# Time course profiling of the kynurenine pathway activity in a pig model of crescentic glomerulonephritis

Zahia Nadour, Julien Dang, Léa Resmini, Virginie Poindessous, Pierre-Louis Tharaux, Nicolas Pallet

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Zahia Nadour, Julien Dang, Léa Resmini, Virginie Poindessous, Pierre-Louis Tharaux, et al.. Time course profiling of the kynurenine pathway activity in a pig model of crescentic glomerulonephritis. BMC Nephrology, 2025, 26 (1), pp.500. 10.1186/s12882-025-04423-6. hal-05296619

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# Time course profiling of the kynurenine pathway activity in a pig model of crescentic glomerulonephritis

Zahia Nadour<fnref n="1" />,<fnref n="2" />†, Julien Dang<fnref n="3" />†, Léa Resmini<fnref n="3" />, Virginie Poindessous<fnref n="1" />, Pierre-Louis Tharaux<fnref n="3" /> and Nicolas Pallet<fnref n="1" />,<fnref n="2" />,<fnref n="4" />*

**Abstract**

**Objective and design** The kynurenine (KYN) pathway is activated in acute kidney disease, including crescentic glomerulonephritis (CGN), an immune-mediated life-threatening syndrome characterized by rapid loss of renal function. The non-invasive measurement of KP metabolites may have clinical diagnostic or prognostic value, but has never been systematically studied using sensitive analytical tools.

**Methods** We have recently developed and validated a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure a comprehensive panel of tryptophan (TRP) catabolic intermediates in blood and urine. We used a novel model of CGN to approximate human biochemical and physiological parameters.

**Results** We show that for a period of 28 days after the onset of the disease, monitoring plasma KP is not informative as it is likely to reflect an accumulation of uremic retention solutes and does not allow to follow the activity of the disease over time. In contrast, the urinary metabolic profile is noteworthy, with an early and significant yet transient increase in urinary KYN/TRP ratio levels. Urinary KYN/TRP ratio is independent of proteinuria.

**Conclusion** These findings suggest that increased urinary KYN/TRP ratio levels by LC/MS/MS in patients with CGN reflect early disease activity.

**Keywords** Crescentic glomerulonephritis, Kynurenine, Pig, Acute kidney injury, Tryptophan

**Introduction**

The kynurenine pathway (KP) activity as a critical determinant of kidney health and disease on its ability to respond to varied stressors in numerous models of acute and chronic kidney injuries, of, but not limited to, autoimmune and inflammatory origins [1–3]. The KP starts with tryptophan 2,3-dioxygenase (TDO) in the liver and indoleamine 2,3-dioxygenase (IDO) elsewhere in the body, which catalyzes the first and rate-limiting step of tryptophan (TRP) catabolism in mammals [4] (Figure S1). IDO1 is mainly expressed in cells of the immune system but also renal epithelial cells, and inflammatory cytokines induce its expression. IDO1 catalyzes the cleavage of the TRP pyrrole ring to N-formyl kynurenine, driving

<footnote>†Zahia Nadour and Julien Dang contributed equally to this work.</footnote>

<footnote>Pierre-Louis Tharaux and Nicolas Pallet are co senior authors.</footnote>

<footnote>*Correspondence:</footnote>

<footnote>Nicolas Pallet
Nicolas.pallet@aphp.fr</footnote>

<footnote n="1">¹Centre de Recherche des Cordeliers, INSERM U1138, Université Paris Cité, 15 rue de l'Ecole de Médecine, Paris 75006, France</footnote>

<footnote n="2">²Department of Clinical Chemistry, Assistance Publique Hôpitaux de Paris, Georges Pompidou European Hospital, Paris, France</footnote>

<footnote n="3">³Paris Cardiovascular Centre (PARCC), Université Paris Cité, 56 rue Leblanc, Inserm, Paris F-75015, France</footnote>

