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RESEARCH ARTICLE
Farnesoid X receptor prevents neutrophil extracellular traps via reduced
sphingosine-1-phosphate in chronic kidney disease
Bryce A. Jones,
1
Komuraiah Myakala,
2
Mahilan Guha,
2
Shania Davidson,
3
Sharmila Adapa,
2
Isabel Lopez Santiago,
2
Isabel Schaffer,
2
Yang Yue,
4
Jeremy C. Allegood,
4
L. Ashley Cowart,
4
Xiaoxin X. Wang,
2
Avi Z. Rosenberg,
5
and Moshe Levi
2
1
Department of Pharmacology and Physiology, Georgetown University, Washington, District of Columbia, United States;
2
Department of Biochemistry and Molecular and Cellular Biology, Georgetown University, Washington, District of Columbia,
United States;
3
Department of Biology, Howard University, Washington, District of Columbia, United States;
4
Department of
Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia, United States; and
5
Department
of Pathology, Johns Hopkins School of Medicine, Baltimore, Maryland, United States
Abstract
Farnesoid X receptor (FXR) activation reduces renal inflammation, but the underlying mechanisms remain elusive. Neutrophil
extracellular traps (NETs) are webs of DNA formed when neutrophils undergo specialized programmed cell death (NETosis). The
signaling lipid sphingosine-1-phosphate (S1P) stimulates NETosis via its receptor on neutrophils. Here, we identify FXR as a nega-
tive regulator of NETosis via repressing S1P signaling. We determined the effects of the FXR agonist obeticholic acid (OCA) in
mouse models of adenosine phosphoribosyltransferase (APRT) deficiency and Alport syndrome, both genetic disorders that
cause chronic kidney disease. Renal FXR activity is greatly reduced in both models, and FXR agonism reduces disease severity.
Renal NETosis and sphingosine kinase 1 (Sphk1) expression are increased in diseased mice, and they are reduced by OCA in
both models. Genetic deletion of FXR increases Sphk1 expression, and Sphk1 expression correlates with NETosis. Importantly,
kidney S1P levels in Alport mice are two-fold higher than controls, and FXR agonism restores them back to baseline. Short-term
inhibition of sphingosine synthesis in Alport mice with severe kidney disease reverses NETosis, establishing a causal relationship
between S1P signaling and renal NETosis. Finally, extensive NETosis is present in human Alport kidney biopsies (six male, nine
female), and NETosis severity correlates with clinical markers of kidney disease. This suggests the potential clinical relevance of
the newly identified FXR-S1P-NETosis pathway. In summary, FXR agonism represses kidney Sphk1 expression. This inhibits renal
S1P signaling, thereby reducing neutrophilic inflammation and NETosis.
NEW & NOTEWORTHY Many preclinical studies have shown that the farnesoid X receptor (FXR) reduces renal inflammation, but
the mechanism is poorly understood. This report identifies FXR as a novel regulator of neutrophilic inflammation and NETosis via
the inhibition of sphingosine-1-phosphate signaling. Additionally, NETosis severity in human Alport kidney biopsies correlates
with clinical markers of kidney disease. A better understanding of this signaling axis may lead to novel treatments that prevent
renal inflammation and chronic kidney disease.
adenine diet; Alport syndrome; farnesoid X receptor; NETosis; sphingosine-1-phosphate
INTRODUCTION
Chronic inflammation is a well-accepted mechanism that
drives chronic kidney disease (CKD) (1–3). Herein, we investi-
gate the anti-inflammatory mechanisms of the farnesoid X re-
ceptor (FXR) in models of adenosine phosphoribosyltransferase
(APRT) deficiency and Alport syndrome. These mouse models
are commonly used to investigate tubulointerstitial fibrosis, the
unifyingmechanismofallformsofCKDinhumans.
APRT is essential for the metabolism of adenine, a product
of the polyamine pathway. In physiological conditions, ade-
nine is first converted to AMP which then enters the xanthine
oxidase pathway to yield uric acid. In patients with APRT defi-
ciency, adenine is directly shunted through the xanthine oxi-
dase pathway to produce 2,8-dihydroxyadenine (2,8-DHA), an
insoluble metabolite that causes crystalline nephropathy (4).
APRT deficiency is treated with xanthine oxidase inhibitors,
but even with treatment, patients often have recurrent epi-
sodes of acute kidney injury that can progress to CKD (5).
