The roles of free-fatty acid receptors in the pathophysiology of renal disorders

Abstract

Kidney diseases represent one of the most prevalent and rapidly growing global health burdens, with rising mortality linked to both acute kidney injury and chronic kidney disease (CKD). Despite the increasing incidence of CKD affecting 8%–16% of the world’s population, therapeutic options remain limited, largely because of an incomplete understanding of underlying pathophysiology and the lack of reliable biomarkers. Recent evidence highlights that free-fatty acids (FFA), particularly short-chain and long-chain fatty acids, are beneficial modulators of renal health, associated with reduced CKD risk and slower kidney function decline. Although effects of FFA have historically been linked to their intracellular activity, they can also engage a family of cell-surface G protein-coupled FFA receptors (FFAR), including FFARs FFA2/GPR43 and FFA3/GPR41, which are activated by short-chain fatty acids, and FFA1/GPR40 and FFA4/GPR120, which are agonized by long-chain fatty acids. This review summarizes current knowledge of FFAR-mediated mechanisms in acute kidney injury, CKD, and associated renal pathologies, emphasizing their potential as therapeutic targets to improve kidney outcomes. We also discuss the therapeutic relevance of dietary FFA related to these FFAR in relation to renal disease.

Significance Statement

Fatty acid sensing G protein-coupled receptors have emerged as important regulators of renal inflammation, fibrosis, and tubular survival across acute and chronic models of kidney injury. This review synthesizes current evidence for short- and long-chain free-fatty acid receptors in kidney disease, highlights translational relevance, and outlines challenges that must be addressed to advance free-fatty acid receptor-targeted therapies.

Graphical abstract

1. Introduction

Globally, kidney disease demonstrates a higher incidence than any other noncommunicable disease identified by the World Health Organization, surpassing chronic respiratory disorders, cardiovascular diseases, malignancies, and diabetes mellitus (DM).^1,2 Over the past 2 decades, mortality associated with kidney dysfunction arising from acute and chronic injuries has shown a consistent upward trend,³ and presently, kidney dysfunction constitutes the seventh leading global risk factor for death, with a burden that is expected to escalate further in the foreseeable future.¹ According to the National Kidney Foundation, approximately 750,000 individuals in the United States are affected by kidney failure each year.⁴ Chronic kidney disease (CKD) is defined as persistent kidney injury or when the estimated glomerular filtration rate remains under 60 mL/min/1.73 m² for a minimum of 3 months, independent of cause,^5,6 and involves a gradual deterioration of kidney function that may ultimately lead to the need for dialysis or transplant. Indicators of kidney damage include uremia, abnormal imaging or biopsy, alterations in urinary sediment, or increased urinary albumin.⁶ A minority of patients with CKD (2%–6%) progress to end-stage renal disease, which affects an estimated 8%–16% of the global population, with DM and hypertension representing the predominant etiological factors.7, 8, 9 On the other hand, acute kidney injury (AKI) is characterized by a rapid rise in serum creatinine, often accompanied by reduced urine output, and typically resolves within 7 days.10, 11, 12 Common complications of AKI include renal vascular disease, glomerulonephritis, acute interstitial nephritis, and acute tubular necrosis.10, 11, 12 The effects of renal impairment are similarly apparent in AKI, which heightens the likelihood of transitioning to CKD and, in severe cases, can result in renal failure.^2,13 So far, only a limited number of pharmacological treatments have been developed specifically for CKD and its related cardiovascular diseases,² and a major challenge in this realm is the limited understanding of the underlying disease mechanisms and the low reliability of existing biomarkers. Consequently, the management of most kidney patients depends largely on therapies aimed at controlling coexisting conditions.¹⁴

Free-fatty acids (FFA) serve as critical cellular energy sources and are essential for maintaining cellular structure and homeostasis, regulating gene expression, and facilitating intracellular signaling processes. A growing body of preclinical, clinical, and epidemiological studies suggests that dietary enrichment with short-chain fatty acids (SCFA) and long-chain fatty acids (LCFAs) confers renal protection, as evidenced by a reduced risk of CKD and a slower decline in renal function.^15,16 SCFA are straight-chain saturated fatty acids with fewer than 6 carbon atoms, including acetate (C2), propionate (C3), butyrate (C4), and valerate (C5), and are primarily produced as metabolites of gut microbiota,¹⁷ whereas LCFA, including ω3 and ω6 fatty acids, contain 12–22 carbon atoms and are widely obtained from dietary sources.¹⁸ Importantly, excess circulating FFA, particularly saturated FFA, can accumulate intracellularly in renal cells including podocytes, proximal tubular epithelial cells (TECs), and mesangial cells, leading to lipotoxicity-induced cellular dysfunction and injury.¹⁹ Impaired FFA metabolism potentiates lipotoxicity and lipid excess promotes mitochondrial dysfunction, endoplasmic reticulum (ER) stress, oxidative stress, and activation of proinflammatory and profibrotic pathways, ultimately contributing to cell death and tissue remodeling, which underlie kidney disease pathogenesis.20, 21, 22 Further, lipid accumulation in renal cells is a key driver of CKD progression, particularly in the context of diabetic nephropathy (DN), where altered lipid metabolism and impaired fatty acid oxidation (FAO) exacerbate renal damage.^23,24 However, proper lipid balance and appropriate ratios of FFA are critical for homeostasis. Although the effects of these FFA have historically been linked to these intracellular effects, emerging evidence indicates that both SCFA and LCFA also exert a myriad of beneficial effects in the kidney by acting on cell-surface free-fatty acid receptors (FFAR) belonging to the G protein-coupled receptor (GPCR) superfamily.

The FFAR family consists of FFA1 (previously termed GPR40), FFA2/GPR43, FFA3/GPR41, and FFA4/GPR120. FFA2 and FFA3 are primarily activated by SCFA,¹⁷ whereas FFA1 and FFA4 respond to medium-to-LCFA,²⁵ including, but not exclusive to, many ω3 FFA. Depending on receptor type and ligand specificity, FFAR signal through distinct G-protein subunits as well as β-arrestin partner proteins to mediate various physiological responses.²⁶ Although FFA1 is more homologous in sequence to FFA2 and FFA3, sharing approximately 30% amino acid homology,^26,27 functionally, it bears more semblance to FFA4, in that both receptors are activated by similar endogenous ligands, such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and primarily, but not exclusively, signal through the Gαq/11-family of G proteins in addition to β-arrestins.²⁸ Although several prior reviews have extensively covered these receptors in terms of their ligands, functional potencies, and nonrenal physiological effects,^25,26,29,30 in this review, we provide a comprehensive assessment of the known roles of both the SCFA and LCFA FFARs in the regulation of chronic and acute kidney injuries, as well as diseases associated with CKD (summarized in Table 1, Table 2, Table 3).31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52

Table 1.

The roles of FFA1 in renal pathophysiology

Renal Disorder	In Vivo Model	In Vitro Model	Drugs Used	KO/KD Used	Effects of FFA1	References
Fibrosis	Adenine-induced CKD model 5/6-Nx rat model of CKD Doxorubicin mouse model of nephropathy Ischemia-reperfusion injury model UUO in mixed	Dermal fibroblasts HK-2	PBI-4050 (200 mg/kg/day)	FFA1−/− mice	↓ Renal fibrosis and inflammation	Gagnon et al31
Fibrosis and inflammation	Adenine-induced renal fibrosis model in mice	—	HWL-088 (100 mg/kg/day) ZLY-032 (100 mg/kg/day)	—	↓ Interstitial fibrosis ↓ Inflammation	Geng et al32
CKI	UUO (CKI model)	NRK52E	GW9508 (10 μM) GW1100 (10 μM)	siRNA	↓ Apoptosis ↑ Cell viability ↓ TNF-α mRNA, AT1R expression, and ROS generation (−) NF-κB translocation	Kim et al33
CKI	Adenine-induced CKD model		PBI-4050 (200 mg/kg/day)	FFA1−/− mice	↓ Anemia ↓ Fibrosis ↓ Inflammation ↓ ER stress ↓ UPR pathway	Thibodeau et al34
CKI	IR-induced AKI model in mice Adenine-AKI model	Bone marrow-derived cells	PBI-4050 (200 mg/kg/day) GW1100	FFA1−/− mice	↓ Anemia	Thibodeau et al34
DN	eNOS−/− db/db hHB-EGFTg/Tg mice	—	PBI-4050 (100 mg/kg/day)	—	↓ DN ↓ Inflammation ↓ Fibrosis ↑ Life span	Li et al35
pRCC	ACHN-cell-derived pRCC xenograft model	ACHN cells	GW9508 (10 μM) AS2034178 (10 μM) GW1100 (1 μM)	—	↑ pRCC proliferation and tumor growth ↑ Src/PI3K/AKT/NF-κB ↓ pRCC migration ↓ EGFR, ERK1/2, STAT3, and EMT	Karmokar et al36

↓, decrease in expression or function; ↑, increase in expression or function.

