenine-induced CKD.

8-week-old rats were fed either a normal chow diet with or without 0.2% adenine-supplementation for 4 weeks. Rats eating an adenine-supplemented diet were treated with vehicle or MARY1 (0.15 mg/kg, IP) daily for 4 weeks, after which the kidneys were weighed and the renal cortices were collected for downstream analyses. Body weight, urine, and serum were collected at the beginning and end of the study. Raw body weight (A) and 12-week kidney-to-body weight ratios (B) were measured. Serum BUN (C), serum creatinine (D), and urinary ACR (E) were measured to assess renal function. KIM-1 (F) and NGAL (G) were measured to evaluate kidney injury. Representative immunoblots are shown in panel H. PAS histological staining (I) was performed to assess 2,8-DHA crystal deposition (J). Statistical comparisons between groups are presented using compact letter display, in which different letters indicate statistically significant differences between means; P < 0.05 by Kruskal–Wallis H-test followed by a Dunn’s post hoc test, Brown–Forsythe one-way ANOVA followed by a Dunnett T3 post hoc test, or Ordinary one-way ANOVA followed by a Tukey’s post hoc test. Scale bar: 100 μm.

Adenine increased protein levels of key kidney injury markers neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule 1 (KIM-1).[35, 36] MARY1 administration reduced these increases by 52% and 68%, respectively, compared to vehicle treatment. KIM1 and NGAL were undetectable in the renal cortices of control rats (Fig 1F–H). PAS histological staining revealed deposition of 2,8-DHA crystals in the tubular lumen of adenine-fed rats. MARY1 administration attenuated the development of 2,8-DHA crystals by 70% compared to vehicle treatment. No crystal deposition was detected in control rats (Fig. 1I–J).

MARY1 administration mitigates adenine-induced renal cortical mitochondrial dysfunction

Nicotinamide adenine dinucleotide (NAD⁺) is an essential coenzyme used for oxidative phosphorylation in the mitochondria. Cellular [NAD⁺] decreased 69% in vehicle-treated adenine-fed rats, which was fully prevented with MARY1 treatment (Fig. 2A). Although renal cortical ATP content was not different from controls in adenine-fed rats, MARY1 increased ATP content 2-fold in adenine-exposed rats compared to vehicle treatment (Fig. 2B). Peroxisomal proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), which is a transcriptional coactivator conventionally known as the “master regulator of MB,” decreased 60% in vehicle treated adenine-exposed rats compared to control rats. MARY1 administration completely prevented the decrease in PGC-1α (Fig. 2C). mtDNA copy number was also reduced by ~50% in adenine-exposed rats that received vehicle (Fig. 2D). Intervention with MARY1 prevented the reduction in mtDNA copy number (Fig. 2D).

Figure 2. MARY1 administration mitigates adenine-induced renal cortical mitochondrial dysfunction.

8-week-old rats were fed either a normal chow diet with or without 0.2% adenine-supplementation for 4 weeks. Rats eating an adenine-supplemented diet were treated with vehicle or MARY1 (0.15 mg/kg, IP) daily for 4 weeks, after which the renal cortices were collected for downstream analyses. NAD+ (A) and ATP (B) were measured to assess mitochondrial health and energy output. PGC-1α protein (C), mtDNA content (D), and protein expression of MFF (E), OPA1 (F), MFN2 (G), and complexes of the ETC: ATP5A (I), UQCRC2 (J), MTCO1(K), SDHB (L), NDUFB8 (M) were measured to assess mitochondrial homeostasis. Representative immunoblot images are shown in panels H and N. Statistical comparisons between groups are presented using compact letter display, in which different letters indicate statistically significant differences between means; P < 0.05 by Kruskal–Wallis H-test followed by a Dunn’s post hoc test, Brown–Forsythe one-way ANOVA followed by a Dunnett T3 post hoc test, or Ordinary one-way ANOVA followed by a Tukey’s post hoc test.

While mitochondria naturally undergo fusion and fission processes to maintain mitochondrial homeostasis [11], excessive fission is generally associated with dysfunctional mitochondrial states [37]. Protein expression of mitochondrial fission factor (MFF), an outer membrane protein involved in mitochondrial fission [38, 39], was elevated over 5-fold in adenine-exposed, vehicle-treated rats (Fig. 2E). MARY1 prevented this increase in MFF expression (Fig. 2E). Markers of mitochondrial fusion, mitofusin 2 (MFN2) and optic atrophy 1 (OPA1) [40, 41] were unchanged across groups (Fig. 2F–G).

