Probiotics-enhanced kynurenic acid mitigates cisplatin-induced nephrotoxicity in mice

Summary

Cisplatin chemotherapy is limited by dose-dependent nephrotoxicity. This study investigates the nephroprotective potential of kynurenic acid (KYNA), a tryptophan metabolite, against cisplatin-induced renal injury. Initial metabolic results revealed significant elevation of serum KYNA levels in cisplatin-treated mice, suggesting endogenous compensatory mechanisms. Systematic pharmacological evaluation demonstrated that intraperitoneal administration (i.p.) of KYNA (100, 250, and 500 mg/kg) dose-dependently attenuated cisplatin-induced nephrotoxicity through multi-modal mechanisms, including the suppression of pro-inflammatory cytokines via NF-κB p65 pathway inhibition and MAPKs dephosphorylation, reduction of renal apoptosis through Bcl-2 family rebalancing and caspase-3 cascade inhibition, and enhancement of antioxidant defenses via Nrf2 pathway activation with concomitant upregulation of downstream effectors. We further established that probiotic supplementation elevated endogenous KYNA production, achieving comparable renoprotection to high-dose KYNA monotherapy. Our findings delineate KYNA’s multi-modal mechanisms against cisplatin nephrotoxicity and demonstrate that the probiotic-mediated modulation of host metabolism represents a viable strategy to enhance endogenous KYNA for renal protection.

Graphical abstract

Highlights

• •Kynurenic acid mitigates cisplatin nephrotoxicity via multi-modal mechanisms
• •Probiotics elevate endogenous kynurenic acid to protect kidneys
• •Study reveals a gut microbiota-KYNA-kidney protective axis
• •KYNA’s renoprotection requires the activation of the Nrf2 pathway

Molecular biology; Neuroscience; Microbiology

Introduction

Cisplatin (CIS) remains a widely prescribed chemotherapeutic agent for treating diverse malignancies, including esophageal, lung, and ovarian cancers, through its DNA-intercalating mechanism that disrupts replication and repair processes in rapidly dividing cells.^1,2 However, its clinical utility is substantially limited by dose-dependent multiorgan toxicity,³ with nephrotoxicity emerging as a particularly debilitating complication.¹ The pathogenesis of cisplatin-induced acute kidney injury (AKI) involves preferential accumulation in renal proximal tubular epithelial cells, triggering a cascade of DNA-adduct formation, excessive inflammatory responses, and reactive oxygen species (ROS) production.^1,4 Current therapeutic strategies emphasize the modulation of redox-sensitive signaling networks, particularly nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways,⁴ alongside activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant response system.^5,6 The latter orchestrates cyto-protection through the upregulation of phase II detoxifying enzymes, including heme oxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GCLM), and NADPH quinone oxidoreductase 1 (NQO1), positioning Nrf2 activation as a pivotal therapeutic target for nephroprotection.^5,7

The tryptophan-kynurenine (KYN) metabolic axis yields biologically active metabolites with divergent pathophysiological roles.⁸ While neurotoxic intermediates such as quinolinic acid and 3-hydroxykynurenine exacerbate oxidative damage, kynurenic acid (KYNA) exhibits pleiotropic protective properties through its antioxidant, anti-inflammatory, and anti-apoptotic capacities.^7,8

Initially, KYNA is characterized as a neuroactive metabolite with dual roles in neuropsychiatric disorders, where cerebral overaccumulation disrupts glutamatergic neurotransmission and exacerbates neurodegeneration⁹ Subsequent research, however, uncovered its therapeutic potential in alleviating headaches and treating neurodegenerative diseases through exogenous administration.^10,11 Emerging evidence has revealed that KYNA is ubiquitous in brain tissue, peripheral blood, and various other tissues, exerting systemic regulatory functions.^8,12 It modulates carcinogenesis through cell-cycle arrest mechanisms, ameliorates metabolic disorders such as diabetes¹³ and osteoporosis.¹⁴ Furthermore, KYNA is closely intertwined with the maintenance of gut microbiota homeostasis across various diseases, playing a pivotal role in preserving the equilibrium of the gut-immune axis.^15,16 Pharmacokinetic studies further identify KYNA as a hepatorenal regulator, undergoing enterohepatic circulation with renal excretion within 24 h post-absorption.^17,18 Clinically, dynamic serum KYNA elevation has been documented across renal pathologies ranging from sepsis-associated multiorgan failure¹⁹ to ischemic²⁰ and chronic kidney disease,^21,22 suggesting its role as both biomarker and endogenous protectant. Despite these advances, the therapeutic potential of KYNA in drug-induced nephrotoxicity remains unexplored.

This study presents the first comprehensive investigation of KYNA’s nephroprotective efficacy against cisplatin-induced AKI, coupled with innovative probiotic-mediated KYNA modulation. We demonstrate that KYNA administration significantly attenuates cisplatin nephrotoxicity through multipronged mechanisms: suppression of NF-κB/MAPK-driven inflammation, enhancement of Nrf2-mediated antioxidant defenses, and inhibition of intrinsic apoptotic pathways. Furthermore, we establish that probiotic-induced KYNA elevation mirrors exogenous administration in renal protection, revealing a novel microbiota-metabolite axis in chemotoxicity management. These findings not only expand KYNA’s therapeutic repertoire to drug-induced nephrotoxicity but also pioneer a dual-intervention strategy combining pharmacological and microbiome modulation for enhanced therapeutic safety.

Results

Alterations in kynurenine metabolites in mice exposed to cisplatin-induced nephrotoxicity

Previous literature has reported significant alterations in kynurenine metabolites in various kidney injury diseases,^7,22 yet there has been no information regarding their levels in cisplatin-mediated nephrotoxicity. To address this gap, we first established an acute cisplatin toxicity model in WT C57BL/6J mice through a single intraperitoneal injection of cisplatin (25 mg/kg). Subsequently, blood samples were collected at 6 h and 12 h post-modeling, respectively, for the detection of biochemical markers, including serum creatinine, blood urea nitrogen (BUN), kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NAGL). As expected, serum levels of creatinine, BUN, KIM-1, and NAGL were significantly elevated after cisplatin exposure (Figures 1A–1E), indicating kidney injury.

Figure 1.

Dynamic changes in kynurenine metabolites during cisplatin-induced nephrotoxicity in mice

Serum and the kidney samples were collected from mice at 0 h, 6 h, and 12 h following the intraperitoneal injection of cisplatin (25 mg/kg). (A and B) Graphs depict blood urea nitrogen (BUN, A) and creatinine (B) levels as serum biochemical markers of renal injury.