Table 2.

The role of FFA4 in renal pathophysiology

Renal Disorder	In Vivo Model	In Vitro Model	Drugs Used	KO/KD Used	Effects of FFA4	References
AKI	Cisplatin-induced AKI model	HK-2	TUG-891 (in vivo-35 mg/kg/day, in vitro-20 nM)	—	↓ ER stress ↓ UPR pathway ↓ Tubular loss	Huang et al37
AKI	Cecal ligation/perforation induced AKI mice	—	TUG-891	—	↑ SirT3 expression ↑ Gq/CaMKKβ/AMPK	Hudson et al38
Pyroptosis	Mouse model of HN	RAW264.7	In vivo-DHA (500 mg/kg/day) EPA (500 mg/kg/day) TUG-891 (5 mg/kg/day) AH-7614 (8 mg/kg/day) In vitro-DHA (50 μM) TUG-891 (10 μM) AH7614 (50 μM)	—	↓ Pyroptosis via (−) β-arrestin-2/NLRP3	Yang et al39
Fibrosis and inflammation	UUO	Macrophages from the peritoneal cavity of UUO rats	TUG-891 (10 μmol/L)	—	↓ Renal injury and fibrosis ↓ Inflammation	Wang et al40
DN	Male C57BLKS/J db/db and db/m mice	MPC5	TUG-891 (35 mg/k/day)	FFA4 siRNA	↓ Podocyte injury ↓ Glomerulosclerosis ↓ Inflammation ↑ β-Arrestin-2/TAB1/TAKI	Wei et al41
DN	A case-control study of 200 subjects (adults) with type 2 diabetes mellitus	—	—	—	↓ Dyslipidemia ↓ Renal injury	Singh et al42
pRCC	ACHN-derived pRCC tumor xenograft model	ACHN cells	GW9508 (10 μM) CpdA (10 μM) AH-7614 (10 μM)	—	↑ Migration and invasion ↑ PI3K/AKT/NF-κB/COX-2/MMP-9 ↑ STAT3 induced EMT ↓ Cell proliferation and tumor growth	Karmokar et al43

↓, decrease in expression or function; ↑, increase in expression or function.

Table 3.

Roles of FFA2 and FFA3 in renal pathophysiology

Renal Disorder	In Vivo Model	In Vitro Model	Drugs Used	KO/KD Used	Effects of FFA2/3	References
Fibrosis and inflammation	—	HRCE	NaAc (25 mM) NaP (1000 μM) NaB (1000 μM) CPC (500 μM) CFMB (25 μM)	FFA2/3 siRNA	↓ TNF-α-induced MCP-1 (−) p38 and JNK phosphorylation	Kobayashi et al44
Fibrosis	KD-fed UUO mice	NRK52E	AR420626 (5 μM)	FFA3 siRNA	↑ FAO ↓ Renal fibrosis	Qiu et al45
CKI	Adenine-induced CKI model in male C57BL/6 mice	—	Propionate	FFA2−/− mice and FFA3−/− mice	↓ Renal fibrosis ↓ Inflammation	Mikami et al46
DN	High-fat diet (HFD) and streptozotocin (STZ)-induced T2D and DN mouse models	SV-40 MES 13	SA SP SB (In vivo: 100 mg/[kg·48 h]; in vitro: 10 mM) Phenylacetamide (1 μM)	FFA2 siRNA	↓ ROS generation ↓ Inflammation	Huang et al47
DN	STZ-induced T2D model	TEC	SA (in vivo: 100 mM; in vitro: 25 mM) SB (in vivo: 50 mM; in vitro: 12 mM) SP (in vivo: 100 mM; in vitro: 3.2 mM)	FFA2 KO mice	↑ SCFA production ↓ Inflammation ↓ Fibrosis	Li et al48
DN	STZ-induced T2D model	Immortalized mouse podocytes	SA	FFA2 KO mice FFA2 siRNA	↑ Lipotoxicity ↑ ERK1/2 and EGR1 ↓ Autophagy	Lu et al49
DN	STZ-induced diabetic mice	Immortalized mouse podocytes	SA GLPG0974 (10 μmol/L)	Global FFA3 KO	↑ Albuminuria and renal injury Restored AMPKα activation	Lu et al50
ORG	HFD-induced diabetic mice	—	ACT001 (200 mg/kg/day)	—	↑ AMPK/NRF2-KEAP1 ↓ Renal inflammation and fibrosis	Zhou et al51
KSD	Renal CaOx stone model in mice	TCMK-1	SA, SP, SB (in vivo: 150 mM; in vitro: 100 μg/mL)	FFA2 KO mice	↓ Kidney CaOx crystals	Jin et al52

(↓) = decrease in expression or function; (↑) = increase in expression or function.

2. Free-fatty acid receptor-1

2.1. Renal tubular ER stress and apoptosis

ER stress and resultant unfolded protein response (UPR) are implicated in the development of CKD and renal fibrosis, suggesting that targeting ER stress could be a promising therapeutic strategy.⁵³ In proximal tubular cells, hypoxic or ischemic injury triggers the ER stress response, directly promoting cell damage.⁵³ The adenine-induced CKD model relies in part on activation of the UPR pathway and ER stress-mediated proapoptotic signaling, which replicates key features of CKD, including fibrosis, inflammation, anemia, and renal impairment.⁵⁴ 3-Pentylbenzeneacetic acid sodium salt (PBI-4050) is a first-in-class, orally active FFA1 agonist/GPR84 antagonist that exhibits antifibrotic effects in various models of organ fibrosis³¹ and has completed phase Ib and II clinical trials in patients with CKD and metabolic syndrome. In the adenine-induced model, FFA1 activation via PBI-4050 prevents renal tubular cell death by inhibiting ER stress and the UPR activation.³⁴ Specifically, PBI-4050 downregulated ER stress-associated proteins involved in translational repression, including C/EBP homologous protein, GRP78, and the phosphorylated forms of the ER stress markers IRE1 and PERK (eukaryotic translation initiation factor 2-α kinase 3).³⁴ Cleaved caspases 7 and 9, as well as the proapoptotic protein Bax, were significantly elevated in adenine-fed mice but markedly reduced in those with PBI-4050 treatment.³⁴ Moreover, markers of kidney stress-induced injury were significantly elevated in the kidneys of adenine-fed FFA1^−/− mice, in which, PBI-4050 had no effect, demonstrating a principal role for FFA1. At the same time, the ER stress-associated proteins XBP1s and GRP78 showed a trend toward upregulation compared with wild-type littermates, suggesting that FFA1 activation prevents proximal tubular apoptosis by inhibiting the ER stress-induced UPR pathway.³⁴

Apoptosis of renal TECs plays a central role in renal injury, which can be initiated by diverse internal and external stimuli and carried out through several molecular pathways.⁵⁵ Kim et al³³ examined the effects of the small molecule dual FFA1/FFA4 agonist GW9508, which has a Ca²⁺-mobilizing potency of ∼50 nM at FFA1 and 1–3 μM at FFA4, in renal tubular apoptosis using the unilateral ureteral obstruction (UUO) chronic kidney injury model.³³ In the UUO model, FFA1 expression was significantly reduced, which was accompanied by an increased expression of apoptotic proteins Bax and Bcl-2, compared with kidneys from control animals.³³ In the NRK52E rat proximal TEC line, activation of FFA1 by the agonist GW9508 attenuated tumor necrosis factor-α (TNF-α)-induced apoptosis, and also decreased cleaved caspase-3, nuclear factor-κB (NF-κB) activity, reactive oxygen species generation, and AT1R expression.³³ In contrast, pharmacological inhibition with the FFA1 antagonist GW1100, or small interfering RNA (siRNA)-mediated knockdown of FFA1, enhanced TNF-α-induced apoptotic signaling, supporting the antiapoptotic role of FFA1 in tubular cells. Of note, the concentration of GW9508 (10 μM) used here would certainly act on FFA4 if it were expressed, however, the authors did not examine FFA4 expression and attribute the activity solely to FFA1 without acknowledging the potential role of the other LCFA FFAR. Although they did show effects of the FFA1 antagonist GW1100 and FFA1-targeted siRNA in rat proximal tubular cell lines, the specific role of FFA1 versus FFA4 was not examined in the in vivo model, making it difficult to gauge the sole effect of FFA1 in this response.