Protein expression of electron transport chain (ETC) subunits ATP5A (ETC complex V), UQCRC2 (ETC complex III), MTCO1 (ETC complex IV), SDHB (ETC complex II), and NDUFB8 (ETC complex I), were decreased 21%, 62%, 34%, 27%, and 42%, respectively, in vehicle-treated adenine-fed rats compared to controls. MARY1 prevented adenine-induced decreases in expression of all five complexes, further increasing ATP5A and MTCO1 beyond control levels. (Fig. 2I–M).

MARY1 administration reduces adenine-induced renal cortical MAPK/ERK and STAT3 phosphorylation

Protein kinase C (PKC) is a known downstream signaling molecule of HTR2B and a regulator of MAPK/ERK and STAT3 signaling pathways [42, 43]. Adenine exposure resulted in a 10-fold increase in PKC expression, while MARY1 prevented this increase (Fig. 3A). To evaluate HTR2B specificity, we assessed expression of protein kinase A (PKA), which is not known to be involved in HTR2B signaling [44] and saw no differences across any treatment groups (Fig. 3B). ERK and STAT3 phosphorylation were increased 2-fold and 5-fold, respectively in the renal cortices of vehicle-treated adenine-fed rats, indicating pathway activation (Fig. 3C–D). Treatment with MARY1 prevented the phosphorylation of ERK and reduced STAT3 phosphorylation by 42% (Fig. 3C–D).

Figure 3. MARY1 administration reduces adenine-induced renal cortical MAPK/ERK and STAT3 phosphorylation.

8-week-old rats were fed either a normal chow diet with or without 0.2% adenine-supplementation for 4 weeks. Rats eating an adenine-supplemented diet were treated with vehicle or MARY1 (0.15 mg/kg, IP) daily for 4 weeks, after which the renal cortices were collected for downstream analyses. Protein expression of PKC (A) and PKA (B) were measured to evaluate HTR2B signal specificity. Phosphorylation and total protein expression of ERK (C) and STAT3 (D) were measured to assess pathway induction. Expression of TNFR2 (E), NF-κB (F), VCAM1 (G), and MCP-1 (H) were assessed to confirm ERK- and STAT3-mediated activation and inflammatory signaling. Representative immunoblot images are shown in panel I. The proposed mechanism is depicted in panel J (created with BioRender.com). Statistical comparisons between groups are presented using compact letter display, in which different letters indicate statistically significant differences between means; P < 0.05 by Kruskal–Wallis H-test followed by a Dunn’s post hoc test, Brown–Forsythe one-way ANOVA followed by a Dunnett T3 post hoc test, or Ordinary one-way ANOVA followed by a Tukey’s post hoc test.

STAT3 and ERK signaling have both been implicated in upregulating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [45, 46], a transcription factor associated with the pathogenesis of renal inflammation and fibrosis in CKD [47–49]. Additionally, tumor necrosis factor receptor 2 (TNFR2), a type I transmembrane glycoprotein that binds TNFα and propagates NF-κB signaling [50, 51], was increased 7-fold in adenine-exposed vehicle-treated rats, but not in MARY1-treated rats (Fig. 3E). Consistent with these findings, NF-κB was elevated 3-fold in the renal cortices of adenine-exposed vehicle-treated rats compared to controls (Fig. 3F). Similarly, expression of vascular cell adhesion molecule 1 (VCAM1), a direct target for upregulation by NF-κB [52–55], was increased 2-fold in vehicle-treated adenine-fed rats compared to controls (Fig. 3G). MARY1 administration prevented the increase in both NF-κB and VCAM1 (Fig. 3G). Monocyte chemoattractant protein 1 (MCP-1) is a chemokine used to recruit and attract monocytes/macrophages to sites of injury [56] and is known to drive pro-inflammatory signaling in CKD [57]. MCP-1 protein was elevated 20-fold in the renal cortices of rats exposed to adenine and treated with vehicle, while MARY1 fully prevented this increase (Fig. 3H).

Based on these data, we propose the mechanism described in Figure 3J, whereby MARY1-mediated antagonism of HTR2B inhibits adenine-induced PKC activity, preventing STAT3 and ERK activation, and subsequently decreasing inflammatory signaling and mitochondrial dysfunction as described in Figure 2.