<footnote n="4">⁴Department of Nephrology, Assistance Publique Hôpitaux de Paris, Georges Pompidou European Hospital, Paris, France</footnote>
the production of kynurenine (KYN). KYN is metabo-
lized to kynurenic acid (KYNA) by kynurenine amino-
transferase (KAT). KYN, as well as KYNA, are activating
ligands of aryl hydrocarbon receptor (AhR), a potent
immunosuppressive transcription factor promoting an
immunosuppressive state in the inflammatory microen-
vironment [5, 6]. Kynurenine 3-monooxygenase (KMO)
is a rate-limiting enzyme for the downstream catabo-
lism of KYN to 3-hydroxykynurenine (3-HK). A decline
in the activity of KMO increases KYN and kynurenic
acid (KYNA) levels and promotes AhR-induced immu-
nosuppression. Kynureninase (KYNU) converts 3-HK to
3-hydroxyanthranilic acid (3-HANA), which is metabo-
lized to quinolinic acid (QUIN) and picolinic acid (PIC)
from the metabolic intermediate 2-amino-3-carboxy-
muconic semialdehyde (ACMS). QUIN is a neurotoxic
uremic retention solute and the substrate for the de novo
synthesis of the cytoprotective molecule NAD⁺ through
the bottleneck enzyme quinolinolate phosphoribosyl
transferase (QPRT). PIC could have antagonistic proper-
ties toward the toxic effects of QUIN. Cellular QUIN and
PIC levels depend on the activity of amino-β-carboxymu-
conate-semialdehyde-decarboxylase (ACMSD), which
is highly expressed in the kidney, and channels the pro-
duction of PIC from ACMS. ACMSD is therefore a key
enzyme directing KYN metabolism towards PIC and tak-
ing it away from de novo NAD⁺ production.

Crescentic glomerulonephritis (CGN) is the histo-
logical manifestation of a spectrum of aggressive kidney
diseases of various etiologies that results from the infil-
tration of inflammatory cells into the glomerular tuft
and accumulation of cells and fibrin in Bowman's space
[7]. Anti-glomerular basement membrane (anti-GBM)
glomerulonephritis, also known as Goodpasture's syn-
drome, one of the earliest recognized autoimmune CGN
in humans [8–10], and anti-neutrophil cytoplasmic anti-
body (ANCA)-associated vasculitis is the most common
cause of CGN worldwide [11]. The most widely used
model for CGN is nephrotoxic serum nephritis (NTN),
in which heterologous anti-GBM antibodies act as a
stimulating antigen in the glomeruli. Experimentally, the
most widely used model for anti-GBM glomerulonephri-
tis and ANCA-associated CGN is NTN, which results in
similar lesions and provides cues to the understanding
CGN mechanisms [12, 13].

Previous studies have shown that the KP is activated in
the kidney upon CGN and that IDO1 plays a protective
role in CGN by regulating immune responses and pre-
venting excessive inflammation [14–16]. Monitoring KP
is a clinically relevant approach to follow the activity of
CGN in the kidney, as the metabolites of TRP produced
in the kidney are released into the urine and circulation.

In this study, we used a novel porcine model of CGN
to investigate the activity of the KP over one month

after disease onset. The domestic pig (Sus domesticus)
provides a valuable opportunity to gain insight into the
human disease. It is close to humans in terms of kidney
anatomy and physiology, and the pig immune system is
well characterised and highly similar to that of humans
[17–19]. To do this, we applied a recently developed and
validated sensitive and rapid liquid chromatography-
tandem mass spectrometry (LC–MS/MS) method for
the simultaneous determination of TRP and 7 metabo-
lites of KYN pathway, including KYN, 3-HK, KYNA,
xanthurenic acid (XA), 3-HANA, QUIN, and PIC in both
plasma and urine [20].

Methods

Animal experiments

Eighteen (18) adult female wild-type domestic pigs (Sus
crofa domesticus) were used in this study. They were
provided by the Maison Alfort Veterinary School in
France.