2.2. Renal fibrosis and inflammation

Renal fibrosis is a common pathological consequence of AKI that can progress to CKD and ultimately end-stage renal disease.⁵⁶ Although fibrosis after AKI is initially protective and integral to the healing process, persistent or severe injury disrupts this balance.⁵⁷ The dysregulated response drives fibroblast activation and differentiation into myofibroblasts, accompanied by the release of proinflammatory cytokines,⁵⁸ along with excessive deposition of extracellular matrix (ECM) proteins.⁵⁹ These changes result in tissue remodeling, progressive loss of renal function, and eventual organ failure.⁶⁰ Despite substantial advances in elucidating the mechanisms of kidney fibrosis, a critical translational gap remains between the discovery of antifibrotic targets and their successful implementation in clinical practice. Emerging evidence suggests that FFAR exert protective effects against fibrosis in multiple organs, including the kidney. Both FFA1 and FFA4 have been extensively investigated for their antifibrotic functions, with diverse in vitro and animal models demonstrating their therapeutic potential in renal fibrosis. FFAR are expressed in various cell types within the “fibrotic niche,” including epithelial cells, fibroblasts, and immune cells such as macrophages.³¹ Activation of these receptors by endogenous ligands or synthetic agonists can modulate key fibrogenic processes, including fibroblast-to-myofibroblast differentiation, epithelial to mesenchymal transition (EMT), macrophage infiltration, and ECM deposition, via GPCR-linked G-protein-dependent and β-arrestin-mediated signaling pathways.^31,32,37 Although we have recently reviewed the roles of FFA1 and FFA4 in detail in organ fibrosis, including the kidney,⁶¹ here, we summarize the roles of FFAR, including the SCFA receptors, in renal fibrosis.

Using multiple kidney fibrosis models with the FFA1 agonist/GPR84 antagonist PBI-4050, Gagnon et al³¹ demonstrated that FFA1 exerts a protective role against renal fibrosis, whereas GPR84 drives fibrosis progression. In the 5/6-Nx CKD model of chronic renal failure, 5/6-Nx rats showed dramatic interstitial fibrosis, inflammation, tubular dilation, and glomerulosclerosis, which were markedly decreased in the presence of PBI-4050.³¹ Moreover, blood pressure and glomerular filtration rate were improved in treated animals. Similarly, in a doxorubicin-induced model of AKI, tubular dilation, glomerulosclerosis, and the presence of histologically-distinct intratubular casts were significantly reduced by PBI-4050, and the agent also performed in a likewise beneficial manner in an adenine-induced mouse model of CKD.³¹ To delineate the roles of FFA1 and GPR84 in renal fibrosis and inflammation, their expression was examined in vitro in human dermal fibroblasts, mouse peritoneal macrophages, human epithelial proximal tubule epithelial cell line HK-2, and human primary podocytes exposed to lipopolysaccharide or transforming growth factor-β (TGF-β).³¹ GPR84 mRNA was detected exclusively in normal human dermal fibroblasts, where PBI-4050 dose-dependently suppressed TGF-β1-induced myofibroblast activation and the expression of profibrotic markers (Fig. 1).³¹ Conversely, FFA1 mRNA was expressed only in human proximal tubule epithelial HK-2 cells, where PBI-4050 inhibited TGF-β1-induced collagen I production.³¹ Both receptors were present in peritoneal macrophages, but under fibrotic and inflammatory conditions, FFA1 expression was downregulated, whereas GPR84 was strongly induced.³¹ Using several renal disease models involving glomerular and/or interstitial fibrosis, the authors further demonstrated differential receptor regulation.³¹ GPR84 mRNA was upregulated in the 5/6 nephrectomy, doxorubicin-induced nephropathy, and adenine-associated nephropathy models, whereas FFA1 mRNA was upregulated only in adenine-induced kidney injury, suggesting potential differential regulation and context-specific roles of these receptors in renal pathology. To assess the role of FFA1 in kidney fibrosis, UUO, long-term postacute ischemic injury, and adenine-induced nephropathy models were compared in both wild-type and FFA1^−/− mice.³¹ Loss of FFA1 exacerbated renal fibrosis across all 3 models, whereas GPR84 knockout attenuated fibrosis and cyst formation (Fig. 1).³¹ In FFA1^−/− mice, PBI-4050 treatment produced only a modest 13% reduction in fibrosis compared with vehicle, whereas in Gpr84^−/− mice, PBI-4050 significantly reduced fibrosis by 36%.³¹ Notably, the combined effect of PBI-4050 in both knockouts mirrored the 50% reduction in fibrosis observed in wild-type adenine-fed mice,³¹ whereas cystic lesions were unaffected by PBI-4050 in Gpr84^−/− mice but tended to be lower in FFA1^−/− mice compared with vehicle.³¹ These findings suggest that PBI-4050 primarily reduces tubulointerstitial fibrosis via FFA1, whereas its effects on cystic lesions are mediated mainly through GPR84 in the adenine-induced CKD model.³¹ Nevertheless, contributions from additional targets of PBI-4050 beyond FFA1 and GPR84 remain unclear, and the downstream signaling mechanisms activated by these receptors were not explored in this study.

Fig. 1.

Antifibrotic and anti-inflammatory effects of FFA1, FFA4, and FFA2/3 in the mouse kidney. FFA1 is expressed in renal epithelial cells, macrophages, and myofibroblasts. In adenine-induced (Ade) fibrosis and UUO models, FFA1 activation reduces excessive ECM deposition and inflammation. In contrast, FFA4 signaling limits fibrosis primarily by promoting macrophage polarization toward an anti-inflammatory phenotype. FFA2 and FFA3 are expressed in distal tubules and collecting tubules in mice, and their knockdown results in increased TNF-α and MCP-1 expression, driving fibrosis progression. Figure created in part under license from Biorender.com.

In a separate study, the same group investigated PBI-4050 in a murine model of spontaneous tubulointerstitial fibrosis caused by selective overexpression of the epidermal growth factor receptor (EGFR) ligand HB-EGF in renal proximal tubules (hHB-EGFTg/Tg).⁶² In this model, EGFR-mediated ERK activation is essential for fibrosis development,⁶³ and MEK inhibition in hHB-EGFTg/Tg mice reduces fibrosis.⁶² ECM accumulation, a hallmark of tubulointerstitial fibrosis, was significantly detected in vehicle-treated hHB-EGFTg/Tg mice, with marked collagen deposition, elevated mRNA and protein levels of α-smooth muscle actin (α-SMA), fibronectin, TGF-β, and connective tissue growth factor, and increased SMAD3 phosphorylation (Fig. 1).³⁵ PBI-4050 markedly decreased phospho-ERK overexpression in proximal tubules of vehicle-treated mice,³⁵ and also attenuated fibrosis, suppressed profibrotic markers, preserved E-cadherin, inhibited Snail2 expression, and reduced renal interstitial macrophage infiltration, indicating protection against both epithelial dedifferentiation and inflammation.³⁵ Although prior studies suggest that FFA1 activation may contribute to the protective effects of PBI-4050 in this model, no FFA1 antagonists or knockout animals were examined, clouding the definitive contributions of the role of FFA1.³¹

Geng et al³² demonstrated that the novel FFA1/peroxisome proliferator-activated receptor delta (PPARδ) dual agonists HWL-088 and ZLY-032 exhibit greater potency as FFA1 agonists compared with PBI-4050.³² ZLY-032 was rationally designed by hybridizing the FFA1 agonist AM-4668 with the PPARδ agonist GW501516, thereby harnessing the synergistic potential of dual FFA1 and PPARδ signaling,⁶⁴ exhibiting favorable pharmacokinetics and higher selectivity for PPARδ over other PPAR subtypes.⁶⁴ An analog of this molecule, HWL-088, demonstrates superior potency with EC₅₀ values of 18.9 nM for FFA1 and 570.9 nM for PPARδ,65, 66, 67 and attenuated streptozotocin-induced diabetic kidney disease by lowering inflammatory mediators, including TNF-α and monocyte chemoattractant protein-1 (MCP-1).^66,67 Given the established roles of FFA1 and PPARδ, dual agonists targeting these pathways may synergistically mitigate renal fibrosis. Indeed, in an adenine-induced fibrosis model, both HWL-088 (100 mg/kg) and ZLY-032 (100 mg/kg) alleviated kidney injury, reducing glomerulosclerosis, tubular dilation, inflammatory infiltration, collagen deposition, and renal hypertrophy (Fig. 1).³² Furthermore, both agonists suppressed macrophage infiltration and downregulated inflammatory and fibrotic mediators, including TNF-α, interleukin 1β (IL-1β), IL-6, Collα1, TGF-β, and TIMP-1 (Fig. 1).³² Although these findings highlight the therapeutic potential of dual FFA1/PPARδ activation in kidney disease, again, no knockdown or antagonist studies were conducted to delineate the distinct contributions of FFA1 or PPARδ to the observed protective effects.³²

2.3. Diabetic nephropathy

Diabetic kidney disease, also known as DN, is a serious complication of DM that often progresses to CKD.^68,69 The Global Burden of Disease Study 2021 estimated that DN accounted for approximately 107.6 million cases, 477,300 deaths, and 11.28 million disability-adjusted life years worldwide.^70,71 DN is defined by persistent albuminuria and characteristic glomerular lesions, with nearly 20% of patients advancing from microalbuminuria to obvious nephropathy.^72,73 Patients with type 1 or type 2 diabetes who experience DN generally experience significantly poorer outcomes than those without renal involvement.⁷⁴