MARY1 administration prevents adenine-induced renal cortical epithelial-mesenchymal transition and induction of fibrotic mediators

Renal fibrosis is the principal pathological endpoint that defines CKD etiology [3]. Transforming growth factor β (TGF-β) is a critical driver of EMT, the transformation of healthy parenchymal cells into migratory myofibroblasts that compromise renal function and deposit extracellular matrix (ECM) proteins [58–60]. A 64-fold increase in TGF-β expression was observed in the renal cortices of adenine-exposed vehicle-treated rats compared to controls. MARY1 treatment prevented this increase by 83% (Fig. 4A,H). Alpha smooth-muscle actin (α-SMA), a key marker for myofibroblast differentiation [61, 62], exhibited a 2-fold increase in expression in adenine-exposed rats, irrespective of MARY1 treatment (Fig. 4B,H).

Figure 4. MARY1 administration prevents adenine-induced renal cortical epithelial-mesenchymal transition and induction of fibrotic mediators.

8-week-old rats were fed either a normal chow diet with or without 0.2% adenine-supplementation for 4 weeks. Rats eating an adenine-supplemented diet were treated with vehicle or MARY1 (0.15 mg/kg, IP) daily for 4 weeks, after which the renal cortices were collected for downstream analyses. Protein expression of TGF-β (A), α-SMA (B), PFKFB3 (C), MMP9 (D), PDGFRβ (E), and Vimentin (I) were evaluated to elucidate the pro-fibrotic and pro-EMT renal cortical environment. Representative immunoblots are shown in panels H. Immunohistochemistry was performed to evaluate spatial expression of PDGFRβ in the renal cortex (F,G). Statistical comparisons between groups are presented using compact letter display, in which different letters indicate statistically significant differences between means; P < 0.05 by Kruskal–Wallis H-test followed by a Dunn’s post hoc test, Brown–Forsythe one-way ANOVA followed by a Dunnett T3 post hoc test, or Ordinary one-way ANOVA followed by a Tukey’s post hoc test. Scale bar: 100 μm.

While 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) is canonically known as a regulatory enzyme in the glycolysis pathway [63–65], recent evidence has implicated PFKFB3 as a key driver of renal fibrosis through the promotion of histone lactylation-mediated NF-κB activation [64]. Renal cortical expression of PFKFB3 increased 4-fold in vehicle-, not MARY1-treated adenine-exposed rats (Fig. 4C,H). Matrix metalloproteinase 9 (MMP9), a pro-inflammatory and pro-fibrotic mediator that is primarily expressed by neutrophils and macrophages [66–68]. increased 3-fold in vehicle-treated adenine-exposed rats compared to controls (Fig. 4D,H). MARY1 administration prevented the adenine-induced increase in expression in MMP9.

Protein expression of platelet derived growth factor receptor beta (PDGFRβ), a regulator of myofibroblast growth, differentiation, proliferation, and migration that is implicated as a potentiator of fibrogenesis [69, 70], increased 4.5-fold in vehicle-treated adenine-fed rats compared to controls. MARY1 treatment attenuated this increase by 45% (Fig. 4E,H). These results were recapitulated using IHC analysis on sectioned renal cortical tissues, which revealed elevated PDGFRβ in adenine-exposed vehicle-treated rats that was prevented with MARY1 administration (Fig. 4F,G).

Vimentin is a type III intermediate filament protein that provides structural support to mesenchymal cells [71] and has been implicated as a driver of EMT [72, 73]. Vimentin increased 5-fold in the renal cortices of adenine-exposed vehicle-treated rats compared to controls. MARY1 administration prevented the adenine-induced increase in expression in vimentin (Fig. 4I).

MARY1 administration prevents adenine-induced renal cortical fibrosis

Collagen 1 and collagen 3, ECM proteins expressed by activated myofibroblasts [74, 75], were increased 1.5- and 1300-fold in the renal cortices of vehicle-treated adenine-exposed rats compared to controls. MARY1 treatment prevented these increases (Fig. 5A–C).

Figure 5. MARY1 administration prevents adenine-induced renal cortical fibrosis.

8-week-old rats were fed either a normal chow diet with or without 0.2% adenine-supplementation for 4 weeks. Rats eating an adenine-supplemented diet were treated with vehicle or MARY1 (0.15 mg/kg, IP) daily for 4 weeks, after which the renal cortices were collected for downstream analyses. Protein expression of COL1 (A) and COL3 (B) were measured to assess extracellular matrix deposition. Representative immunoblots are shown in panel C. Masson’s trichrome staining (D) was used to measure collagen deposition (E). Immunohistochemical staining was used to measure AQP1 (F,G). Statistical comparisons between groups are presented using compact letter display, in which different letters indicate statistically significant differences between means; P < 0.05 by Kruskal–Wallis H-test followed by a Dunn’s post hoc test, Brown–Forsythe one-way ANOVA followed by a Dunnett T3 post hoc test, or Ordinary one-way ANOVA followed by a Tukey’s post hoc test. Scale bar: 100 μm.