Preparation of sheep anti-pig GBM serum (Nephrotoxic serum)

Sheep nephrotoxic serum (NTS) containing polyclonal
anti-pig glomerular basement membrane (GBM) anti-
bodies were obtained by xeno-immunization as previ-
ously described [21, 22]. Whole kidneys were obtained
from healthy pigs. Cortical tissues were dissected and
digested in type I collagenase (17100-017, GIBCO). The
tissue samples were then filtered through several sieves.
Samples with adequate purification (> 80% of glomeruli)
were mixed, centrifugated, and resuspended in ultrapure
water to induce hypotonic cell swelling and lysis and to
eliminate intracellular proteins from the supernatant. The
remaining proteins, essentially from GBM, were dissoci-
ated from the cellular debris by ultrasonication (Vibra-
Cell, Sonics & Materials, Inc., Newtown, CT, USA) on
ice. Six sheep were immunized with the GBM extract by
four weekly intradermal injections (1 mg of proteins, in
complete Freund's adjuvant), followed by one intravenous
infusion (1 mg of proteins, without Freund's adjuvant) 4
weeks after the last intradermal injection.

Serum was taken from living sheep one week later
before euthanization. Immunofluorescence tested each
serum for GBM specificity in the pig's kidney section.
Specific sera were pooled and centrifugated. Finally, the
supernatant was heat-inactivated in a water bath and fil-
tered twice with 0.45 and 0.20 µm membranes consecu-
tively. Aliquots of NTS were stored frozen (-80 °C) until
the experiment.

Anesthesia and euthanasia

Premedication was administered with a 1:1 mixture of
tiletamine-zolazepam (Zoletil100, 100 mg/mL, Virbac,
Carros, France) 3.0 mg/kg IM and morphine (Morphine
Aguettant, 10 mg/mL, Laboratoire Aguettant, Lyon, France) 0.2 mg/kg IM. A 20G peripheral catheter was inserted into the marginal auricular vein after sedation. The pigs in the basal group were euthanised after premedication using a euthanasia solution (T61(embutramide, mebezonium and tetracaine solubilised in a mixture of N, N-dimethylformamide/water (60/40, v/v), MSD Santé Animale, Beaucouze, France).

### Induction of crescentic glomerulonephritis

We used the accelerated anti-GBM CGN model as described in rodents [23, 24]. Two to three-month-old pigs (n=18) were pre-immunized with a subcutaneous injection of 500 µg of purified sheep IgG (I5131, Sigma) diluted in 1 mL of sterile PBS and 1 mL of complete Freund's adjuvant (F5881, Sigma), 7 days before the beginning of the protocol. Each individual received an intramuscular injection of NTS on two consecutive days (0.15 mL/kg of body weight on day 0 and 0.3 mL/kg of body weight on day 1) to induce an accelerated CGN model as previously described. The animals were euthanized on day 28. One control non-immunized pig was sacrificed on day 20 for histological analysis.

### Plasma and urine samples

Blood and urine samples were collected from the pigs' treatment group and control group by ear vein and trans parietal bladder puncture at day 0 and then once a week (days 7, 14, 21, and 28) until the pigs died or were euthanized. Blood samples were obtained by ear vein and collected into spray-coated lithium heparin-containing tubes (BD Vacutainer ® PSTTM, Becton Dickinson and Company, Le Pont de Claix, France). Samples were centrifuged at 2000 g at +4 °C for 8 min and stored at -80 C. The urine was obtained by transparietal puncture of the urinary bladder and collected into BD V-Monovette ® Urine (Sarstedt Company, Nümbrecht, Germany), then centrifuged at 2000 g at +4 °C for 8 min and stored at -80 C.

### Biochemistry analyses

The urine and plasma measurements were performed at the Clinical Chemistry Department of the Georges Pompidou European Hospital (Paris, France). Blood concentrations of creatinine, urea, total proteins, albumin, sodium, and potassium, and urinary concentrations of creatinine, proteins and albumin were determined by enzymatic methods using AU 5800 analyzer (Beckman Coulter France S.A.S, Villepinte, France).