In eNOS^−/− db/db mice, a murine model of accelerated type 2 diabetes, early treatment with PBI-4050 (100 mg/kg) prevented further increases in established albuminuria and mitigated reductions in glomerular filtration rate, glomerulosclerosis, interstitial injury, and podocyte loss.³⁵ In a model of established DN, where treatment began at 16 weeks of age, PBI-4050 halted further albuminuria progression,³⁵ an effect that was enhanced when combined with the angiotensin-converting enzyme inhibitor captopril, resulting in a pronounced and sustained reduction in albuminuria.³⁵ Even with delayed initiation, PBI-4050 reduced glomerulosclerosis, tissue injury, and collagen deposition, while also decreasing Smad3 phosphorylation, macrophage infiltration, and oxidative stress in eNOS^−/− db/db mice.³⁵ Given that FFA1 is highly expressed in pancreatic islets and promotes insulin secretion,⁷⁵ the authors also examined hyperglycemia in these mice, and as expected, PBI-4050 lowered blood glucose, improved glucose tolerance, and preserved plasma insulin levels by maintaining islet function and limiting fibrosis.³⁵ Notably, even with delayed administration, PBI-4050 sustained insulin production and mitigated both pancreatic and renal fibrosis via FFA1-mediated signaling.³⁵ Importantly, PBI-4050 treatment also significantly improved lifespan in eNOS^−/− db/db mice.³⁵ These findings suggest that PBI-4050 protects against DN by preserving kidney function, potentially through the activation of renal FFA1 and inhibition of GPR84, which reduces immune cell infiltration, oxidative stress, and fibrosis and also supports islet function, lowers blood glucose, enhances autophagy, and decreases ER stress.³⁵ Importantly, although these findings are clearly demonstrable of antifibrotic effects, it remains possible that the observed improvements in renal fibrosis are secondary to broader renoprotective mechanisms, for example, simply lowering blood glucose and improving insulin sensitivity. In particular, reductions in oxidative stress, macrophage infiltration, and metabolic dysfunction may collectively alleviate the profibrotic milieu, thereby indirectly limiting fibrotic progression rather than reflecting a direct antifibrotic action of PBI-4050.

2.4. Anemia associated with renal injuries

Anemia is common yet substantially undertreated in patients with nondialysis-dependent CKD, with less than one-quarter receiving therapy, and treatment rates increasing only in advanced stages.76, 77, 78 Undertreatment contributes to greater transfusion requirements, which in transplant candidates increases the risk of allosensitization and transplant rejection.⁷⁹ The pathogenesis of CKD-associated anemia is multifactorial, involving elevated hepatic hepcidin, impaired erythropoietin synthesis, and reduced red blood cell integrity and survival.80, 81, 82 Current reliance on erythropoietin (EPO)-stimulating agents is limited by suboptimal efficacy and adverse effects, underscoring the need for novel therapies.

Adenine-fed mice developed microcytic anemia,³⁴ and PBI-4050 significantly improved red blood cell counts and mean corpuscular volume, with a trend toward higher hemoglobin, and markedly elevated plasma EPO levels compared with adenine controls, indicating enhanced renal EPO production.³⁴ Consistently, EPO levels were ∼3.5-fold lower in adenine-fed FFA1^−/− mice relative to both healthy and adenine-fed wild-type mice, supporting a critical role of FFA1 activation in maintaining EPO production and mitigating anemia in CKD.³⁴ FFA1 activation may also serve as a novel therapeutic strategy for treating CKD-associated anemia through EPO-independent mechanisms⁸³ as PBI-4050 significantly increased colony-forming unit-erythrocyte generation, an effect abrogated by the FFA1 antagonist GW1100, whereas FFA1 antagonism alone reduced colony-forming unit-erythrocyte counts.⁸³ PBI-4050 exhibited efficacy comparable to EPO in promoting colony-forming unit-erythrocyte formation⁸³ and also preserved hematocrit after ischemia-reperfusion-induced AKI. Moreover, the agonist alleviated anemia in adenine-induced CKD, as reflected by improvements in hematocrit, hemoglobin, mean corpuscular volume, and plasma EPO levels.⁸³ Importantly, anemia severity was similar between wild-type and FFA1^−/− CKD mice, and PBI-4050 failed to restore hematocrit in FFA1-deficient animals, confirming that its antianemic effects require FFA1.⁸³ Collectively, these findings highlight a role for FFA1 in EPO and EPO-independent mechanisms for correcting anemia in CKD, with PBI-4050 providing therapeutic potential by sustaining erythropoietic parameters while also supporting renal and cardiovascular outcomes.⁸³

2.5. Renal cell carcinoma

Renal cell carcinoma (RCC) is the most common type of kidney cancer, with clear cell RCC (ccRCC) accounting for 75%–80% of cases and papillary RCC (pRCC) representing 15%–20%.^84,85 RCC subtypes are defined by distinct histological and genetic features, with pRCC frequently harboring alterations in c-MET, FH, CDKN2A, and NF2.^86,87 Advanced pRCC is highly metastatic and associated with poor prognosis, however, most therapies and clinical studies have focused on ccRCC, leaving limited effective options for patients with pRCC,^86,88 underscoring the need to identify novel molecular pathways to better understand pRCC carcinogenesis and to develop more effective, subtype-specific therapies.

Although previous studies have shown that FFAR can exert either protumor or antitumor effects depending on the tissue context, we previously demonstrated that FFA1 and FFA4 were specifically expressed in the human epithelial adenocarcinoma pRCC cell line ACHN but were undetectable in human epithelial adenocarcinoma ccRCC cell lines Caki-2 and 786-O.^36,43 Furthermore, FFA1 and FFA4 exhibited opposing roles in regulating oncogenic properties, including proliferation and migration, in ACHN pRCC cells (Fig. 2).^36,43 FFA1 expression was elevated in human pRCC tissues compared with patient-matched adjacent noncancerous kidney tissue and was associated with pathological progression of pRCC.³⁶ In contrast, FFA1 expression appeared to decrease with advancing ccRCC pathology.³⁶ Using the selective FFA1 agonist AS2034178 and antagonist GW1100, we demonstrated that FFA1 agonism promoted serum-induced proliferation of pRCC cells,³⁶ whereas GW1100 alone or combined with AS2034178 suppressed cell proliferation.³⁶ Conversely, FFA1 agonism suppressed wound healing, transwell migration, and invasion, indicating that FFA1 negatively regulated the migratory capacity of metastatic pRCC cells.³⁶ These effects were reversed by the FFA1 antagonist GW1100, alone or in combination with AS2034178.³⁶

Fig. 2.

Oncomodulatory properties of FFA1 and FFA4 in pRCC ACHN cell line. Negative crosstalk between FFA1 and EGFR suppresses EGFR-ERK1/2 signaling, partially inhibiting pRCC cell invasion, whereas FFA4 promotes invasion via EGFR-mediated ERK activation. FFA1 also drives pRCC cell proliferation through Src-dependent PI3K/AKT signaling, leading to NF-κB translocation and COX-2 transcription. In contrast, FFA4 activates PI3K/AKT in a Src-independent manner, promoting NF-κB translocation and upregulation of MMP-9 and COX-2, thereby enhancing cell invasion. Figure created in part under license from Biorender.com.