Although transgenic Aprt-null mice exist, APRT deficiency is
most frequently modeled by placing rodents on a diet supple-
mented with adenine, referred to herein as adenine mice (6).
Alport syndrome is a hereditary disease caused by a muta-
tion in the collagen IV a3a4a5 heterotrimer, a component of
Correspondence: M. Levi (Moshe.Levi@georgetown.edu).
Submitted 15 September 2023 / Revised 10 October 2023 / Accepted 10 October 2023
F792 1931-857X/23 Copyright ©2023 the American Physiological Society. http://www.ajprenal.org
Am J Physiol Renal Physiol 325: F792–F810, 2023.
First published October 12, 2023; doi:10.1152/ajprenal.00292.2023
Downloaded from journals.physiology.org/journal/ajprenal at Virginia Commonwealth Univ (128.172.077.159) on April 18, 2024.
the glomerular basement membrane. If left untreated, 90%
of males and 15% of females with X-linked Alport syndrome
will progress to end-stage renal failure (ESRF) by the age of
40 yr old, with some as early as 10 yr old (7). Fewer data are
available from patients with autosomal recessive Alport syn-
drome, but disease progression in both sexes can be severe
(8,9). The current standard of care is treatment with angio-
tensin-converting enzyme inhibitors (ACE-i) or angiotensin
receptor blockers (ARB) (10). Nevertheless, patient outcomes
remain bleak. In a recent cohort of patients, early intervention
with ACE-i/ARBs delayed the onset of ESRF by 13 yr com-
pared with sibling controls. However, over 50% of the early
intervention group still progressed to ESRF by the age of 40 yr
old (11). These data underscore the importance of developing
novel treatments for Alport syndrome. Numerous preclinical
models of Alport syndrome have been developed, all based on
the genetic deletion of a component of the collagen IV a3a4a5
heterotrimer (12). We use autosomal recessive Col4a3-null
mice, referred to herein as Alport mice (13,14).
FXR is a nuclear receptor that is endogenously activated
by bile acids, and obeticholic acid (OCA) is an FXR agonist
that is clinically approved to treat primary biliary cholangitis
(15–17). Numerous preclinical studies have shown that FXR
protects against kidney disease (18–25). Briefly, FXR activa-
tion decreases renal fibrosis, inflammation, lipotoxicity, oxi-
dative stress, and endoplasmic reticulum stress (26,27).
However, mechanistic studies on how FXR agonism reduces
renal inflammation are lacking. We address this gap herein.
The inflammatory milieu in CKD consists of many cell
types, each with unique roles. Some immune cells are nephro-
protective, but many cell types aberrantly exacerbate disease
(28–30). Neutrophils are innate immune cells that are best
known for being the first line of defense against pathogens.
One mechanism whereby neutrophils protect the host from
infection is by immobilizing microbes within neutrophil
extracellular traps (NETs). NETs are stringy webs of decon-
densed DNA and antimicrobial proteins that are released into
the extracellular milieu during NETosis, a form of pro-
grammed cell death (31,32). However, aberrant NETosis has
been reported in sterile inflammation, including some kidney
diseases (33). There is accumulating evidence that renal
NETosis contributes to kidney disease arising from lupus,
hypertension, diabetes, vasculitis, renal calculi, and acute kid-
neyinjury(34–41).NostudythusfarhasinvestigatedNETsin
models of APRT deficiency or Alport syndrome, nor has the
role of FXR in NETosis been investigated.
Sphingosine-1-phosphate (S1P) is a pro-inflammatory sig-
naling lipid produced from sphingosine by its two kinases:
Sphk1 and Sphk2. S1P is best understood in the context of T-
cells (42–45), but emerging research indicates that S1P also
promotes neutrophilic inflammation. S1P increases neutro-
phil chemotaxis and activates NETosis via its G protein-
coupled receptors on neutrophils (46–51). Also, inhibiting
S1P signaling stimulates neutrophil apoptosis and clearance
by macrophages, promoting the resolution of neutrophilic
inflammation (48,52). Although a few initial studies indi-
cated that S1P may be beneficial to the kidney (53,54), many
recent studies using Sphk1-null mice showed that inhibition
of SPHK1 protects the kidney (55–59).