### Targeted metabolites analysis by LC/MS-MS

Plasma metabolites analysis could be performed on 6 to 10 NTS pigs per group, and urine metabolites analysis was done on 8 to 13 NTS pigs per group. The protocol

has been previously described [20]. 50 µL of pure plasma or urine samples diluted at 1:1 in 0.1% formic acid in water were spiked with 25 µL of internal standard solution and precipitated with 250 µL of ice-cold methanol. The solution was then homogenized and centrifuged for 10 min at 13,000 g at 4°C. 200 µL of the supernatant was then evaporated under a nitrogen stream and the dry residue was reconstituted with 80 µL of 0.1% formic acid in water. 60 µL were directly transferred to a 96-well plate for LC-MS/MS analysis of QUIN, 3-HK, XA, PIC, and 3-HANA. To analyze TRP, KYN and KA, 10 µL were transferred to a 96-wells plate containing 90 µL of 0.1% formic acid in water. The 96-wells plate was homogenized, centrifuged for 3 min at 4000 g at 4 °C then analyzed by LC-MS/MS. 3 µL of injected volume was separated on an ACQUITY UPLC HSS T3 C18 column (100 × 2.1 mm i.d., 1.8 µm particle size, Waters, Saint-Quentin-en Yvelines, France) preheated at 40 °C. The metabolites were separated over a gradient from 0% of 0.1% formic acid in water B to 90% of 0.1% formic acid in MeOH at a flow rate of 0.5 mL min⁻¹ for 17 min. Compounds were detected using an LCMS-8060 triple quadrupole mass spectrometer (Shimadzu Corporation, Marne-la-Vallée, France) as described [20]. Data acquisition and data post-processing were performed using LabSolutions™ LCMS Software, version 5.97 SP1 (Shimadzu Corporation, Marne-la-Vallée, France). Limits of detection and quantification of KP metabolites measured by LC-MS/MS are presented in Table S1.

### Kidney histological staining (Masson's trichrome stain)

Kidney biopsies were fixed in alcohol-formalin-acetic acid, dehydrated with ethanol and xylene, embedded in paraffin, and cut into 3 µm sections. Samples were then deparaffinized, rehydrated in graded alcohols and then stained with Masson's Trichrome.

### Kidney mRNA extraction and RT-qPCR

12 kidney biopsies from euthanized pigs were conserved in 5mL conical tubes and conserved at -80 °C. 2 mm³ of kidney biopsy were disrupted and homogenized in 600 µL of RLT buffer (Qiagen) using TissueLyser II (Qiagen). Supernatants were stored at -80 °C until mRNA extraction. Total RNA from whole kidneys was extracted from the Supernatants using the RNeasy Mini Kit (Qiagen) and reverse-transcribed into cDNAs using TaqMan Reverse Transcription Reagents (Applied Biosystems) according to the manufacturer's protocols. Transcript expression levels were quantified through SYBR Green RT-qPCR using a Kit QuantStudio 12 K (Applied Biosystems). Vehicle-treated samples were used as controls, and the fold changes for each tested gene were normalized to the ribosomal protein L13a housekeeping gene.
The relative expression levels were calculated using the 2⁻ΔΔCT method [25]. Primer sequences are listed in Table S2.

## Statistical analyses

Graphs and statistical analyses were generated using GraphPad Prism 9 Software (GraphPad Software, Inc.). Data are reported as mean ± standard error of the mean. Number of samples assayed in each experiment is indicated in the Figure Legends. ANOVA (Mixed effects model analysis) was used to perform repeated measures over time with missing values and was followed by a Dunnett's multiple comparisons test for multiple comparisons against the control condition (Day 0). Pearson correlation coefficient was used to measure of linear correlation between two sets of data. A p value < 0.05 was considered to represent a statistically significant difference.