We also delineated the mechanism by which FFA1 signaling promoted proliferation and suppressed migratory capacity in the ACHN pRCC cell line (Fig. 2). As upstream regulators of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling cascade, GPCRs play a pivotal role in governing key RCC processes such as proliferation, migration, and survival.⁸⁹ Our findings demonstrated that FFA1 activation by the AS2034178 upregulated expression of the PI3K p85α subunit, induced phosphorylation of AKT at Ser473, and promoted phosphorylation of c-SRC at Tyr416 in a time-dependent manner.³⁶ PI3K/AKT signaling induces nuclear translocation and transcriptional activation of NF-κB via downstream stimulation of the inhibitor of nuclear factor-κB (IκB) kinase (IKK) complex, thereby suppressing apoptosis and promoting tumor growth in cancer models90, 91, 92 and indeed, FFA1 agonism enhanced the expression of NF-κB p65 and its downstream transcriptional target cyclooxygenase-2 (COX-2), which together drive proliferation.³⁶ We further demonstrated that the AKT inhibitor MK2206,⁹³ the IκB-α/IKK inhibitor BAY 11-7802,⁹⁴ which blocks NF-κB activity, and the COX-2 selective inhibitor celecoxib⁹⁵ each suppressed AS2034178-induced proliferation of ACHN pRCC cells in serum. These findings suggest that FFA1 agonism activates signaling through the c-Src/PI3K/AKT/NF-κB/COX-2 axis in ACHN pRCC cells (Fig. 2).³⁶

EGFR, a member of the receptor-tyrosine kinase superfamily, is overexpressed in up to 60% of RCC tumors.⁹⁶ Because GPCRs frequently transactivate EGFR, we investigated whether FFA1 agonism affects EGFR signaling in pRCC cells. The FFA1 agonist AS2034178 rapidly and consistently suppressed EGFR phosphorylation at Tyr1068 and reduced downstream ERK1/2 activation, indicating negative regulation of the EGFR/mitogen-activated protein kinase pathway (Fig. 2).³⁶ In contrast, EGF strongly induced EGFR and ERK1/2 phosphorylation, which was completely blocked by the EGFR inhibitor AG1478 and partially inhibited by AS2034178, suggesting that FFA1 dampens both basal and ligand-induced EGFR signaling.³⁶ Functionally, EGF significantly promoted pRCC cell invasion in Matrigel assays, whereas AG1478 fully, and AS2034178 partially, reversed this effect.³⁶ Together, these findings show that FFA1 limits pRCC cell invasion, at least in part, through inhibition of EGFR/ERK1/2 signaling (Fig. 2). EMT, marked by loss of E-cadherin and upregulation of mesenchymal markers such as N-cadherin, vimentin, and fibronectin (FN1), drives cancer cell migration and metastasis,97, 98, 99, 100 and FFA1 activation significantly reduced E-cadherin and vimentin expression, strongly suppressed FN1, but had no effect on N-cadherin.³⁶ These changes occurred independently of EGFR, as EGF and AG1478 failed to alter EMT markers.³⁶

Finally, we demonstrated that FFA1 agonism promotes pRCC tumor growth in a xenograft mouse model in which subcutaneous-flank tumor-bearing mice were treated daily for 28 days with vehicles, AS2034178, GW1100, or both.³⁶ AS2034178 significantly accelerated tumor growth from day 16 onward, whereas GW1100 suppressed tumor growth in a time-dependent manner and abrogated the effects of AS2034178.³⁶ Consistently, tumor weights were elevated in the AS2034178 group but markedly reduced by GW1100 alone or in combination.³⁶ These findings align with our in vitro data, confirming that FFA1 signaling drives pRCC tumor progression, although the effects on tumor migration were unexamined in this study. Although this work has only been performed in a single pRCC cell line that is driven by mutant c-MET, further work in other pRCC cell lines is currently ongoing to better characterize the role of FFA1 in heterogeneously-derived pRCCs from distinct mutagenic etiologies. Nonetheless, this work from the well characterized ACHN cell line demonstrates a robust role of FFA1 in mediating proliferation and migratory capacity.

Although evidence discussed here shows a clear renoprotective and antifibrotic role for FFA1 in preclinical models of CKD, recent findings in pRCC indicate that FFA1 activation may promote tumor cell growth and survival, raising potential safety concerns. These observations suggest that the beneficial effects of FFA1 agonists in nonmalignant kidney disease may not fully translate in settings where oncogenic pathways may be active. Therefore, careful consideration of disease context, along with further investigation into tissue- and pathway-specific signaling mechanisms, will be critical for the safe therapeutic development of FFA1-targeted agents. An additional layer of complexity arises from evidence that FFA4 agonism may exert opposing effects in various cancer cell types, as we describe in detail below, underscoring the need for further studies to delineate the distinct and potentially divergent roles of FFA1 and FFA4 in modulating oncogenic processes, particularly in the context of coexisting pathologies such as fibrosis.

3. Free-fatty acid receptor-4

3.1. Renal tubular apoptosis

Similar to FFA1, activation of FFA4 also protects against renal tubular apoptosis by modulating ER stress and UPR signaling pathways in kidney injury models. In a mouse model of cisplatin-induced kidney injury, oral administration of the FFA4-selective agonist TUG-891 restored the expression of FFA4, which was decreased by cisplatin in proximal TECs, and alleviated the symptoms of tubular damage.³⁷ Moreover, TUG-891 treatment downregulated proapoptotic biomarkers Bax and cleaved Caspase-3, and proinflammatory cytokines IL-1β, IL-6, and TNF-α in epithelial cells, suggesting that FFA4 activation prevents tubular apoptosis and inflammation induced by cisplatin.³⁷ In vivo, TUG-891 reduced extensive rough ER degranulation, smooth ER dilation, and other ER morphological abnormalities. FFA4 stimulation also decreased the expression of UPR pathway sensors including PERK, IRE1, and ATF6 in proximal TECs, indicating that FFA4 activation helps prevent tubular loss and injury in cisplatin-induced AKI by suppressing ER stress and downstream UPR signaling.³⁷ TUG-891 further suppressed both the expression and transcription of C/EBP homologous protein, a downstream stress-related transcription factor that regulates proapoptotic protein activity.³⁷ Likewise, TUG-891 treatment restored FFA4 expression, inhibited apoptosis, and reduced UPR protein levels in response to chemically induced ER stress in human epithelial proximal tubule HK-2 cells in vitro.³⁷ It is important to note that although TUG-891 is profoundly more selective for human FFA4 than human FFA1 (∼50–300-fold), this selectivity is significantly reduced (∼3–61-fold) at the mouse ortholog,³⁸ and given the concentrations of the agonist used in this study and the lack of FFA1-investigation, a role for FFA1 in these animal studies cannot be ruled out. FFA4 expression was reported to be reduced in TECs of cecal ligation/perforation sepsis-induced AKI mice,¹⁰¹ where systemic or TEC-specific knockout of FFA4 worsened renal dysfunction and injury, whereas activation with TUG-891 alleviated cisplatin-induced AKI.¹⁰¹ FFA4 also regulated TEC senescence, as shown by changes in SA-β-gal activity, p21, p53, Lamin B1, γH2A.X, phospho-Rb, and IL-6.¹⁰¹ Mechanistically, FFA4 activation or overexpression restored SirT3 via Gq/CaMKKβ/AMPK signaling, exerting antisenescent effects in cisplatin-induced AKI.¹⁰¹

Hyperuricemic nephropathy (HN) is a clinical condition characterized by elevated uric acid levels that result in renal inflammation,¹⁰² which has been linked to pyroptosis, a proinflammatory form of programmed cell death,¹⁰³ although the exact underlying mechanisms remain unclear. In a mouse model of HN, ω-3 fatty acids (DHA and EPA) protected against renal injury by downregulating the mRNA and protein levels of pyroptosis- and inflammation-related markers, including NLRP3, cleaved gasdermin, caspase-1, and IL-1β.³⁹ In vitro, FFA4 activation by TUG-891 or DHA reduced uric acid- and lipopolysaccharide-induced pyroptosis in the RAW264.7 mouse macrophage cell line, an effect reversed by the antagonist AH-7614.³⁹ In HN mice, TUG-891 reduced tubular dilation, epithelial atrophy, and interstitial inflammation, whereas DHA plus AH-7614 worsened these lesions compared with DHA alone.³⁹ In murine RAW264.7 macrophages, DHA induced FFA4 internalization and its association with β-arrestin-2, which sequestered NLRP3 to disrupt inflammasome assembly, reduced NLRP3/caspase-1 interaction, and suppressed inflammation, revealing the mechanism of FFA4 activation in HN.³⁹

3.2. Renal fibrosis and inflammation

FFA4, expressed on macrophages, adipocytes, and selected endocrine cells, regulates metabolism and immune responses.104, 105, 106, 107 In peritoneal and RAW264.7 macrophages, it signals via Gαq/11 and β-arrestin-2, the latter of which blocks TAK1-IKK/NF-κB signaling to suppress inflammasome priming and inflammatory transcription.³² FFA4 activation also promotes M2 polarization and inhibits reactive oxygen species, cyclooxygenase activity, prostaglandin synthesis, and NLRP3 inflammasome activation,^108,109 highlighting its potential as a therapeutic target in fibrosis (Fig. 1).

In isolated rat peritoneal macrophages that were repolarized in vitro with the FFA4 agonist TUG-891 and autologously transplanted into obstructed kidneys via intrarenal injection for 7 days post-UUO surgery,⁴⁰ TUG-891 markedly increased M2 and decreased M1 marker expression compared with vehicle-treated cells (Fig. 1).⁴⁰ Moreover, reprogrammed macrophages alleviated renal injury and interstitial fibrosis, evidenced by preserved parenchymal thickness and reduced Masson trichrome staining,⁴⁰ and they also suppressed renal inflammation by downregulating TNF-α and IL-1β and attenuated the UUO-induced upregulation of TGF-β1 and SMAD2 (Fig. 1).⁴⁰ Furthermore, FFA4-repolarized macrophages inhibited EMT and myofibroblast differentiation, reducing vimentin, α-SMA, and β-catenin in UUO mice,⁴⁰ highlighting their therapeutic potential for renal fibrosis. However, the mechanisms underlying these antifibrotic effects have not been fully explored, and given the species-based selectivity of TUG-891 noted above, without knockdown studies, the possible synergistic roles of FFA1 remain unclear, despite its known protective functions in renal fibrosis.