These findings were further validated by histological analysis using Masson’s Trichrome staining for collagen deposition, which revealed a 4-fold increase in cortical fibrotic burden in adenine-exposed vehicle-treated rats, which was blunted by 43% with MARY1 treatment (Fig. 5D,E). Additionally, aquaporin 1 (AQP1), a crucial transporter in the kidney whose downregulation is known to promote renal fibrosis [76], was decreased 20% in the renal cortex of vehicle- treated adenine-fed rats, but not in those treated with MARY1 (Fig. 5F,G).

We also examined glomerular size and count. In CKD, glomerular dysfunction often coincides with tubular dysfunction, both of which contribute to the decline in GFR that defines CKD [2, 77]. We showed that adenine-exposure resulted in a reduction in glomerular size by ~10% compared to controls (Fig. 6A,B). Similarly, the number of glomeruli observed declined by ~30% in adenine-exposed rats compared to controls (Fig. 6C). Treatment with MARY1 prevented both of these reductions (Fig. 6A–C).

Figure 6. MARY1 administration prevents glomerular loss.

8-week-old rats were fed either a normal chow diet with or without 0.2% adenine-supplementation for 4 weeks. Rats eating an adenine-supplemented diet were treated with vehicle or MARY1 (0.15 mg/kg, IP) daily for 4 weeks, after which the renal cortices were collected for downstream analyses. PAS histological staining (A) was performed to assess mean glomerular size (B) and number of glomeruli (C). Statistical comparisons between groups are presented using compact letter display, in which different letters indicate statistically significant differences between means; P < 0.05 by Kruskal–Wallis H-test followed by a Dunn’s post hoc test, Brown–Forsythe one-way ANOVA followed by a Dunnett T3 post hoc test, or Ordinary one-way ANOVA followed by a Tukey’s post hoc test. Scale bar: 25 μm.

Discussion

While there are many contributing factors to CKD, a commonly understood cellular cause is mitochondrial dysfunction [10, 11, 78]. The tubular cells of the kidney, which perform the bulk of the reabsorption of the glomerular filtrate, are mitochondrially dense and highly metabolically active [11, 78]. Loss of mitochondrial number combined with reductions in mitochondrial efficiency can hinder the ability of tubule cells to effectively filter and reabsorb, leading to a gradual loss of kidney function and ultimately CKD [10–12, 79]. Research has shown that pharmacological restoration of mitochondrial function via MB can decrease the progression of renal injury and improve recovery [11, 80]. This study aimed to evaluate the therapeutic potential of HTR2B antagonism with MARY1 in preventing CKD pathology, potentially through MB induction and restoration of mitochondrial homeostasis.

Prolonged adenine-enriched diet resulted in a robust CKD phenotype characterized by elevated BUN, SCr, and ACR, increased renal injury markers, mitochondrial dysfunction, inflammatory signaling, and cortical fibrosis [1, 2, 81]. Furthermore, deposition of 2,8-DHA crystals, the primary driver of renal pathology in this model [30], was observed in the tubular lumen. Administration of MARY1 during adenine supplementation mitigated BUN elevation, decreased 2,8-DHA crystal deposition, suppressed KIM-1 and NGAL, prevented loss of glomeruli, preserved renal cortical mitochondrial markers, and attenuated multiple inflammatory and fibrotic pathways. Together, these findings demonstrate that MARY1 mitigates renal injury and fibrosis in a rat model of adenine-induced CKD. Interestingly, MARY1 did not alter SCr or urinary ACR. This may reflect the limited sensitivity of SCr to modest changes in renal function and the complex regulation of urinary albumin excretion, which is influenced by both glomerular filtration and tubular albumin reabsorption [82–84]. Consistent with this, experimental models of kidney disease demonstrate that changes in BUN, SCr, and albuminuria can occur at different stages of disease progression [85, 86]. Future studies at additional time points will determine whether the improvements in renal pathology with MARY1 translate to broader improvements in kidney function.

Supraphysiological exposure to adenine overwhelms the purine salvage pathway mediated by adenine phosphoribosyl transferase (APRT), and reliance on xanthine oxidase increases, producing the insoluble 2,8-DHA metabolite that occludes the tubules of the kidney leading to inflammation and fibrosis [30, 81]. MARY1 administration reduced the overall burden of 2,8-DHA crystals within the renal cortex, which may reflect decreased crystal formation, enhanced clearance of the pathological metabolite, or altered crystal morphology. Future studies will define the mechanisms of this effect, including whether MARY1 facilitates natural clearance by promoting tubular dilation or endocytosis, or by reducing crystal size [30].