## Results

### Validation of a porcine CGN model

Eighteen pigs were initially included in the study. Two pigs died of anaphylactic shock during the day after NTS injection and were excluded from the study. Three pigs did not have plasma and urine samples available for KP metabolic profiling and were also excluded from the analyses. Therefore, a total of 13 pigs were analyzed for monitoring of renal function parameters and KP monitoring in blood or urine. In this cohort, 2 pigs died on days 21 and 28 and blood and urine samples for KP metabolite profiling could not be collected at that time (Fig. 1A). The cause of death is unknown, but their last plasma creatinine was 2089 µmol/l and 3630 µmol/l (reference values 88.8 µmol/l to 238 µmol/l), respectively.

Kidney function parameters (plasma creatinine, plasma urea, and proteinuria) were measured 7, 14, 21 and 28 days after NTS injection (day 0) (Fig. 1B). NTS injection triggered a significant and transient rise in plasma creatinine and urea with a peak at day 7 and a partial resolution thereafter (Fig. 1C and D), albeit the response was heterogeneous. As expected, NTS injection was followed by the appearance of high-grade proteinuria, testifying to the glomerular lesions induced by the inflammatory reaction. On average, this proteinuria was at its maximum on day 7 and tended to persist throughout the follow-up period, although to a lesser extent.

Glomerular injury was confirmed by histological analysis of Masson's trichrome-stained renal biopsies from NTS pigs on day 28. Normal renal histology was observed in non-immunized pigs (Fig. 1E). Conversely, pigs immunized with sheep anti-pig GBM serum exhibited extracapillary proliferation and signs of tubular and glomerular lesions and interstitial inflammation (Fig. 1D). Histological quantification of the 10 renal biopsies highlighted the severity of the model and reflects the

chronicity of the lesions, as we assessed that 6 out of 8 kidneys had grade 3 IFTA, 5 out of 8 had more than 50% sclerotic glomeruli, and 3 out of 8 had more than 50% glomerular crescents. These results indicate that NTS injection in pigs was responsible for a CGN with variable severity, with an acute kidney injury and high-grade proteinuria.

### Blood metabolic profiling of KP intermediates after the induction of CGN

Numerous metabolites (KA, XA, 3-HANA, and PIC) were detected in plasma but could not be quantified with our highly sensitive LC-MS/MS method (Table S2 for limits of detection and quantification). Serum TRP and KYN levels decreased (without this being statistically significant) on day 7 and returned to normal levels after that (Fig. 2A and B). Conversely, HK and QUIN levels showed a non-significant upward trend on day 7 and returned to normal values thereafter (Fig. 2C and D). These variations were not statistically significant, perhaps due to a lack of power, but are consistent with similar profiles described in acute or chronic renal failure of various etiologies since KP intermediates are known uremic retention solutes [26]. The plasma KYN/TRP ratio, a potent marker IDO activity, was not affected in our CGN model (Fig. 2E).

### Urine metabolic profiling of KP intermediates after CGN induction

In urine, all 8 KP metabolites targeted by our method were successfully quantified and expressed as a ratio to urinary creatinine. Urinary levels of TRP, KYN, KA, 3-HK and QUIN peaked on day 7 and declined thereafter (without this variation being statistically significant), but only urinary levels of TRP and KYN were significantly increased on day 7 compared to baseline (Fig. 3A to H). We therefore monitored the urinary KYN/TRP ratio, which may reflect IDO activity in the kidney [16].