3.3. Diabetic nephropathy

FFA4 is expressed in glomerular podocytes in both patients with DN and db/db mice, where activation with the agonist TUG-891 improved the urine albumin-to-creatinine ratio, reduced glomerulosclerosis, and alleviated podocyte injury and foot process effacement, indicating mitigation of renal damage.⁴¹ TUG-891 also decreased glomerular collagen deposition and ECM components, including α-SMA, FN1, collagen IV, and TGF-β1, and suppressed profibrotic and proinflammatory signaling via Smad3, JAK2/STAT3, and IKKβ/NF-κB and JNK/c-Jun pathways.⁴¹ As noted above the FFA4/β-arrestin-2/TAB1/TAK1 cascade and downstream inhibition of NF-κB, JNK, and p38 mitogen-activated protein kinase, reduces inflammatory cytokine production^105,110 and these effects were confirmed in HG-cultured human MPC5 podocytes, where TUG-891 suppressed ECM and inflammatory markers in an FFA4-dependent manner.⁴¹ Collectively, these findings establish that FFA4 agonism via TUG-891 activates the β-arrestin-2/TAB1/TAK1 axis, dampens downstream signaling, and alleviates inflammation and fibrosis in DN.⁴¹

The role of FFA4 in DN was also examined by Singh et al⁴² in a case-control study that included 100 patients diagnosed with type 2 diabetes and DN, and 100 patients with type 2 diabetes without DN. Although the manuscript does not mention the source of FFA4 as a circulating serum biomarker, diabetic patients with nephropathy exhibited significantly lower serum FFA4 levels, which were closely linked to dyslipidemia and renal dysfunction.⁴² Regression analysis showed that high-density lipoprotein cholesterol positively correlated with serum FFA4, whereas low-density lipoprotein cholesterol (LDL-C), triglycerides, cystatin C, and microalbuminuria were inversely associated with FFA4.⁴² These findings suggest a protective synergy between serum FFA4 and high-density lipoprotein cholesterol, whereas elevated LDL-C or triglycerides may counteract this effect and promote renal injury.⁴² Higher EGFR also predicted greater FFA4 levels, indicating a possible compensatory role.⁴²

3.4. Renal cell carcinoma

Similar to FFA1, FFA4 was found to be upregulated in pRCC tissue compared with patient-matched normal kidney tissue, with its expression rising alongside tumor pathological grade.⁴³ Selective FFA4 activation by highly selective FFA4 agonist CpdA inhibits serum-induced human ACHN pRCC cell proliferation in vitro and suppresses tumor growth in vivo.⁴³ Conversely, CpdA treatment enhanced pRCC cell migration, wound healing, and basement membrane invasion, effects that were reversed by the FFA4 antagonist AH-7614.⁴³ Using the EGFR inhibitor AG1478 and the MEK inhibitor PD98059, we showed that FFA4 drives pRCC invasion partly via EGFR activation and directly through ERK1/2 signaling (Fig. 2).⁴³ FFA4 agonism with CpdA also upregulated PI3K, pAKT1, NF-κB, COX-2, and matrix metalloproteinase-9 (MMP-9) in pRCC cells and, importantly, CpdA failed to promote invasion in the presence of inhibitors targeting AKT (MK2206), NF-κB (BAY11-7802), COX-2 (celecoxib), or MMPs (GM6001), indicating that FFA4 promotes pRCC cell invasion via the PI3K/AKT/NF-κB/COX-2/MMP-9 axis (Fig. 2).⁴³ Moreover, FFA4 agonism activated STAT3-driven EMT signaling in pRCC cells, highlighting its key role in promoting cell invasion.⁴³ In contrast to FFA1 agonism, FFA4 agonism in mouse flank xenografts showed a substantial reduction in tumor size, an effect that was reversed in the presence of AH-7614. Together, these results suggest that FFA1 and FFA4 play opposing roles in the regulation of pRCC and likely share this regulatory role in homeostatic renal regulation.

4. Free-fatty acid receptors-2 and -3

4.1. Renal fibrosis and inflammation

Kobayashi et al⁴⁴ demonstrated that both FFA2 and FFA3 are expressed in the distal renal tubules and collecting tubules in the human kidney. MCP-1 is a potent chemoattractant found in renal cells, particularly in renal epithelial cells, where its elevation contributes to the progression of fibrosis.¹¹¹ In human renal cortical epithelial (HRCE) cells, SCFA significantly reduced TNF-α-induced MCP-1 mRNA and protein levels in a dose-dependent manner.⁴⁴ Using the selective agonists CFMB and CPC for FFA2 and FFA3, respectively, the authors demonstrated that activation of FFA2 and FFA3 inhibits TNF-α-induced MCP-1 production in HRCE cells.⁴⁴ Silencing FFA2 and FFA3 also reversed NaP-mediated suppression of TNF-α-induced MCP-1 in HRCE cells, confirming that the inhibitory effects of SCFA are FFA2/FFA3-dependent.⁴⁴ In HRCE cells, SCFA attenuated TNF-α-induced MCP-1 expression by inhibiting p38 and JNK phosphorylation.⁴⁴ FFA2 and FFA3 were shown to be mainly expressed in distal and collecting tubules in mouse kidneys, consistent with findings from renal biopsies of patients with minor glomerular abnormalities.⁴⁶ In an adenine-induced CKD mouse model, propionate alleviated tubular damage, inflammation, and fibrosis. These effects were abolished when FFA2 and FFA3 were knocked out, suggesting that SCFA exert renoprotective effects at least in part via FFA2 and FFA3 activation (Fig. 1).⁴⁶

Qiu et al⁴⁵ demonstrated that a ketogenic diet (KD), high in fat and extremely low in glucose, exerts antifibrotic effects in renal fibrosis via an FFA3-dependent mechanism.⁴⁵ KD enhances the metabolism of ketone bodies, including acetoacetate, β-hydroxybutyrate (β-OHB), and acetone, and attenuates structural remodeling in solid organs.112, 113, 114 In the UUO mouse model, KD preserved tissue structure, reduced excessive matrix deposition, and upregulated FAO rate-limiting enzymes Cpt1a and Acox1.⁴⁵ In the UUO mouse model, the KD preserved tissue structure, reduced excessive matrix deposition, and upregulated the FAO rate-limiting enzymes Cpt1a and Acox1. FAO is a mitochondrial metabolic process in which fatty acids are broken down to generate energy, and its enhancement is critical for maintaining renal tubular energy homeostasis.¹¹⁵ These protective effects were abolished by the Cpt1a inhibitor etomoxir, leading to more severe renal injury and fibrosis, suggesting KD mitigates renal fibrosis through FAO enhancement.⁴⁵ Furthermore, KD alleviates renal inflammation by suppressing IL-6 secretion and macrophage infiltration in an FAO-dependent manner.⁴⁵ In rat renal epithelial NRK52E cells, the most abundant ketone body, β-OHB, significantly downregulated TGF-β-induced profibrotic markers and restored the impaired expression of FAO enzymes and FFA3.⁴⁵ Similar effects were observed with the selective FFA3 agonist AR420626.⁴⁵ Knockdown of FFA3 using siRNA reversed the effects of β-OHB, indicating that β-OHB mitigates fibrosis by enhancing cellular FAO through an FFA3-dependent pathway.⁴⁵ However, the study did not investigate the detailed mechanism of FAO in renal fibrosis, nor did it examine the effects of exogenous ketone body supplementation or the involvement of other FFAR.

4.2. Diabetic nephropathy

Butyrate, an SCFA, improves insulin resistance and alleviates diabetes in DN models via FFA2 activation in a β-arrestin-2-dependent manner.⁴⁷ In type 2 diabetes models, butyrate restores FFA2 expression, downregulates β-arrestin-2, and inhibits p-NF-κB p65 and MCP-1, and protects against DN via FFA2-mediated NF-κB inhibition (Fig. 3).⁴⁷ In glomerular mesangial cells, both butyrate and FFA2 agonists inhibited high-glucose-induced NF-κB signaling and reactive oxygen species generation (Fig. 3),⁴⁷ and FFA2 silencing partially reversed these effects, whereas FFA2 overexpression enhanced the protective action.⁴⁷

Fig. 3.

Dual effects of FFA2 signaling in DN. In diabetic mice, SCFA-mediated FFA2 activation in glomerular mesangial cells (GMCs) suppresses NF-κB signaling and reduces proinflammatory mediators, thereby alleviating renal inflammation. Conversely, in the same model, acetate-induced FFA2 activation in podocytes enhances LDL cholesterol uptake and impairs insulin signaling, which can exacerbate DN. Figure created in part under license from Biorender.com.