Similar to prior studies from our laboratory that demonstrate MB and restoration of mitochondrial function as key drivers of renal recovery and protection [14, 17, 18, 87], we showed that the renal protective effects of MARY1 occurred concurrently with MB induction and preservation of mitochondrial homeostasis. Adenine-induced CKD reduced renal cortical NAD+, PGC-1α, mtDNA, and ETC protein expression, and increased MFF expression, indicating mitochondrial fission. MARY1 prevented these reductions and increased ATP levels above that of the controls. These findings are consistent with MARY1 as an inducer of MB and regulator of mitochondrial function. Because mitochondrial dysfunction is a central driver of CKD onset/progression, this may represent a central mechanism by which MARY1 prevents downstream renal injury and dysfunction [10].

Canonical HTR2B signaling operates through a G_q/11 protein-coupled mechanism that activates PKC [88]. PKC then acts as a central signaling hub that activate signal cascades such as MAPK/ERK and STAT3 [42, 88–90]. MARY1 prevented ERK phosphorylation and reduced STAT3 phosphorylation in the adenine-fed rats, indicating reduction of these signaling pathways[91, 92]. More specifically, ERK signaling has been implicated as a key driver of inflammatory cytokine release and fibrotic deposition in other rat models of kidney disease [43, 93, 94]. Additionally, it has been shown that ERK signaling inhibits PGC-1α mediated MB [95]. STAT3 is a key signaling hub for inflammation and fibrosis in CKD, primarily through the upregulation of pro-inflammatory cytokines [91, 94]. Our results showed that MARY1 prevented adenine-induced elevation of TNFR2, NF-κB, and VCAM1, and MCP-1, suggesting reduced inflammatory activation and immune cell infiltration and supporting inhibition of STAT3 signaling [48, 49, 55, 96]. Together, these data suggest a novel mechanism where antagonism of HTR2B with MARY1 inhibits STAT3 and ERK signaling, resulting in retained mitochondrial homeostasis and reduced inflammation, representing another possible mechanism by which MARY1 reduces CKD-associated renal injury [1].

The cytokine TGF-β is a central mediator of pro-fibrotic signaling [58, 97]. In CKD, pathological overexpression of TGF-β can lead to the transformation of fibroblasts into activated myofibroblasts, which results in deposition of extracellular matrix proteins that cause fibrosis [58, 97, 98]. MARY1 prevented the upregulation of TGF-β and other pro-fibrotic mediators like PDGFRβ and MMP9 [68, 69]. MARY1 also reduced the collagen deposition in the renal cortex. These data show that MARY1 reduced the overall fibrotic burden in the renal cortex and suggest that it may be acting as an anti-fibrotic agent primarily through inhibition of pro-fibrotic TGF-β signaling.

PFKFB3 is a glycolysis enzyme that has been implicated as a key driver of fibrosis in the kidney by promoting histone lactylation-mediated NF-κB activation [20, 64]. PFKFB3 is a rate-limiting glycolytic enzyme that primarily generates fructose-2,6-bisphosphate, an allosteric activator of phosphofructokinase-1. Fructose-2,6-bisphosphate increases phosphofructokinase-1 activity, which accelerates the conversion of fructose-6-phosphate to fructose-1,6-biphosphate and enhances glycolytic throughput. In the context of renal physiology, PFKFB3 may facilitate a transition in metabolic programming from beta-oxidation to glycolysis [64]. This metabolic reprogramming has been known to promote CKD [99]. We have shown that in the context of renal aging, elevated PFKFB3 is implicated as well [20]. Moreover, we showed that MARY1 reduced the expression of PFKFB3 in the context of renal aging and CKD [20]. While we did not assess the role of PFKFB3-mediated metabolic reprogramming in this study, our evidence suggests that it may be driving some of the observed pathogenesis and it should therefore be assessed in future studies.

This study revealed that MARY1 prevents multiple hallmarks of CKD, including renal injury, mitochondrial dysfunction, inflammation, and fibrosis. Additional studies are needed to determine whether MB is the primary driver of MARY1-induced renal protection. Importantly, this work demonstrates the potential of MARY1 in preventing the onset/progression of CKD, as opposed to reversal of CKD pathology. Future research will assess the effectiveness of MARY1 when treatment is initiated after CKD is established and use genetic or molecular inhibition of HTR2B to confirm receptor-dependent effects. Nonetheless, these findings support MARY1 as a promising therapeutic candidate for CKD.

Data Availability Statement

The original contributions presented in this manuscript are included within this article. Further inquiries can be directed to the corresponding author.