(C–E) Representative immunoblots (C) and quantitative analysis of kidney injury molecule-1 (KIM-1, D) and N-acetyl-β-D-glucosaminidase (NAGL, E) protein expression (n = 3).

(F) Schematic representation of kynurenic acid (KYNA) biosynthesis within the kynurenine (KYN) pathway.

(G–I) Quantification of serum KYNA (G), KYN (H), and tryptophan (I) levels.

(J–L) Metabolic ratios of KYNA/KYN (J), KYN/tryptophan (K), and KYNA/tryptophan (L). Data are presented as the mean ± SEM. Statistical significance was determined by one-way ANOVA with Bonferroni’s post hoc test (A, D, E, G, H, J, and L) or Welch’s ANOVA with Games-Howell post hoc test (B). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus 0 h control group (n = 9).

In the kynurenine metabolite pathway,⁸ KYNA is primarily derived from KYN under the action of KAT enzymes (Figure 1F). Our results indicate that there was a significant increase in serum KYNA levels following 12 h of exposure to cisplatin-induced toxicity (Figure 1G). Although an upward trend was also observed in the levels of KYN (Figure 1H) and tryptophan (Figure 1I), these changes were not statistically significant. Importantly, the ratios of KYNA/KYN and KYNA/tryptophan increased markedly (Figures 1J–1L), suggesting that more tryptophan and KYN were converted into KYNA after cisplatin treatment. Overall, these findings suggest that KYNA plays a role in the pathological processes associated with cisplatin-mediated nephrotoxicity.

Protective effects of exogenous kynurenic acid on cisplatin-induced nephrotoxicity in mice

Despite exhibiting a complex dual effect in the field of neuropsychiatric disorders, KYNA is predominantly beneficial to the organism in most instances.^13,23 Multiple studies have suggested that KYNA can alleviate kidney injury induced by ischemia-reperfusion, and multi-organ damage mediated by sepsis.^19,24 Here, we hypothesize that the gradual increase in KYNA levels during cisplatin-induced kidney injury may represent a negative feedback mechanism activated by the body to counteract the damage. However, it is difficult to assess KYNA’s individual effects due to the absence of a specific KYNA-neutralizing agent. Additionally, agents capable of modulating KYNA levels, such as inhibitors of indoleamine 2,3-dioxygenase (IDO) and kynurenine aminotransferase (KAT) enzymes, also impact other kynurenine metabolites.^25,26

In this study, we employed the strategy of administering exogenous KYNA (100, 250, and 500 mg/kg, i.p.) 2 h prior to cisplatin treatment (Figure 2A). This adjustment yielded significant results: exogenous KYNA (250 and 500 mg/kg) mitigated cisplatin-induced nephrotoxicity, evidenced by a substantial reduction in mortality rates (Figure 2B) and decreased levels of BUN and serum creatinine (Figures 2C and 2D), as well as the protein levels of KIM-1 and NAGL (Figures 2E–2G). Conversely, a KYNA dose of 100 mg/kg failed to yield meaningful improvements in these parameters (Figures 2B–2G).

Figure 2.

Exogenous KYNA protects against cisplatin-induced nephrotoxicity in mice

(A) Experimental design: Mice were pretreated with exogenous KYNA (100, 250, or 500 mg/kg, i.p.) 2 h prior to cisplatin administration (25 mg/kg, i.p.), followed by survival analysis and serum/tissue collection.

(B) Survival rates for each group (n = 6).

(C and D) Serum BUN (C) and creatinine (D) levels 12h post-cisplatin (n = 6).

(E–G) Representative immunoblots (E) and quantitative analysis of KIM-1 (D) and NAGL (E) protein expression (n = 3).

(H) Representative hematoxylin and eosin (HE)-stained kidney sections (scale bars, 200 μm) show the effect of KYNA on cisplatin-induced renal injury.

(I) Semiquantitative histopathological injury scores assessed based on tubular necrosis, cast formation, and glomerular degeneration (n = 15). Data are presented as the mean ± SEM. Statistical significance was determined by one-way ANOVA with Bonferroni’s post hoc test (F), Welch’s ANOVA with Games-Howell post hoc test (C, D, G, I), or log rank test (B). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus control; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 versus cisplatin model (CIS-MOD) group.

Histopathological analysis revealed notable morphological distinctions among the experimental groups. The renal tissue of both the control group and the K500 group (KYNA 500 mg/kg, i.p.) maintained their normal histological architecture (Figure 2H). In stark contrast, the CIS-MOD group (cisplatin 25 mg/kg, i.p.) exhibited pronounced renal pathology, characterized by tubular atrophy with narrowed lumina, unclear glomerular capillary structures, increased mesangial cell proliferation, and a resultant reduction or obliteration of the Bowman’s capsule with indistinct borders (Figures 2H and 2I). Notably, the KYNA pre-treatment groups demonstrated a dose-dependent preservation of histological features. Improvements were particularly evident in the K250+CIS (250 mg/kg KYNA, i.p., 2h before 25 mg/kg cisplatin, i.p.) and K500+CIS (500 mg/kg KYNA, i.p., 2h before 25 mg/kg cisplatin, i.p.) groups. Compared to the CIS-MOD group, these KYNA pre-treated groups showed partial but statistically significant restoration of renal tubule and glomerular morphology, as well as clearly visible renal capsular cavities (Figures 2H and 2I). In summary, these results demonstrate that exogenous KYNA supplementation, particularly when administered prior to injury, can significantly mitigate the cisplatin-induced nephrotoxicity.

Exogenous kynurenic acid attenuates cisplatin-induced renal apoptosis

The TUNEL colorimetric assay was performed on kidney tissues to evaluate the impact of exogenous KYNA on renal apoptosis, which is a critical aspect of renal injury. As illustrated in Figures 3A–3C, the results demonstrated a significant increase in the number of TUNEL-positive cells in the cisplatin-treated group. In contrast, pretreatment with exogenous KYNA markedly reduced the number of these TUNEL-positive cells, with the K500+CIS group showing particularly notable improvement (Figures 3A–3C).

Figure 3.

Exogenous KYNA attenuates cisplatin-induced renal apoptosis

(A) Representative TUNEL staining of kidney sections showing apoptotic cells (red fluorescence, scale bars, 10 μm).

(B and C) Quantitative analysis of apoptotic cell density in 10 x (B, n = 12) and 20 x (C, n = 24) magnification fields.

(D) Immunoblot analysis of apoptosis-related proteins: cleaved caspase-3, Bax, and Bcl-2.