Dietary fiber reduces inflammation and mortality in CKD, likely via SCFA production, although the underlying mechanism remains unclear.^116,117 Li et al⁴⁸ reported that fiber-rich diets increase SCFA production and protect against DN through SCFA acting via FFA2 and the GPCR GPR109A, which is not an official FFAR family member, but is agonized by SCFA in addition to niacin. In diabetic mice, high-fiber diets decreased albuminuria, glomerular hypertrophy, podocyte injury, and interstitial fibrosis compared with normal or fiber-free diets.⁴⁸ Fiber reshaped the gut microbiota, promoting SCFA-producing bacteria such as Prevotella and Bifidobacterium, thereby increasing fecal and systemic SCFA levels.⁴⁸ It also downregulated renal genes associated with inflammation (CXCL10, CCL2, and TGF-β) and fibrosis (TNF-α and FN1) (Fig. 3).⁴⁸ In vitro, SCFA attenuated hyperglycemia-induced inflammation in primary murine renal TECs and reduced injury to primary podocytes.⁴⁸ Using FFA2 and GPR109A knockout diabetic mice, the study demonstrated that these receptors, which bind acetate and butyrate, are necessary for fiber- and SCFA-mediated protection against DN,⁴⁸ however, the detailed contribution of individual receptors has not been clearly elucidated.

Although most studies report on the protective effects of FFA2 agonism in DN, Lu et al⁴⁹ demonstrated that FFA2 activation can also promote DN progression. Lipid molecules are essential for podocyte integrity and function, whereas excessive cholesterol accumulation causes lipotoxicity and podocyte injury, a hallmark of DN.^118,119 In diabetic mice, FFA2 deletion reduced plasma LDL cholesterol and renal cholesterol accumulation (Fig. 3).⁴⁹ In vitro, acetate promoted excessive cholesterol deposition in high glucose-stimulated immortalized mouse podocytes by enhancing LDLR-mediated uptake and impairing autophagic degradation, as evidenced by reduced LC3 maturation, p62 degradation, and autophagosome formation.⁴⁹ These effects were prevented by FFA2 knockdown or inhibition.⁴⁹ Moreover, FFA2 activation increased ERK1/2 activity and early growth response protein 1 expression, leading to greater cholesterol influx and autophagy suppression, whereas FFA2 deletion ameliorated these alterations both in vivo and in vitro.⁴⁹

FFA2 expression is elevated in kidney biopsies from patients with DN and in diabetic mice and is associated with reduced murine podocyte insulin sensitivity.⁵⁰ In vitro, acetate increased FFA2 mRNA and protein expression in podocytes in a dose-dependent manner⁵⁰ and also induced a phenotypic shift, with elevated α-SMA and collagen I and reduced nephrin expression, demonstrating activation of myofibroblasts and fibrotic remodeling as well as podocyte dysfunction.⁵⁰ These effects were markedly diminished by FFA2 siRNA treatment.⁵⁰ Acetate also impaired insulin signaling and glucose uptake in podocytes, effects that were reversed by FFA2 knockdown or antagonism, suggesting a role for FFA2 in insulin sensitivity within renal podocytes.⁵⁰ In FFA2 knockout mice, FFA2 deficiency alleviated podocyte and kidney injury associated with insulin resistance in DN.⁵⁰ The authors further demonstrated that gut microbiota dysbiosis in DN elevates acetate, activating FFA2 and impairing podocyte insulin signaling, whereas microbiota correction reduces acetate, FFA2 activity, and kidney injury.⁵⁰ Acetate dose-dependently inhibited AMPKα phosphorylation in murine immortalized podocytes, an effect reversed by FFA2 knockdown or antagonism.⁵⁰ FFA2 deficiency in diabetic mice restored AMPKα activation, indicating FFA2 as a key suppressor of AMPKα signaling in DN (Fig. 3).⁵⁰

ACT001, an orphan drug derived from the natural sesquiterpene micheliolide, exerts anti-inflammatory effects by inhibiting NF-κB and STAT signaling pathways.^120,121 In a mouse model of high-fat diet–induced obesity-related glomerulopathy, ACT001 alleviates chronic kidney injury via an FFA2/AMPK-dependent mechanism.⁵¹ In high-fat diet–induced obesity-related glomerulopathy mice, ACT001 reduced kidney fibrosis and improved renal structure and filtration function.⁵¹ ACT001 restored intestinal valproic acid levels, enhanced SCFA-FFA2 interaction in the kidney, and activated AMPK, which in turn stimulates Nrf2-Keap1 to inhibit NF-κB signaling.⁵¹ Additionally, SCFA increased colonic FFA2, suppressed NLRP3 inflammasome, and restored zonula occludens-1 and occludin expression.⁵¹

4.3. Nephrolithiasis/kidney stone diseases

Nephrolithiasis, or kidney stone disease, is a common global urological disorder, affecting over 35 million North Americans annually and imposing substantial healthcare burdens.¹²² In 2021, 106 million new cases were reported worldwide, predominantly in men, representing a 27% increase since 2000.^122,123 Calcium oxalate (CaOx) is the most frequent stone component, accounting for approximately 80% of cases.¹²⁴ Although nephrolithiasis has traditionally been considered an acute condition, growing evidence suggests it is a chronic, systemic disorder that can progress to end-stage renal disease.^122,123,125 Various factors, such as metabolic syndrome,¹²⁵ diet, vitamins, hydration, and sex hormones, are associated with CaOx stone formation,¹²⁶ however, the underlying mechanisms remain unclear, limiting the development of effective therapies and preventive measures. Genomic studies link CaOx stones to genes regulating oxidative stress, immunity, inflammation, and complement activation, driving recruitment of macrophages, dendritic cells, and T cells.127, 128, 129 However, the immune cell landscape in CaOx-affected kidneys remains poorly characterized. In this regard, administration of SCFA, including acetate, propionate, and butyrate, for 4 weeks significantly reduced crystal formation in a renal CaOx stone mouse model.¹³⁰ Moreover, taxonomic analysis of gut microbiota in patients with kidney stone versus nonstone controls revealed decreased absorption and utilization of acetic acid by gut bacteria or other organs, suggesting a potential link between SCFAs and kidney stone formation.¹³⁰

In a follow-up study, the same authors demonstrated a pivotal role for SCFA and their receptor FFA2 in attenuating CaOx kidney stone formation through immune modulation.⁵² In a murine CaOx nephrolithiasis model, SCFA significantly reduced renal crystal deposition, accompanied by decreased inflammatory cytokines (IL-1β, IL-6, and TNF-α) and suppression of NLRP3 inflammasome activation.⁵² SCFAs reshaped renal immune cell populations by increasing anti-inflammatory CX3CR1⁺CD24⁻ macrophages while reducing infiltration of proinflammatory GR1⁺ neutrophils.⁵² Mechanistically, SCFA enhanced FFA2 expression on CX3CR1⁺CD24⁻ macrophages, promoting anti-inflammatory activity and limiting crystal adhesion, while suppressing FFA2 expression on GR1⁺ neutrophils, thereby inhibiting their migration and inflammatory responses.⁵² These immunomodulatory effects were gut microbiota-dependent, as antibiotic-mediated bacterial depletion abolished immune regulation despite preserved reductions in crystal deposition. Furthermore, Jin et al⁵² identified a novel FFA2-mediated macrophage-neutrophil crosstalk, wherein CX3CR1⁺CD24⁻ macrophages suppressed FFA2 expression on GR1⁺ neutrophils. Collectively, these findings highlight SCFA and FFA2 as promising therapeutic targets for CaOx nephrolithiasis via immune and microbiota-dependent mechanisms and underscore the importance of the gut-kidney axis, whereby microbiota-derived SCFA orchestrate systemic immune responses that directly influence renal inflammation and crystal pathogenesis.

5. Current challenges and knowledge gaps

Short-chain (FFA2/FFA3) and long-chain (FFA1/FFA4) FFAR have been implicated in renoprotection across multiple models of acute and chronic kidney injury, however, the magnitude, endurance across disease state progression, and translatability of these effects remain incompletely established. The reported benefits of FFAR function, including preservation of tubular integrity, attenuation of fibrosis and inflammation, and protection from nephropathy, are context and model-dependent and have yet to be replicated across species or in humans. For example, oral administration of TUG-891 restores FFA4 expression and attenuates ER stress-induced apoptosis by suppressing maladaptive UPR signaling,³⁷ however, the durability of this response, and its disease stage dependence are still unknown. Similarly, FFA1 modulates ER stress and UPR-linked proapoptotic pathways in chronic kidney injury models such as UUO, but whether this reflects a shared or redundant protective effect or FFA1-specific role remains unknown, underscoring its broader role in maintaining tubular cell survival.³⁴ Moreover, the distinct cellular and molecular signals that modulate these effects remain uncharacterized. For instance, although both FFA1 and FFA4 have been shown to protect against renal tubular apoptosis through modulation of ER stress and UPR, it remains unclear whether these receptors converge on identical downstream signaling cascades or whether they activate distinct, context-specific protective mechanisms.