The urinary KYN/TRP ratio was significantly increased at day 7 and returned to baseline values thereafter (Fig. 4A), suggesting that IDO activity is high in the early phases of CGN. Importantly, the urinary KYN/TRP ratio was independent of proteinuria levels (as TRP and KYN bind to albumin) at day 7 [27]) (Fig. 4H). In turn, the ratio of TRP to urinary metabolites downstream of KYN, XA/TRP and 3-HANA/TRP moderately decreased, which may indicate a decrease in KMO activity as described in inflammatory responses (Fig. 4B to F). Finally, we observed that urinary QUIN/TRP and urinary PIC/TRP levels were significantly decreased in the early phase of CGN (Fig. 4G), which may reflect increased activity of QPRT and decreased activity of ACMSD in the early phase of CGN.
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Validation of the model of CGN in pigs A. Graphic description of the CGN model. 18 pigs were pre-immunised with a subcutaneous injection of purified sheep IgG 7 days before the start of the protocol, and 13 pigs were included in the urine and plasma metabolites analyses. Each individual received an intramuscular injection of NTS on two consecutive days to induce an accelerated CGN model. Blood and plasma were collected on day 1, day 7, day 14, day 21 and day 28. B, C, D. Serial measurements of plasma urea, plasma creatinine and proteinuria over time in pigs after NTS injection. (n = 8 to 11 per group). Bars represent mean ± sem. p values were calculated using ANOVA (mixed model analysis) followed by Dunnett's multiple comparison test. E. Representative images (left, control pig and right, NTS pig) showing renal lesions associated with CGN at late stage (day 28): glomerular crescents, interstitial inflammation and fibrosis, tubular dilation and atrophy in pigs challenged with NTS (Masson Trichrome, x 40)</figcaption></figure>

## KP enzyme expression in whole kidneys in late stage CGN

We then tested whether CGN affects the activity of KP enzymes in the kidney in the late phase of the disease. To do this, we measured the expression of 4 critical enzymes in this pathway: IDO, KMO, QPRT and ACMSD in whole kidneys from 10 pigs sacrificed on day 28. We observed that IDO transcript levels were significantly increased with sCr, suggesting that the severity of chronic renal lesions correlates with increased IDO activity (Fig. 5A). KMO and QPRT levels did not appear to be affected by renal injury (Fig. 5B and C). Finally, we observed a robust decrease in ACMSD expression in the most severely injured kidneys (as reflected by sCr levels) (Fig. 5D).

Since ACSMD converts AMCS into PIC, this suggests that KP may be channeled into de novo NAD+ synthesis in the late phase of CGN.

## Discussion

We performed, for the first time, a comprehensive metabolic profiling of KP metabolites over time in the plasma and urine of pigs with NTS-induced CGN. We show that the urinary KYN/TRP ratio is a biomarker for the early phase of CGN activity and that monitoring plasma KP intermediates is of limited informative value. Urinary KYN/TRP ratio does not correlate with proteinuria,
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 2** Blood metabolic profiling of KP intermediates after CGN induction. Serial measurements of plasma TRP, KYN, 3-HK, QUIN and KYN/TRP in pigs after NTS injection (n=6 to 10 per group). Bars represent mean ± sem. p values were calculated using ANOVA (mixed model analysis) followed by Dunnett's multiple comparison test</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 3** Urine metabolic profiling of KP intermediates after CGN induction. Serial measurement of urinary TRP, KYN, KA, 3-HK, XA, 3-HANA, PIC and QUIN in pigs after NTS injection (n = 8 to 13 per group). Bars represent mean ± sem. p values were calculated using ANOVA (mixed model analysis) followed by Dunnett's multiple comparison test</figcaption></figure>

which strengthens its specificity as a disease activity biomarker.

Our results suggest that monitoring plasma KP in this porcine model may not be a promising source of biomarkers of CGN activity, as half of the metabolites in the panel could not be quantified, despite the high sensitivity of the LC-MS/MS method, and the levels of the quantifiable metabolites were not significantly affected. A reduction in plasma TRP is classically observed in many models of AKI and CKD, primarily due to IDO consumption, and is often associated with increased concentrations

of downstream metabolites, including KYN and QUIN [1–3]. Specifically, high serum KYN levels with decreased TRP concentrations have been found in patients with antineutrophil cytoplasmic antibody-associated vasculitis, one of the causes of CGN in humans [28]. However, the major difficulty in this situation is that the metabolites of KP are also primarily considered to be uremic retention solutes or even uremic toxins. Therefore, it is not possible to deconvolute the effects of increased IDO activity and reduced renal clearance, as CGN promotes both IDO activity and renal failure at the same time.
A