(E–G) Densitometric quantification of cleaved caspase-3 (E), Bax (F), and Bcl-2 (G) protein levels normalized to β-actin (n = 3). Data are presented as the mean ± SEM. Statistical significance was determined by one-way ANOVA with Bonferroni’s post hoc test (E–G) or Welch’s ANOVA with Games-Howell post hoc test (B, C). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus control; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 versus CIS-MOD group.

Furthermore, our analysis of protein levels provided additional insights into the mechanisms underlying the protective effect of KYNA. Specifically, we found that the cisplatin-induced upregulation of cleaved caspase-3 and BAX, which are key markers of apoptosis, was reversed by exogenous KYNA (Figures 3D–3F). This reversal was particularly pronounced in the K500+CIS group. Similarly, we observed that the cisplatin modulated downregulation of Bcl2-associated X (Bcl2), an anti-apoptotic protein, was also mitigated by KYNA pretreatment (Figures 3D and 3G). Collectively, these results suggest that KYNA may play a protective role in cisplatin-induced renal apoptosis by modulating the expression of key apoptotic proteins.

Exogenous kynurenic acid attenuates cisplatin-induced renal inflammation

The inflammation associated with cisplatin-induced renal toxicity was also evaluated by examining pro-inflammatory cytokines and inflammatory pathways.^1,4 As illustrated in Figures 4A–4I, the results indicated a significant elevation in the gene levels of pro-inflammatory cytokines, such as Interleukin--1β (IL-1β), IL-6, Cyclooxygenase-2 (Cox2) and IL-18, as well as chemokines including C-C motif ligand- 2 (CCL2), CCL4, CXC chemokine ligand-1 (CXCL1), CXCL2, and CXCL10, in the CIS-MOD group. The administration of exogenous KYNA demonstrated a potent, dose-dependent attenuation of cisplatin-induced expression for the majority of inflammatory cytokines and chemokines measured. This effect was most robust in the K500+CIS group, where expression levels approached those of the Ctrl group, indicating a substantial inhibition of the cisplatin-induced inflammatory response (Figures 4A–4I). Cisplatin promotes inflammatory responses by activating the NF-κB p65 and MAPK signaling pathways. Our findings revealed that exogenous KYNA could suppress the phosphorylation of NF-κB p65, p38 MAPK, and ERK1/2 (Extracellular regulated protein kinases 1/2), as demonstrated in Figures 4J–4M. However, KYNA did not affect cisplatin-mediated JNK (C-Jun N-terminal kinase) activation (Figures 4J and 4N). Similarly, the K500+CIS group exhibited a notable performance in this regard, demonstrating a marked suppression of the targeted phosphorylation events. Taken together, these findings suggest that exogenous KYNA, particularly in the K500+CIS group, has the potential to ameliorate cisplatin-induced renal toxicity by significantly inhibiting the inflammatory responses and the associated signaling pathways, thereby providing a promising therapeutic approach for managing cisplatin-related side effects.

Figure 4.

Exogenous KYNA attenuates cisplatin-induced renal inflammatory responses

(A–I) Renal mRNA expression profiles of pro-inflammatory mediators, including interleukin-1β (IL-1β, A), IL-6 (B), C-C motif chemokine ligand 2 (CCL2, C), CCL4 (D), C-X-C motif chemokine ligand 1 (CXCL1, E), CXCL2 (F), CXCL10 (G), cyclooxygenase-2 (Cox2, H), and IL-18 (I) quantified by RT-qPCR.

(J) Immunoblot analysis of phosphorylated NF-κB p65 (p-p65), p38 mitogen-activated protein kinase (p-p38 MAPK), extracellular signal-regulated kinase 1/2 (p-ERK1/2), and c-Jun N-terminal kinase (p-JNK).

(K–N) Densitometric quantification of p-p65/NF-κB p65 (K), p-p38/p38 (L), p-ERK1/2/ERK1/2 (M), and p-JNK/JNK (N) ratios normalized to total protein. Data are presented as the mean ± SEM. Statistical significance was determined by one-way ANOVA with Bonferroni’s post hoc test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus Ctrl group; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 versus CIS-MOD group (n = 3).

The effect of exogenous kynurenic acid on cisplatin-induced oxidative stress

Previous studies have demonstrated that oxidative stress play crucial role in the mechanisms underlying cisplatin-induced nephrotoxicity.¹ Given the antioxidant properties of KYNA,^8,13 it is plausible that KYNA may exert a beneficial effect through this mechanism. Our results clearly show that cisplatin treatment markedly diminishes renal levels of superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT) (Figures 5A–5C), which are essential antioxidants for combating oxidative stress. This reduction was accompanied by an elevation in malondialdehyde (MDA) levels (Figure 5D), a critical marker of oxidative stress, further indicating a decline in renal antioxidant defenses. Notably, pre-administration of exogenous KYNA in the K500+CIS group significantly preserved the renal antioxidant capacity of mice (Figures 5A–5D), highlighting its protective role against cisplatin-induced oxidative stress. Intriguingly, when compared to the Ctrl group, KYNA treatment alone was also capable of enhancing the levels of SOD, GSH, and CAT, while simultaneously reducing MDA levels (Figures 5A–5D). This underscores the antioxidant capabilities of KYNA.

Figure 5.

Exogenous KYNA alleviates cisplatin-induced renal oxidative stress

(A–D) Renal oxidative stress biomarkers, including superoxide dismutase (SOD, A), glutathione (GSH, B), catalase (CAT, C), and malondialdehyde (MDA, D) levels (n = 4).

(E) Immunoblot analysis of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream antioxidants, heme oxygenase-1 (HO-1) and glutamate-cysteine ligase modifier subunit (GCLM), and its upstream negative regulator Kelch-like ECH-associated protein 1 (KEAP1).

(F–I) Densitometric quantification of Nrf2 (F), HO-1 (G), GCLM (H), and KEAP1 (I) protein levels normalized to β-actin (n = 3).

(I and J) Serum BUN (J) and creatinine (K) levels 12h post-cisplatin (n = 6).

(L) Survival rates for each group (n = 8).

(M) Representative HE-stained kidney sections (scale bars, 200 μm).

(N) Semiquantitative histopathological injury scores assessed based on tubular necrosis, cast formation, and glomerular degeneration (n = 15). Data are presented as the mean ± SEM. Statistical analyses: one-way ANOVA with Bonferroni’s post hoc test (A, F–K); Welch’s ANOVA with Games-Howell post hoc test (B, D, N); one-way ANOVA (C, no post hoc test performed as the overall effect was not significant); log rank test (L). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus control; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 versus CIS-MOD group; ˆp < 0.05, ˆˆp < 0.01, and ˆˆˆp < 0.001 versus K500+CIS group.