Taken together, we hypothesized that FXR agonism
represses kidney Sphk1 expression and lowers S1P, thereby
reducing neutrophilic inflammation and NETosis in models
of APRT deficiency and Alport syndrome.
MATERIALS AND METHODS
Animal Models
Animal studies were approved by the Institutional Animal
Care and Use Committee of Georgetown University. All mice
were maintained on a 12:12-h light-dark cycle and fed a grain-
based chow (Cat. No. 5053, LabDiet) unless otherwise speci-
fied. C57BL/6J mice were obtained from The Jackson
Laboratory (Bar Harbor, ME). Col4a3
tm1Jhm
mice on the 129S1/
SvImJ background were obtained as a gift from Dr. Jeffrey H.
Miner (Washington University in St. Louis) (13). Col4a3
tm1Dec
miceontheC57BL/6Jbackgroundwereobtainedasagiftfrom
Sanofi(Framingham, MA) (14,60). Col4a3
/
(Alport mice)
was the disease genotype, and both Col4a3
þ/þ
and Col4a3
þ/
were the control genotypes. Consistent with the original
reports and recent publications (13,14,61), we did not observe
adiseasephenotypeinCol4a3
þ/
mice at the timepoints we
studied (data not shown). Genotyping was performed by
Transnetyx (Cordova, TN) using RT-qPCR. For the FXR-null
experiment, male Nr1h4
tm1Gonz
(FXR-null) mice and age-
matched male C57BL/6 mice were used (62). For all animal
studies, heparinized plasma and organs were collected
upon euthanasia. All chemicals used in the animal studies
are commercially available: adenine (CAS No. 73-24-5),
corn oil (CAS No. 8001-30-7), dimethyl sulfoxide (DMSO,
CAS No. 67-68-5), myriocin (CAS No. 35891-70-4), and OCA
(CAS No. 459789-99-2).
FXR Expression and Function Study in Adenine Mice
Eight male C57BL/6J mice were purchased at 12 wk of age,
and equal numbers were fed chow alone or admixed with ad-
enine (0.2% wt/wt) for 7 wk. Mice were euthanized at 19 wk
of age.
FXR Expression and Function Study in Alport Mice
Male 129S1/SvImJ control and Alport mice were eutha-
nized at 8 wk of age. Urine was collected during the week of
euthanasia. A satellite cohort of male 129S1/SvImJ control
and Alport mice was euthanized at 11–12 wk of age.
FXR Agonism Study in Adenine Mice
Twenty-fourmaleC57BL/6Jmicewerepurchasedat10wk
of age and maintained on a purified control diet (D19120401i,
ResearchDiets).At12wkofage,12micewereswitchedtothe
control diet admixed with adenine (0.2% wt/wt, D19120402i,
Research Diets), referred to as the adenine diet. Also starting
at 12 wk of age, mice were treated with either vehicle (98%
corn oil, 2% DMSO) or OCA (10 mg/kg body wt) by oral gavage
5 days per week for 7 wk. We have previously validated the
delivery of this dose of OCA by oral gavage (22). Body weights
and food weights were recorded semiweekly. Mice were eu-
thanized at 19 wk of age.
FXR Agonism Study in Alport Mice
Male 129S1/SvImJ Alport mice and littermate controls were
fed chow alone or admixed with OCA (30 mg/kg body wt)
FXR PREVENTS NETs VIA REDUCED S1P
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from 3 wk to 11 wk of age. We have previously validated the
delivery of this dose of OCA by admixture with the diet (20).
Urine (if present) was collected directly from the bladder after
euthanasia at 11 wk of age. Most Alport mice did not have
urine to collect.
Inhibition of De Novo Sphingosine Synthesis in Alport
Mice
Male C57BL/6J Alport mice and heterozygous controls were
used.Startingat31wkofage,littermate-matchedAlportmice
Figure 1. Authentication of key biological
resources. A: relative transcript level of
Nr1h4 in the kidney using primers specific
for the C-terminal ligand-binding domain
(LBD) of FXR. C
t
values over 40 could not be
quantified and are arbitrarily set to zero. B:
immunoblot of total-kidney lysate shows that
FXR is not expressed in FXR-null mice. Total
protein (Ponceau S) was used as a loading
control. C: immunohistochemistry of control
and FXR-null kidneys shows absence of nu-
clear staining in the tubules of FXR-null
mice. Glomerular signal in FXR-null kidneys
does not exclusively localize to the nucleus,
and it arises from binding of the secondary
antibody to endogenous immunoglobulin.