<figure><img src="image_10.png" /></figure>

B

<figure><img src="image_11.png" /></figure>

C

<figure><img src="image_12.png" /></figure>

D

<figure><img src="image_13.png" /></figure>

E

<figure><img src="image_14.png" /></figure>

F

<figure><img src="image_15.png" /></figure>

G

<figure><img src="image_16.png" /></figure>

H

<figure><img src="image_17.png" /></figure>

**Fig. 4** Time-course monitoring of KP urine metabolites ratio during CGN. **A-G.** Serial measurement of urinary KYN/TRP, KA/TRP, KA/TRP, 3-HK/TRP, XA, 3-HANA/TRP, PIC/TRP, and QUIN/TRP in pigs after NTS injection (n=8 to 13 per group). Bars represent mean ± sem. p values were calculated with an ANOVA (mixed model analysis) followed by Dunnett's multiple comparison test. **H.** Correlation analysis (Pearson correlation) between urinary KYN/TRP and proteinuria in 8 pigs for which urine samples were available for both metabolic profiling and urine protein dosage at day 7

Our results indicate that monitoring urine KP metab-
olites may provide useful information on the activity of
CGN. The rationale for using metabolite-to-creatinine
ratios in this specific disease context is that we are look-
ing for urinary biomarkers that would appear in elevated
concentrations reflecting CGN activity in addition to glo-
merular filtration rate decrease, and that these concen-
trations can predict the ultimate severity of CGN. In such

cases, there is often significant variability in both urinary
biomarker and creatinine concentrations. The ability
to detect kidney injury is enhanced by the requirement
for an increase in biomarker concentration in relation
to a decrease in creatinine clearance. However, in cases
of severe AKI, the urinary creatinine concentrations
decrease in proportion to the magnitude of the decrease
in glomerular filtration rate. This results in a sudden
A

<figure><img src="image_18.png" /></figure>

$R^2=0.50, p=0.02$

B

<figure><img src="image_19.png" /></figure>

$R^2=0.18, p=0.21$

C

<figure><img src="image_20.png" /></figure>

$R^2=0.05, p=0.53$

D

<figure><img src="image_21.png" /></figure>

$R^2=0.68, p=0.003$

<caption>**Fig. 5** Kidney expression of KP enzymes transcripts in the late phase of CGN. Correlation analysis (Pearson correlation) between IDO, KMO, QPRT, and ACMSD transcripts measured by q-PCR in the kidney cortex of 10 pigs 28 days after NTS injection and plasma creatinine</caption>

increase in normalized biomarker levels despite constant production and excretion rates of the biomarker. Therefore, the standardization of urinary AKI biomarkers to urine creatinine may lead to an overestimation of acute kidney damage resulting from acute and unstable changes in creatinine clearance. However, with regard to our study and model, the impact of normalizing urinary metabolites is limited and does not affect the main message of our work, which is that the KYN/TRP ratio (i.e. unadjusted for urinary creatinine) is the candidate biomarker for CGN activity. Since the turnover of KP metabolites is highly determined by tubular excretion [15], they may serve as promising biomarkers of tissue KP activity in the diagnosis and prognosis of CGN. An important result of our study is that the kinetics of the levels of most urinary metabolites affected by CGN is transient (in this

model with a single trigger). This means that (1) inflammatory disease activity is not self-sustaining in the long term; (2) elevated urinary metabolites are markers of inflammatory activity in the kidney and could therefore indicate the need to initiate immunosuppressive therapy; and (3) baseline levels do not indicate the absence of CGN when measured in the late phase of the disease. Critically, our data support the notion that urinary KYN/TRP ratio reflects IDO activity and as such could be a potent non-invasive biomarker of the early phase of CGN. These findings are supported by experimental evidence. For example, in a mouse model of CGN induced by NTS, increased IDO activity was suggested by an increased KYN/TRP ratio in renal tissue and upregulated IDO gene expression in glomeruli and tubular epithelial cells [16]. In addition, inhibition of IDO-mediated TRP
catabolism using 1-methyl-tryptophan (1-MT) resulted in increased glomerular crescent formation, macrophage infiltration and intrarenal cell proliferation, significantly stimulating nephritis progression.