Nrf2 plays a central role in maintaining redox homeostasis by regulating the expression of various signaling proteins and enzymes.⁵ Numerous Nrf2 activators have been reported to alleviate cisplatin-induced nephrotoxicity by upregulating antioxidants and inhibiting MDA production.⁵ Therefore, we conducted an investigation to evaluate the effect of exogenous KYNA on this process. Our western blot assays revealed that exogenous KYNA was capable of activating the Nrf2 pathway by regulating the protein levels of Nrf2, HO-1, GCLM, and KEAP1 in the kidney in a dose-dependent manner (Figures 5E–5I). Additionally, in mice treated with cisplatin, KYNA was also able to activate the Nrf2 pathways (Figures 5E–5I), thereby enhancing its antioxidant capacity in cisplatin-induced nephrotoxicity. ML385, an inhibitor of Nrf2, has been shown to exert potent effects in blocking the protective actions of Nrf2 activators. ML385 alone does not affect basal BUN or creatinine levels (Figure S3). When administered prior to KYNA intervention, ML385 significantly abrogated the ameliorative effects of KYNA on cisplatin-induced renal damage. This was evident from the failure of KYNA to reduce BUN and creatinine levels (Figures 5J and 5K), as well as the lack of improvement in survival rates (Figure 5L) and renal injury scores (Figures 5M and 5N). Overall, our findings indicate that KYNA preserves renal antioxidant defenses and alleviates cisplatin-induced nephrotoxicity.

Probiotics enhance kynurenic acid levels and protect against cisplatin-induced nephrotoxicity

While the direct clinical application of KYNA requires further investigation,²³ there remains an urgent need to develop strategies for elevating KYNA levels to mitigate cisplatin-induced nephrotoxicity. Considering that dietary intake serves as the principal source of tryptophan in humans and that gut microbiota critically regulate its metabolic pathways,^15,16 along with established evidence demonstrating microbiota modulation can alleviate cisplatin toxicity,^27,28 we hypothesized that targeted microbial interventions might influence kynurenine pathway dynamics to mitigate cisplatin-related renal damage.

To assess this, we first conducted a cross-database analysis using PubMed to identify microbial targets by intersecting two distinct categories of gut bacteria: those reported to exhibit potential in mitigating cisplatin nephrotoxicity (cisplatin-related, CIS-R) and those strongly correlated with KYNA (KYNA-related, KYNA-R). This intersectional analysis identified seven qualifying bacterial genera, and interestingly, Lactobacillus and Bifidobacterium, two well-known probiotics, were found to be predominant among them (Figures 6A and 6B). Therefore, we opted to employ probiotics as an intervention strategy. We first formulated a probiotic mixture (Lactobacillus rhamnosus, Bifidobacterium longum, and Lactobacillus reuteri; 1 × 10⁸ CFU/mL) for daily oral gavage administration to mice over a sustained period. As expected, our results indicated a significant elevation in KYNA levels following the probiotic mixture administration, with a notably larger increase observed on day 14 (PD14 group) compared to day 7 (PD7 group) (Figure 6C). Additionally, increases in serum KYN and tryptophan levels were also detected (Figures 6D and 6E). By calculating the ratios of KYNA/KYN and KYNA/tryptophan, we confirmed the enhanced biosynthesis of KYNA via the kynurenine pathway (Figures 6F–6H).

Figure 6.

Probiotic supplementation increases KYNA biosynthesis and synergistically protects against cisplatin nephrotoxicity

(A) Experimental workflow: Mice received daily oral gavage of probiotic mixture (1 × 10⁸ CFU/mL) for 14 days. Subgroups were either analyzed for kynurenine pathway metabolites or co-treated with KYNA (100/250/500 mg/kg, i.p.) 2 h prior to cisplatin challenge (25 mg/kg, i.p.).

(B) A cross-database analysis was conducted using PubMed to identify microbial targets.

(C–E) Serum concentrations of KYNA (C), kynurenine (KYN, D), and tryptophan (E).

(F–H) Metabolic flux ratios: KYNA/KYN (F), KYN/tryptophan (G), and KYNA/tryptophan (H).

(I) Survival curves following combinatorial treatment (n = 15).

(J and K) Serum BUN (J) and creatinine (K) levels 12 h post-cisplatin (n = 6).

(L–N) Representative immunoblots (L) and quantitative analysis of KIM-1 (M) and NAGL (N) protein expression (n = 3). Data are presented as the mean ± SEM. Statistical analyses: Welch’s ANOVA with Games-Howell post hoc test (C, F); one-way ANOVA with Bonferroni’s post hoc test (E, H, J–N); one-way ANOVA (D, G, no post hoc tests performed as the overall effects were not significant); log rank test (I). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus Ctrl group; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 versus CIS-MOD group; ˆp < 0.05, ˆˆp < 0.01, and ˆˆˆp < 0.001 versus K100+CIS group.

On the other hand, following consecutive periods of administering the probiotic mixture, cisplatin was utilized to induce a toxicity model. The findings revealed that a 14-day administration of probiotics (PD14+CIS group) markedly mitigated cisplatin-induced nephrotoxicity, which was not only evident in enhancing the survival rate of mice but also in decreasing cisplatin-mediated levels of BUN and creatinine, as well as reducing the protein levels of KIM-1 and NAGL (Figures 6I–6N). In contrast, a 7-day administration (PD7+CIS group) period exhibited a more modest effect. When compared to the CIS-MOD group, it showed statistical significance only in reducing BUN and KIM-1 levels (Figures 6J and 6M), lacking statistical significance in altering creatinine and NAGL levels (Figures 6K and 6N).

As previously mentioned, there currently lacks a specific antagonist for KYNA, and alternative methods are hindered by interference from other metabolites.²⁶ In Figure 2, we observed that 100 mg/kg of KYNA alone (K100+CIS group) exhibited a relatively modest ameliorative effect on cisplatin-induced nephrotoxicity. Therefore, we devised a strategy where, after consecutive periods of gavage administration of the probiotic mixture, the mice were intraperitoneally injected with 100 mg/kg of KYNA prior to cisplatin exposure to induce nephrotoxicity (Figure 6A). The results revealed that when combining the two moderately effective conditions—“7 days of probiotic gavage” and “100 mg/kg KYNA injection” (PD7+K100+CIS group)—there was a significant improvement in cisplatin-induced nephrotoxicity (Figures 6I–6N). Additionally, the effect of the PD14 + K100+CIS group was also superior to that of the PD14+CIS group and the K100+CIS group (Figures 6I–6N). Combining these findings that showed that probiotics can elevate serum KYNA level in mice (Figures 6C–6H), it suggests that when the KYNA produced by probiotics was able to effectively alleviate cisplatin-induced nephrotoxicity.