Another unresolved issue relates to the interpretation of antifibrotic effects attributed to FFAR acting agents and the true selectivity of the drugs used. For example, PBI-4050, a FFA1 agonist, has been shown to slow fibrosis progression in adenine-induced kidney fibrosis and spontaneous tubulointerstitial fibrosis models,^31,35 however, the relative contribution of FFA1 versus still-to-be defined targets of this, and other agents remains uncertain. Interestingly, dual FFA1/PPARδ agonists such as HWL-088 and ZLY-032, exhibit strong renoprotective effects in UUO mice,³² suggesting functional crosstalk between FFAR and other receptor pathways. Although evidence for antifibrotic roles of FFA2/FFA3 in the kidney is limited, both receptors are expressed in distal tubules and collecting ducts in mice, and their deletion accelerates fibrosis progression.⁴⁴ Overall, FFAR appear to reduce proinflammatory mediators such as TNF-α and key fibrotic regulators, including TGF-β1 and SMADs, but the receptor-specific mechanisms by which they modulate the central fibrosis driver TGF-β1 remain unclear (Fig. 1).^32,35,40,44

A key limitation across this field is the heavy reliance on pharmacological ligands presumed to be receptor-selective, despite accumulating evidence that many of these compounds engage multiple FFAR. For instance, GW9508, often used as an FFA1 agonist, also activates FFA4, an activity that is not consistently acknowledged and complicates attribution of receptor-specific effects.¹³¹ Similarly, although TUG-891 is widely used to probe FFA4 function in CKD models, its FFA4 over FFA1 selectivity at the rodent receptor is greatly reduced compared with its selectivity at the human receptor³⁸ and ligand-dependent issues, which can greatly confound the interpretation of results, should be considered when using these agents.

Similarly, PBI-4050, an FFA1 agonist described here to attenuate renal apoptosis, fibrosis, and CKD-associated anemia, does not demonstrate effects mediated exclusively through a single receptor, as it also acts at GPR84. Although some studies suggest that FFA1 may represent a more promising therapeutic target than GPR84, given its broader impact on fibrosis, anemia, and ER stress, these conclusions are difficult to disentangle from ligand polypharmacology, and notably, the reduced efficacy of PBI-4050 in the absence of FFA1 supports a central role for this receptor, but does not exclude contributions from other targets. Likewise, newer agents such as HWL-088 and ZLY-032 demonstrate greater potency in reducing renal fibrosis and inflammation in adenine-induced models, however, their dual activity as FFA1/PPARδ agonists further complicates mechanistic interpretation. Collectively, these studies underscore the need for cautious interpretation of pharmacological data and highlight the importance of incorporating complementary genetic approaches (eg, receptor knockout models) or rigorously validated, highly selective antagonists to definitively establish receptor-specific mechanisms in renal fibrosis.

Another key knowledge gap relates to the coexpression of multiple FFAR subtypes within the same renal cell populations and the harmonized actions of FFA at these receptors in vivo. To date, most studies have examined the role of individual FFAR in isolation, or with agonists that are demonstrated to be selective for one FFAR over others, despite growing evidence that multiple FFAR are expressed within the same renal compartments, including TECs, immune cells, and interstitial fibroblasts. Because many endogenous FFA exhibit overlapping affinity and functional agonism across several FFAR (eg, FFA1 and FFA4 or FFA2, FFA3 and GPR109A), physiological and pathological FFA signaling in the kidney is likely to involve the simultaneous or synchronous activation of multiple receptors rather than engagement of pathways linked to a single receptor at a single point in time. Whether FFAR function in a complementary, synergistic, or antagonistic manner when coactivated remains completely unexplored, and this gap has important translational implications, as pharmacologic strategies that selectively target individual FFAR may fail to recapitulate the integrated signaling elicited by dietary or endogenous FFA, potentially limiting efficacy or producing unanticipated outcomes. A more comprehensive understanding of FFAR coexpression, receptor crosstalk, and signal integration will therefore be essential for designing therapeutic approaches that more accurately reflect the complexity of fatty acid signaling in kidney disease.

6. Therapeutic potential of dietary FFA in kidney diseases

Accumulating preclinical, clinical, and epidemiologic-based results suggest that the type of dietary fat, rather than simply the total fat intake, plays meaningful roles in kidney disease progression and related cardiovascular and metabolic downstream effects. For example, observational studies demonstrate that higher intake of LCFA, such as ω3 polyunsaturated fatty acids, are associated with lower prevalence of albuminuria, slower decline in glomerular filtration rate, and reduced risk of CKD, particularly in patients with DM.^132,133 Moreover, interventional studies with ω3 FFA in patients with CKD show that ω3 supplementation improves dyslipidemia, reduces systemic inflammation, and lowers proteinuria.^133,134 In contrast, higher intake of saturated fats and certain ω6 fatty acids has been associated with greater albuminuria, endothelial dysfunction, and adverse renal outcomes.132, 133, 134, 135 Although results such as these have not been demonstrated to definitively involve FFAR, given the roles of LCFA FFAR such as FFA1 and FFA4 in recognizing structurally-distinct FFA, and the biased nature of FFA responses at these receptors,136, 137, 138 it is within reason that FFAR mediate at least some of these responses.

From a translational standpoint, regardless of the yet to be determined involvement of specific FFAR in mediating responses to dietary FFA in this context, these findings support dietary fat modification as a feasible, low-risk adjunct to pharmacotherapy in CKD, diabetic kidney disease, and perhaps even patients with AKI at risk of further progression. Diets enriched in ω3 fatty acids and unsaturated fats align with improved cardiovascular outcomes in patients with CKD, a population in whom cardiovascular disease remains the leading cause of mortality.^135,139 In this regard, pharmacologic formulations of ω3 FFA, such as icosapent ethyl, a high-purity EPA product available by prescription in the United States (Vascepa), as well as many other countries, demonstrates significant cardiovascular benefit in high-risk populations, suggesting potential translational relevance to CKD, DN, or other patient populations where cardiovascular burden is significant. However, it is not known whether its benefits extend to renal endpoints, and no definitive mechanistic role of FFAR in its renal or other effects have been established, even though EPA is an agonist of FFA4 and FFA1.

In parallel, fiber-rich dietary patterns that increase SCFA production have been associated with reduced inflammation, improved metabolic profiles, and lower mortality in CKD cohorts, highlighting a gut-kidney axis that may act, at least in part, through SCFA-responsive receptors such as FFA2, FFA3, and GPR109A.^4,6 Despite these promising associations, several pharmacological and clinical limitations remain, including variability in bioavailability and tissue distribution of dietary FFA, challenges in achieving therapeutically effective concentrations at target tissues, and serum protein binding, which is significant for most LCFA. An additional layer of complexity in SCFA-mediated effects arises from interindividual variability of the gut microbiome, which plays a central role in the fermentation of dietary fiber and subsequent SCFA production. The composition and functional capacity of the microbiota can vary widely based on diet, age, medication use, and comorbidities, including CKD and DN, which is associated with gut dysbiosis.140, 141, 142, 143 This variability can significantly influence both the quantity and profile of SCFA FFAR agonists, including acetate, propionate, and butyrate, thereby impacting downstream activation of SCFA-responsive receptors such as FFA2, FFA3, and GPR109A. Consequently, individuals consuming variable diets may exhibit markedly different metabolic and inflammatory responses due to differences in microbiome composition and function. Furthermore, uremic conditions in CKD may alter microbial metabolism, shifting production away from beneficial SCFA toward potentially harmful metabolites, further complicating therapeutic strategies.¹⁴⁴ These considerations warrant integration of microbiome profiling into future studies and suggest that modulation of the gut microbiota, through prebiotics or probiotics, may represent a complementary approach to optimize SCFA production and FFAR-mediated signaling in kidney disease.

Importantly, heterogeneity in clinical responses to dietary FFA interventions also likely reflect variability in disease stage, underlying metabolic context such as fat and inflammatory conditions, as well as cell-type-specific FFAR expression, underscoring the need for further research delineating the role of FFAR in FFA responses that may align toward precision nutrition strategies rather than uniform dietary prescriptions. Future efforts will require development of receptor-selective FFAR agonists with improved pharmacokinetic properties, as well as well controlled clinical trials that integrate dietary, pharmacologic, and biomarker-driven approaches to better define patient populations most likely to benefit. Such strategies may ultimately bridge the gap between observational associations and targeted therapeutic interventions in kidney disease.

Conflict of interest

The authors declare no conflicts of interest.