Our findings highlight the potent effect of CGN on the final steps of the TRP catabolic pathway, which involves the production of QUIN and PIC and drives de novo synthesis of NAD+, a cytoprotective pathway. In the early phase of CGN, decreased urinary QUIN/TRP levels are compatible with increased activity of QPRT and potentially the production of intermediates for NAD + synthesis. Previous metabolomic studies have suggested that an elevated urine QUIN/TRP ratio is an indicator of reduced QPRT activity and a predictor of septic/ischemic AKI and other adverse outcomes in critically ill patients [29]. As we observed that urine QUIN/TRP levels were significantly decreased (instead of increased) in the early phase of CGN, this suggests that the interpretation of the change in urine QUIN/TRP ratio during AKI depends on its causes and mechanisms. In addition, a decreased urine PIC/TRP ratio may reflect decreased ACMSD activity [30]. As ACMSD drives ACMS away from QUIN production and de novo NAD+ synthesis, these results are in line with an increased metabolic flow through the de novo NAD+ biosynthetic pathway.

This study has several limitations. The number of pigs included in the study was relatively small, which may limit the power of the study. Critically, the non-congenic genetic background of the animals partly explains the different responses to NTS observed in this study, but it mimics the heterogeneity of disease severity in patients with CGN. Another limitation comes from transcript quantification which was performed on whole kidney tissue, which may dilute cell-specific signals (glomerular vs. tubular). Finally, it is important to note that transcript measurements were not performed in the early phase of the disease when metabolites were significantly different from day 0, which may limit the understanding of disease progression. Rather, the histological and transcript correlation study was conducted at a late, scarring stage of CGN and therefore reflects very little of the inflammatory activity of the glomerular disease. A time course profiling of kidney histology and transcripts expression study would have allowed for a better understanding of the possible correlation between transcript expression over time, metabolic profiles, and inflammatory activity in kidney disease.

In conclusion, we show for the first time with a detailed analysis of KP metabolites in blood and urine over time in a porcine CNG model that plasma monitoring is of little use in this context, but that urine analyses are a potential source of markers of glomerular disease activity. In particular, an increased urinary KYN/TRP ratio may reflect early inflammatory activity of the disease, and a

decreased urinary QUIN/TRP ratio may reflect increased metabolic flux through the NAD + de novo synthesis pathway.

## Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12882-025-04423-6.

Supplementary Material 1

Supplementary Material 2

Supplementary Material 3

## Acknowledgements

None.

## Author contributions

Zahia Nadour performed LC-MS/MS experiments Julien Dang and Léa Resmini generated the NTS model Virginie Poindessous performed qPCR Pierre-Louis Tharaux and Nicolas Pallet designed the study and wrote the manuscript.

## Funding

This project has been funded by a grant from the Département Médico Universitaire (DMU) Biophygen.

## Data availability

No datasets were generated or analysed during the current study.

## Declarations

### Ethical approval

All animal procedures were performed following the European Community guidelines (L358-86/609EEC) and were approved by the Institut National de la Santé et de la Recherche Médicale and the Ministry for Higher Education and Scientific Research (APAFIS#25526-2020061917538762). The animals were housed individually in the Center for Biomedical Research facilities from the National Veterinary School of Maison-Alfort (France) (authorization G-94-046-02). The study is reported in accordance with ARRIVE guidelines.

### Competing interests

The authors declare no competing interests.

Received: 29 April 2025 / Accepted: 21 August 2025

Published online: 29 August 2025

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