Discussion

In the present study, KYNA has exhibited significant effectiveness in alleviating cisplatin-induced nephrotoxicity in mice through apoptosis suppression, oxidative stress mitigation, and inflammation modulation. Notably, sustained probiotic supplementation over consecutive days demonstrated the capacity to upregulate endogenous KYNA production, creating a preventive metabolic milieu against nephrotoxic insults. Previous studies have reported the therapeutic spectrum of KYNA in various chronic renal pathologies (diabetic nephropathy,¹³ sepsis-associated multi-organ dysfunction¹⁹) and certain acute injury models (ischemia-reperfusion,²⁰ heatstroke-induced AKI²⁹). To our knowledge, the present study extends the application of KYNA to the realm of protecting against drug-induced injuries, thereby broadening its potential therapeutic scope in the medical field.

Pathological elevation of serum KYNA has emerged as a consistent biochemical feature in renal injury, demonstrating quantitative correlations with conventional renal function markers.^8,24 This phenomenon may originate from dual mechanistic pathways: (1) compromised renal clearance capacity due to impaired tubular secretion function, and (2) activation of endogenous protective responses against oxidative stress and inflammatory cascades.^8,13 Notably, this feedback protective mechanism finds parallel evidence in oncology research, where KYNA upregulation has shown tumor-suppressive effects through the dual modulation of cellular proliferation arrest and apoptosis induction.^19,24 Our systematic investigation in cisplatin-induced nephrotoxicity models delineates a sophisticated protective network orchestrated by KYNA. Experimental evidences have revealed that KYNA exhibits multi-dimensional nephroprotective effects, including attenuated cellular apoptosis, suppressed inflammatory responses, and ameliorated oxidative damage (Figure 7). Mechanistically, KYNA exerts its therapeutic efficacy by restoring renal function, normalizing classical renal injury biomarkers (such as serum creatinine, BUN, KIM-1 and NAGL), improving glomerular filtration rate and tubular secretion capacity, modulating apoptosis through rebalancing Bcl-2 family dynamics (downregulating pro-apoptotic BAX and upregulating anti-apoptotic Bcl-2) and inhibiting the caspase-3 cleavage cascade, suppressing pro-inflammatory factors (such as IL-1β, IL-6, CCL2, and CXCL) and blocking NF-κB nuclear translocation, attenuating p38 MAPK and ERK phosphorylation cascades, enhancing antioxidant defense by augmenting endogenous antioxidants (SOD, GSH, and CAT), reducing lipid peroxidation marker MDA, and activating the Nrf2 signaling axis with the subsequent upregulation of downstream targets (HO1 and GCLM). Moreover, the renoprotective efficacy of high-dose KYNA (500 mg/kg) was comparable to that of curcumin (100 mg/kg), a recognized nephroprotective agent (Figure S3). Our findings establish a molecular-to-tissue level framework elucidating KYNA’s nephroprotective pharmacology. Beyond validating its diagnostic potential as a renal injury biomarker, this study highlights KYNA’s translational value as a pleiotropic nephroprotective agent.

Figure 7.

Schematic model of KYNA-mediated protection against cisplatin nephrotoxicity

KYNA alleviates cisplatin-induced renal injury through multi-modal mechanisms: restoration of renal function and biomarkers, inhibition of apoptosis via Bcl-2 family rebalancing and caspase-3 suppression, attenuation of inflammation via NF-κB and MAPK pathways, and enhancement of antioxidant defenses via Nrf2 pathway activation and reduction of oxidative stress.

Emerging preclinical evidence highlights KYNA as a multifaceted therapeutic agent with demonstrated efficacy against inflammatory and neuropsychiatric disorders.⁹ Although other works have validated its renalprotective efficacy against cisplatin-induced injury, clinical translation remains limited by unresolved pharmacodynamic complexities and safety risks associated with exogenous KYNA administration. Fortunately, our cross-database analysis showed that Lactobacillus and Bifidobacterium, two renowned probiotics, exhibited a marked decrease in a cisplatin-induced nephrotoxicity model and demonstrated a strong positive correlation with KYNA levels. Therefore, we administered a probiotic consortium to mice via oral gavage and confirmed its dual therapeutic efficacy: it notably increases serum KYNA levels in murine models and effectively mitigates cisplatin-induced renal injury. On the other hand, our metabolic profiling revealed a striking convergence between probiotic intervention and cisplatin exposure in modulating tryptophan metabolism - both conditions enhanced tryptophan catabolism through the kynurenine axis, driving increased KYNA biosynthesis. This parallelism suggests that KYNA elevation during cisplatin nephrotoxicity may represent an endogenous compensatory mechanism. Notably, beyond KYNA modulation, probiotics may exert pleiotropic effects through gut microbiota remodeling and enhancement of butyrate synthesis to attenuate renal tubular oxidative stress. These complementary pathways could synergize with KYNA-mediated effects to establish a multi-layered nephroprotective barrier. Notably, beyond KYNA modulation, probiotics may exert pleiotropic effects through gut microbiota remodeling and enhancement of butyrate synthesis to attenuate renal tubular oxidative stress. These complementary pathways could synergize with KYNA-mediated effects to establish a multi-layered nephroprotective barrier. Collectively, our data showed that probiotic administration circumvents the limitations associated with direct KYNA administration, holding significant clinical relevance and utility.

Conventional validation of the “probiotic-KYNA-renoprotection” axis would require selective KYNA pathway inhibition. However, methodological constraints persist due to the absence of KYNA-specific antagonists and the inherent cross-reactivity of pharmacological inhibitors targeting upstream enzymes (IDO/KAT), which disrupt broader tryptophan metabolic homeostasis.^26,30 Intriguingly, our dose-response studies identified two distinct therapeutic tiers: moderate efficacy with 7-day probiotic supplementation or 100 mg/kg KYNA administration, versus high efficacy with 14-day probiotics or 500 mg/kg KYNA. Notably, combinatorial treatment with suboptimal interventions (7-day probiotics +100 mg/kg KYNA) synergistically achieved maximal nephroprotection, indicating a threshold-dependent mechanism where probiotic-derived KYNA accumulation potentiates exogenous KYNA therapy. This proof-of-concept study establishes a paradigm-shifting strategy to enhance KYNA’s therapeutic index through probiotic coadministration. Our findings not only resolve critical gaps in KYNA’s translational roadmap but also pioneer a biocompatible approach for managing chemotherapy-induced toxicity.