Isotype and secondary antibody control
slides are presented in the Supplemental
Material. D: this subfigure is best viewed
with a computer. Immunofluorescence
shows absence of tubule staining in FXR-
null mice. Glomerular signal arises from
binding of the secondary antibody to endog-
enous immunoglobulin (data not shown).
Scale bars represent 50 lm. Data are
expressedasthemeans±SD,andeachda-
tum represents one mouse. FXR, farnesoid
Xreceptor.
Figure 2. Renal FXR signaling is reduced
in adenine mice. A: experimental design:
Mice were fed a control diet or an adenine-
enriched diet to induce kidney disease. B:
renal Nr1h4 expression was reduced in
adenine mice compared with healthy con-
trols. Cand D: renal FXR protein expres-
sion was unchanged on immunoblot in
adenine mice compared with healthy con-
trols. Total protein (Ponceau S) was used
as a loading control. E: renal transcription
of the canonical FXR target genes are
reduced in adenine mice, thus implying
reduced FXR activity. Significance was
determined by Student’stwo-tailedttest,
and exact Pvalues are shown. Data are
expressed as the means ± SD, and each
datum represents one mouse. FXR, farne-
soid X receptor.
FXR PREVENTS NETs VIA REDUCED S1P
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were treated with vehicle [1% DMSO in phosphate-buffered
saline (PBS)] or myriocin (1 mg/kg body wt) by intraperitoneal
injection 6 days per week for 2 wk. Heterozygous controls
were injected with vehicle alone.
Plasma and Urine Chemistries
Blood urea nitrogen (BUN) was determined using the Jung
method (63). Plasma/urine creatinine (Cat. No. DICT-500
BioAssay Systems) and urine albumin (Cat. No. 1011, Ethos
Biosciences) were determined, according to the manufac-
turers’instructions.
Analysis of Published RNA-Sequencing Datasets
Datasets were downloaded from the Sequence Read
Archive and analyzed with BioJupies using the default set-
tings (64,65). Within each experiment, healthy mice were
compared with diseased mice (adenine or Alport) (66–69).
Descriptions of the experiments are provided in the Supple-
mental Material (Supplemental Table S1).
Differentially expressed genes were obtained from BioJupies
and processed in R using the Cluster Profiler package, precisely
the enrichGO function, to perform the gene ontology relating
only to biological processes on the datasets. The bitr function
was used to convert gene symbols to ENTREZID and ENSEMBL
types. The graphs were created using the ggplot2 package in R.
Data presented are from the 250 most upregulated differential
genes. Principal component analysis was performed using the
prcomp method from the R Stats package. The data were nor-
malizedusingtheedgeRupperquartilemethod.
RNA Isolation and Quantitative RT-PCR
The kidney cortex was homogenized in a bead mill homoge-
nizer, and total RNA was isolated with spin columns (Cat. No.
74104, Qiagen). cDNA was synthesized from 100 ng total RNA
(Cat. No. 4387406, Applied Biosystems). Real-time quantitative
PCR was performed (Cat. No. 4385610, Applied Biosystems),
and target gene transcript expression was normalized to Rn18s
using the DDC
T
method. Primer sequences are provided in the
Supplemental Material (Supplemental Table S2).
Tissue Homogenization and Immunoblots
A mid-transverse kidney piece was homogenized in T-PER
(Cat. No. 78510, Thermo Scientific) with protease and phos-
phatase inhibitors using a Potter-Elvehjem tissue grinder.
Samples of equal protein concentration were prepared in
reducing and denaturing conditions (Cat. No. 7722, Cell
Signaling Technology).
Twenty-five micrograms of protein from each sample were
run (100 V, 100 min) on polyacrylamide gels (Cat. No.