The pharmacokinetic characterization of exogenous KYNA revealed a non-monotonic profile, where a significant systemic elevation occurred after the critical 2-h pretreatment window for nephroprotection. This temporal dissociation indicates that the protective mechanism is initiated prior to peak systemic exposure. Furthermore, probiotic pretreatment not only elevated basal KYNA levels but, when combined with a sub-therapeutic KYNA dose, yielded a pharmacokinetic synergy that established a sustained, protective metabolite milieu at the time of cisplatin challenge. The dynamic pharmacokinetics may involve a complex homeostatic interplay between exogenous and endogenous KYNA pools, potentially mediated by tissue distribution and compensatory feedback mechanisms.

In conclusion, our study establishes KYNA as a multi-target nephroprotective agent that counteracts cisplatin-induced renal injury through the coordinated regulation of apoptosis, oxidative stress, and inflammation, while pioneering a probiotic-based strategy to amplify endogenous KYNA biosynthesis. These findings not only expand KYNA’s therapeutic scope to drug-induced toxicity but also provide a biocompatible blueprint for enhancing chemotherapeutic safety through metabolite-microbiome crosstalk modulation.

Limitations of the study

While our findings demonstrate the promising nephroprotective role of KYNA and its enhancement by probiotics, this study has several limitations that warrant consideration. First, the measured plasma KYNA levels are assay-dependent. Our quantification using ELISA, while consistent with the reported ranges from studies employing similar methodologies,^31,32 highlights the need to interpret absolute concentrations within the context of the detection platform. Different methods (e.g., high-performance liquid chromatography, HPLC, liquid chromatography-tandem mass spectrometry, LC-MS/MS) can yield varying absolute values due to differences in sensitivity, specificity, and calibration standards, which is a known variability in the field.^33,34,35 Future studies employing standardized or cross-platform validation would strengthen the comparability of KYNA measurements across different research settings. Second, although we observed elevated serum KYNA levels following probiotic supplementation and proposed a gut-kidney axis, we did not directly measure kynurenine pathway metabolites in key tissues such as the intestine, liver, or brain. Consequently, the precise tissue origins and inter-organ trafficking of KYNA, which are critical for fully understanding the “gut-microbiota-KYNA-kidney” axis, remain to be fully elucidated. Third, the exact mechanism by which probiotics enhance systemic KYNA levels is not definitively established. The elevation could result from direct microbial metabolism of tryptophan, probiotic modulation of host enzymatic activity (e.g., IDO or KAT), or compositional changes in the gut microbiota favoring KYNA-producing commensals. Further studies are needed to dissect these potential mechanisms. Furthermore, the definitive establishment of causal links within the “probiotic-KYNA-renoprotection” axis is currently constrained by the lack of a specific KYNA-neutralizing agent or antagonist. The use of upstream enzyme inhibitors (e.g., for IDO or KAT) affects multiple metabolites in the pathway, limiting the precision of mechanistic conclusions. Finally, our study utilized a single high-dose cisplatin model to induce acute kidney injury, which, while well-established, does not fully recapitulate the repeated, lower-dose chemotherapy regimens common in clinical practice. The efficacy and safety of KYNA and probiotic interventions in chronic or multi-cycle cisplatin treatment scenarios remain to be investigated.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Shu-Lan Qin (1094821826@qq.com).

Materials availability

This study did not generate new unique reagents. All commercial reagents and kits are listed in the method details section.

Data and code availability

• •Data: All data reported in this article are available from the lead contact upon request.
• •Code: This article does not report original code.
• •Additional information: Any additional information required to reanalyze the data reported in this article is available from the lead contact upon request.

Acknowledgments

We express our gratitude to Mr. Haihua Luo for generously supplying the reagents and facilities. We also thank all members of Southern Medical University and its affiliated hospitals for their invaluable support, which enabled the successful completion of our experiments. This project was supported by the National Natural Science Foundation of China grant (82241061), Guangdong Basic and Applied Basic Research Foundation grant (2022B1515120024), and Shenzhen Science and Technology Planning Project (No. JCYJ20220530165014033). The graphical abstract was created with BioRender.com.

Author contributions

K.X.L. and H.Y. contributed equally. K.X.L. and J.Y.W. conceived and designed the study. K.X.L. developed the methodology, performed the software analysis, and wrote the original draft. H.Y., with assistance from J.X.O., carried out most of the experiments and was responsible for data curation, visualization, and investigation. G.M.C. performed validation and provided the method for biochemical index detection. Q.N.D. provided bacterial resources. S.Y.L. collected samples. Q.D.Q. contributed to the construction of the mouse model. S.L.Q. administered the project and provided supervision. K.X.L. and J.Y.W. acquired funding. All authors have read and approved the final version of the article.

Declaration of interests

The authors declared no conflict of interest.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used DeepSeek to assist with language polishing, refinement of responses to reviewers’ comments, and improving the logical flow of textual explanations. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Cleaved Caspase-3 (Asp175) Antibody	Cell Signaling Technology (CST)	Cat# 9661, RRID: AB_2341188
Bax Antibody	CST	Cat# 2772, RRID: AB_10695870
Bcl-2 (124) Mouse Monoclonal Antibody	CST	Cat# 15071, RRID: AB_2744528
NF-kappaB p65 (D14E12) Rabbit Monoclonal Antibody	CST	Cat# 8242, RRID: AB_10859369
Phospho-NF-kappaB p65 (Ser536) (93H1) Rabbit Monoclonal Antibody	CST	Cat# 3033, RRID: AB_331284
p38 MAPK Antibody	CST	Cat# 9212, RRID: AB_330713
Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) Rabbit Monoclonal Antibody	CST	Cat# 4511, RRID: AB_2139682
p44/42 MAPK (Erk1/2) (137F5) Rabbit Monoclonal Antibody	CST	Cat# 4695, RRID: AB_390779
Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) Rabbit Monoclonal Antibody	CST	Cat# 4370, RRID: AB_2315112
SAPK/JNK Antibody	CST	Cat# 9252, RRID: AB_2250373
Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit Monoclonal Antibody	CST	Cat# 4668, RRID: AB_823588
NRF2 (D1Z9C) Rabbit Monoclonal Antibody	CST	Cat# 12721, RRID: AB_2715528
HO-1 Antibody	CST	Cat# 70081, RRID: AB_2799772
Anti-GCLM antibody [EPR6667]	Abcam	Cat# ab126704, RRID: AB_11127439
Anti-NAGL antibody [EPR21092]	Abcam	Cat# ab216462, RRID: AB_3665857
Anti-beta Actin antibody	Abcam	Cat# ab8226, RRID: AB_306371
KIM-1/HAVCR1 Polyclonal antibody	Proteintech	Cat# 30948-1-AP, RRID: AB_3669790
KEAP1 Monoclonal antibody	Proteintech	Cat# 60027-1-Ig, RRID: AB_2132623
Bacterial and virus strains
Lactobacillus rhamnosus	ATCC	ATCC: 53103
Bifidobacterium bifidum	ATCC	ATCC: 29521
Lactobacillus reuteri	ATCC	ATCC: 23272
Chemicals, peptides, and recombinant proteins
Kynurenic acid (KYNA)	Yuanye	Cat# S47549
Cisplatin (CIS)	MedChemExpress	Cat# HY-17394
	Sigma-Aldrich	Cat# P4394
Curcumin	Sigma-Aldrich	Cat# 458-37-7
ML385	MedChemExpress	Cat# HY-100523
Critical commercial assays
Blood Urea Nitrogen (BUN) assay kit	JianCheng	Cat# C013-2-1
Creatinine (CR) assay kit	JianCheng	Cat# C011-2-1
Glutathione (GSH) assay kits	JianCheng	Cat# A006-2-1
Superoxide dismutase (SOD) assay kits	JianCheng	Cat# A001-3-2
Catalase (CAT) assay kits	JianCheng	Cat# A007-1-1
Malondialdehyde (MDA) assay kits	JianCheng	Cat# A003-1-2
Mouse tryptophan (Trp)ELISA kit	Enzyme-linked Biotechnology	Cat# MM-0756M1
Mouse kynurenine (KYN) ELISA kit	Enzyme-linked Biotechnology	Cat# MM-0755M1
Mouse kynurenic acid (KYNA) ELISA kit	Enzyme-linked Biotechnology	Cat# MM-45731M1
TMR (red) Tunel Cell Apoptosis Detection Kit	Servicebio	Cat# G1502
Oligonucleotides
Primer sequences for quantitative PCR of target genes, see Table S1	This paper	–
Software and algorithms
ImageJ	National Institutes of Health	https://imagej.net/ij/
SPSS software (version 20.0)	IBM	https://www.ibm.com/cn-zh/products/spss

Experimental model and study participant details

Animals

Wild-type (WT) male C57BL/6J mice aged 6-8 weeks were purchased from the experimental animal center of Southern Medical University (Guangzhou, China). The use of male mice was based on established literature³⁶ demonstrating their higher and more consistent susceptibility to cisplatin-induced nephrotoxicity compared to females, thereby reducing model variability.

Housing and ethical approval

All animal experiments complied with ARRIVE guidelines. Prior to the commencement of the experiment, all animals were given a two-week acclimatization period to adapt to the new environment. During this time, they were housed under standard conditions and had unlimited access to both food and water. The experimental protocol and procedures were approved by the Ethics of Animal Experiments Committee of Southern Medical University (Approval No: SYXK-2021-0167), ensuring adherence to ethical guidelines for animal research.

Method details

Preparation of the probiotic mixture

The Institute of Biological and Medical Engineering, Guangdong Academy of Sciences (Guangzhou, China), kindly provided Lactobacillus rhamnosus, Bifidobacterium bifidum and Lactobacillus reuteri. These strains were grown separately on solid media under anaerobic conditions at 37°C for 24 hours. Subsequently, they were transferred into de Man, Rogosa and Sharpe (MRS) broth and their concentrations were determined through serial dilution plating and colony counting. The bacteria were then diluted to achieve a concentration of 1 x 10ˆ8 CFU/mL.³⁷ Prior to administration to the mice, each type of bacteria (0.5 mL per type) was individually centrifuged at 5,000 × g for 2 minutes. Subsequently, the precipitates from these three types of bacteria were combined and resuspended in 1.5 mL of PBS, resulting in a mixed bacterial suspension.

Cisplatin-induced nephrotoxicity model and interventions

Acute toxicity was induced in mice via a single intraperitoneal injection of cisplatin, which was dissolved in sterile 0.9% saline and administered at a dose of 25 mg/kg after an 8-hour fasting period.^38,39 For pretreatment, KYNA was dissolved in saline, pH-adjusted to 7.4, and injected intraperitoneally at graded doses (100, 250, and 500 mg/kg) 2 hours before cisplatin challenge. To comparatively evaluate the nephroprotective efficacy of KYNA, curcumin (100 mg/kg), dissolved in corn oil, was included as a positive control and administered intraperitoneally 2 hours before cisplatin. To examine the role of Nrf2 signaling, specific experimental groups received the Nrf2 inhibitor ML385, dissolved in saline containing 5% dimethyl sulfoxide (DMSO), at a dose of 30 mg/kg (i.p.) 4 hours prior to cisplatin administration. Additionally, a probiotic mixture (1 × 10ˆ8 CFU/mL in PBS) was orally administered for 7 or 14 consecutive days before cisplatin treatment.

Part 1. Group Ⅰ is the normal control (NC) group, where the mice received an intraperitoneal injection of isotonic normal saline. Group Ⅱ to Ⅲ are cisplatin-treated groups, further categorized into 6-hour and 12-hour subgroups based on the timing of cisplatin administration.

Part 2. Group Ⅰ and Ⅱ are respectively the normal control (Ctrl) group and the cisplatin-model (CIS-MOD) group, while Group Ⅲ to Ⅴ are the K100, K250 and K500 group where mice in each only receive exogenous KYNA. Groups Ⅵ to Ⅷ are KYNA pretreatment groups, comprising the K100+CIS group, the K250+CIS group, and the K500+CIS group, respectively.

Part 3. Group Ⅰ and Ⅱ are respectively the Ctrl group and the CIS-MOD group, respectively. Group Ⅲ, termed K500+CIS group, consists of mice that receive KYNA at a dose of 500 mg/kg in conjunction with cisplatin. Group Ⅳ, ML385+CIS, includes mice pretreated with ML385, an inhibitor of Nrf2 signaling, four hours prior to cisplatin exposure. Lastly, Group V, ML385+K500+CIS, involves mice pretreated with both ML385 and KYNA (500 mg/kg) before being administered cisplatin.