5671095, Bio-Rad) using Tris-glycine SDS running buffer (25
mM Tris base, CAS No. 77-86-1; 192 mM glycine, CAS No. 56-
40-6;0.1%wt/volSDS,CASNo.151-21-3;pH8.3).Proteinwas
transferred(400mAh,60min,4
C) in Towbin buffer (25 mM
Tris base; 192 mM glycine; 20% methanol, CAS No. 67-56-1;
pH 8.3) onto PVDF membrane (Cat. No. 88518, Thermo
Scientific). Total protein was quantified with Ponceau S (1%
wt/vol, CAS No. 6226-79-5) in 0.5% acetic acid (CAS No. 64-19-
7). Membranes were washed with 0.1% vol/vol Tween 20 (CAS
No. 9005-64-5) in Tris-buffered saline (20 mM Tris base; 150
Figure 3. Renal FXR signaling is reduced in Alport mice. A: experimental design: Alport mice on the fast-progressing 129S1/SvImJ background rapidly
develop kidney disease compared with littermate controls. Two timepoints were investigated. B:renalNr1h4 expression was unchanged in 8-wk Alport
mice compared with healthy controls. C,D: renal FXR protein expression was unchanged on immunoblot in 8-wk Alport mice compared with healthy
controls. Total protein (Ponceau S) was used as a loading control. Eand F: renal transcription of some FXR target genes are decreased at the 8-wk time-
point, but all FXR target genes are decreased by the 11- to 12-wk timepoint. Significance was determined by Student’stwo-tailedttest, and exact Pval-
ues are shown. Data are expressed as the means ± SD, and each datum represents one mouse. FXR, farnesoid X receptor.
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mM NaCl, CAS No. 7647-14-5; pH 7.4) (TBST) between each of
the following steps. Membranes were blocked (1 h, 20C) with
5% non-fat dry milk (NFDM, CAS No. 68514-61-4) in TBST.
They were then incubated (overnight, 4C) with primary anti-
bodies diluted 1:1000 in 5% bovine serum albumin (CAS No.
9048-46-8) in TBST. Antibody information is provided in the
Supplemental Material (Supplemental Table S3). The next
day,membraneswereincubatedwiththeappropriatesecond-
ary antibody diluted 1:5000 in 5% NFDM in TBST. Blots were
incubated in enhanced chemiluminescence substrate (Cat.
No. 34580, 34096, or A38554, Thermo Scientific) and imaged
(Azure Imager c300, Azure Biosystems).
Histopathology and Immunohistochemistry of Mouse
Kidneys
Tissues were drop-fixed in 10% neutral-buffered forma-
lin (Cat. No. SF98-4, Fisher Scientific) for 24 h at 4Cand
then transferred to 70% ethanol (CAS No. 64-17-5) for stor-
age at 4C before processing and embedding into paraffin
(CAS No. 8002-74-2). Tissues were thinly sectioned (3–5lm)
onto glass slides. Picrosirius red (PSR) staining was per-
formed according to standard histological procedures (Cat.
No. SO-674, Rowley Biochemical). Polarized microscopy on
PSR-stained adenine and Alport kidneys was performed
using an Olympus model IX83 and a Nikon BioPipeline
SLIDE, respectively.
Immunohistochemistry for FXR (1:100, 25C, 1 h) was
performed following antigen retrieval (pH 9, 95C, 1 h).
Immunohistochemistry for CD45, Ly-6G, and myeloper-
oxidase (MPO) (1:200, 25C, 1 h) was performed following
antigen retrieval (pH 6, 95C, 20 min). Staining was
performed by Histoserv (Germantown, MD) and the
Histopathology & Tissue Shared Resource at Georgetown
University. Antibody information is provided in the
Supplemental Material (Supplemental Table S3).
Immunofluorescence of Mouse Kidneys
Mouse kidneys were snap-frozen in liquid nitrogen, em-
bedded in O.C.T. (Cat. No. 23-730-571, Fisher Scientific), and
cryosectioned (5 lm) onto glass slides. Sections were fixed
Figure 4. FXR agonism prevents kidney disease in adenine mice. A: experimental design: OCA treatment was investigated in the adenine diet model. B
and D: representative images and quantification of picrosirius red (PSR) stained kidneys imaged with polarized light show that OCA reduced renal fibrosis.
C,Eand F: oil red O staining shows that OCA reduced renal lipid accumulation (top row). OCA does not affect 2,8-DHA crystals (bottom row, arrowheads).
Scale bars represent 100 lm. Significance was determined by one-way ANOVA with the Holm–
Sídák correction for multiple comparisons, and exact Pval-
ues are shown. Data are expressed as the means ± SD, and each datum represents one mouse. FXR, farnesoid X receptor; OCA, obeticholic acid.