Part 4. Group Ⅰ and Ⅱ are respectively the normal control (Ctrl) group and the cisplatin-model (CIS-MOD) group. Group Ⅲ is the K100+CIS group, where mice receive KYNA at a dose of 100 mg/kg in combination with cisplatin. Group Ⅳ is the 7 days of probiotic pretreatment + cisplatin (PD7+CIS) group. Group Ⅴ is the 14 days of probiotic pretreatment + cisplatin (PD14+CIS) group. Group VI is the 7 days of probiotic pretreatment + KYNA (100 mg/kg) pretreatment + cisplatin (PD7+K100+CIS) group. Group Ⅶ is the 14 days of probiotic pretreatment + KYNA (100 mg/kg) pretreatment + cisplatin (PD14+K100+CIS) group.

Part 5. Mice were respectively administered cisplatin obtained from MedChemExpress or Sigma-Aldrich at doses of 0, 5, 15, 25, 35, and 45 mg/kg (i.p.).

Part 5. Group I and II served as the normal control (Ctrl) and cisplatin-induced model (CIS-MOD), respectively. Group III (K500+CIS) received KYNA (500 mg/kg, i.p.) 2 hours prior to cisplatin; Group IV (ML385+CIS) was pretreated with the Nrf2 inhibitor ML385 4 hours before cisplatin exposure; and Group V (Curcumin+CIS) was administered curcumin 2 hours preceding cisplatin. Additionally, three monotherapy control groups were included: Group VI (ML385 alone), Group VII (K500 alone), and Group VIII (Curcumin alone), which respectively received only ML385, KYNA (500 mg/kg), or curcumin to assess their basal effects.

Serum collection and tissue sampling

At the end of the experiments, whole blood (approximately 400-600μl) of the mice was collected by performing direct heart puncture. The blood samples were left to clot at room temperature for about 30 minutes, and then centrifuged at 3,500 × g for 15 minutes at 4°C. The resulting serum supernatants were used for performing biochemical analysis.

After blood collection, the mice were sacrificed, and their kidneys were promptly excised, washed with 0.9% sodium chloride solution, patted dry using filter paper, and then divided into four parts. One part of the isolated kidney tissues was stored in 4% paraformaldehyde for histopathological analysis. Another part was stored at −80°C for the preparation of tissue homogenates to assess oxidative stress and antioxidant status. The third part was lysed using TRIZOL and stored at −80°C for subsequent qPCR experiments. Additionally, a separate part was rapidly cooled using liquid nitrogen and then stored at −80°C for WB experiments.

Measurement of biochemical makers and kynurenine metabolites

The concentrations of blood urea nitrogen (BUN), and serum creatinine, which are indicators of kidney function, were quantitated using specific enzyme-linked immunosorbent assay (ELISA) kits. Additionally, the levels of glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA), which reflect oxidative stress and antioxidant status in kidney tissues, were also measured using corresponding ELISA kits.

Furthermore, the contents of tryptophan, KYN (kynurenine), and KYNA, which are important metabolites in the kynurenine pathway, were quantified using dedicated kits. This method for quantification is consistent with that reported in the literature,^17,30,40 ensuring accuracy and reproducibility of the results, as all analytical procedures were rigorously followed in accordance with the manufacturer's guidelines.

Biochemical and histopathological assays

Mouse kidney tissues were fixed in 4% neutral-buffered formalin for 24 hours, then dehydrated in a graded ethanol series (75–100%) and embedded in paraffin. Sections were cut using a rotary microtome and mounted on slides. For histochemical analysis, slides were dewaxed, stained with hematoxylin and eosin (HE), and assessed for hepatic steatosis using a microscope (Leica Application Suite v4.0). For TUNEL staining, slides were incubated with protease K (20 μg/mL, DNase-free) at 37°C for 30 minutes, followed by 50 μL of TUNEL reaction mixture for 60 minutes at 37°C in the dark. After washing with PBS, stained sections were observed under a microscope. The number of TUNEL-positive cells was counted in one randomly selected field from six sections using Image J software for comparison.

RNA extraction, expression analysis, and sequencing

Total RNA was extracted from tissues and cultured cells using TRIzol reagent and quantified by a spectrometer. A reverse transcription kit and SYBR Green real-time PCR master mix were used to analyze mRNA expression using a 7500 real-time PCR system (Applied Biosystems, Foster City, CA). All primer sequences used in the study are provided in Table S1 in the supplemental information. The relative transcription level was calculated using the 2^−ΔΔCt method.

Western blot analysis

Protein samples from each group were collected and lysed with RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were measured using a BCA Protein Assay kit. Subsequently, 35 μg of proteins were resolved on 10% or 12% SDS-PAGE gels and transferred to PVDF membranes. The membranes were probed with primary antibodies, including cleaved caspase3, BAX, Bcl2, NF-κB p65, phospho-NF-κB p65, p38 MAPK, phospho-p38 MAPK, p44/42 MAPK, phospho-p44/42 MAPK, SAPK/JNK, phospho-SAPK/JNK, Nrf2, HO-1, GCLM, KIM-1, NAGL, KEAP1 and β-actin and secondary antibodies conjugated with horseradish peroxidase. Proteins were visualized using Super ECL Detection Reagent and the ChemiDoc Imaging System (Bio-Rad). Band density was analyzed with Image J software, and the expression levels were normalized to β-actin. The blot shown is representative of three independent experiments. Molecular weight markers (in kDa) are indicated on the right.

Quantification and statistical analysis

All quantitative data are presented as the mean ± SEM, with the sample size (n) detailed in the figure legends. Statistical analyses were performed using GraphPad Prism and SPSS 20.0. Normality was assessed using the Shapiro-Wilk test. For two-group comparisons, an unpaired two-tailed Student’s t-test was used. For multiple groups, variance homogeneity was first tested (Brown-Forsythe test). Data with equal variances were analyzed by one-way ANOVA followed by Bonferroni’s post hoc test if the overall comparison was significant (p < 0.05); data with unequal variances were analyzed by Welch’s ANOVA followed by Games-Howell post hoc test. Survival data were analyzed by the log-rank test. Significance was set at p < 0.05, denoted as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 in figures, with additional symbols (#, ˆ) defined in respective legends.

Additional resources

Supplementary figures and tables are provided in the supplemental information. Primer sequences for qPCR are listed in Table S1. Kinetic data for KYNA, KYN, and tryptophan after administration are summarized in Table S2.

Published: February 17, 2026