Table 1. Plasma markers of kidney disease from the FXR agonism study in adenine mice
Control 1Veh Control 1OCA Adenine 1Veh Adenine 1OCA
N66 4 5
BUN, mg/dL 21.1 ± 2.8 23.2 ± 2.4 69.1 ± 9.5†69.9 ± 27.2†
Plasma Cr, mg/dL 0.23 ± 0.06 0.24 ± 0.05 0. 80 ± 0.16†0.62 ± 0.15†,‡
†P<0.0001 vs. control þveh. ‡P<0.05 vs. adenine þveh. Significance was determined by one-way ANOVA with the Holm–
Sídák
correction for multiple comparisons. Data are expressed as means ± SD. BUN, blood urea nitrogen; Cr, creatinine; N, number of surviving
mice; OCA, obeticholic acid; Veh, vehicle; FXR, farnesoid X receptor.
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(15 min, 25C) in 4% paraformaldehyde (CAS No. 50-00-0),
blocked (1 h, 25C) in 5% normal donkey serum (Cat. No. 017-
000-121, Jackson ImmunoResearch) with 0.3% Triton X-100
(CAS No. 9036-19-5), incubated (1:200, overnight, 4C) with
their respective primary antibodies, incubated (1:400, 1 h,
25C) with their respective highly cross-absorbed secondary
antibodies, incubated (1:2,000, 15 min, 25C) with DAPI (Cat.
No. EN62248, Fisher Scientific), and then mounted with
Prolong Diamond Antifade Mountant (Cat. No. P36961,
Invitrogen). Sections were washed (5 min, 25C) three times
in PBS (137 mM NaCl; 2.7 mM KCl, CAS No. 7447-40-7; 4.3
mM Na
2
HPO
4
, CAS No. 7558-79-4; 1.47 mM KH
2
PO
4
, CAS No.
7778-77-0; pH7.4) in between each of the steps. Antibody
information is provided in the Supplemental Material
(Supplemental Table S3). Kidney sections were imaged in
their entirety with a Leica SP8 laser scanning confocal micro-
scope equipped with 20 and 63 objectives.
Immunofluorescence of Human Biopsies
Human Alport kidney biopsies were obtained from
the Johns Hopkins University Renal Pathology Archive.
Specimens were collected during the course of clinical
care and de-identified before use in research. Multiplex
immunofluorescence for NETosis was performed follow-
ing antigen retrieval (20 min, pH6, 95C). Samples were
blocked in 5% normal donkey serum with 0.3% Triton X-
100 (1 h, 25C), incubated with their respective primary
antibodies(1:100,overnight,25
C), incubated with their
respective highly cross-absorbed secondary antibodies
(1:200, 3 h, 25C),incubatedwithDAPI(1:2,000,15min,
25C), and then mounted with Prolong Diamond Antifade
Mountant. Sections were washed three times in PBS (5
min, 25C) in between each of the steps. Spectral and auto-
fluorescence control slides were processed alongside the
Figure 5. FXR agonism prevents kidney disease in Alport mice. A: experimental design: control and Alport mice on the fast-progressing 129S1/SvImJ
background were treated with or without obeticholic acid (OCA). B: OCA treatment reduced blood urea nitrogen (BUN), plasma creatinine (trend), and
urinary albumin-to-creatinine ratio (ACR) in Alport mice. As described in the MATERIALS AND METHODS, urinary ACR was quantified for all available Alport
samples. C: representative images and quantification of picrosirius red (PSR) stained kidneys imaged with polarized light show that OCA reduced renal
fibrosis. D: representative images and quantification of fibronectin immunofluorescence further confirm that OCA reduced renal fibrosis. Scale bars rep-
resent 100 lm. Significance was determined by one-way ANOVA with the Holm–
Sídák correction for multiple comparisons, and exact Pvalues are
shown. Data are expressed as the means ± SD, and each datum represents one mouse.
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test slides. Antibody information is provided in the Supple-
mental Material (Supplemental Table S3).
Multispectral microscopy of the human biopsies was per-
formed using a Vectra 3.0 (PerkinElmer) equipped with 20
and 40 objectives. Spectral and autofluorescence control
slides were used to construct a spectral library. Using this
library, multispectral images were unmixed with inForm
(v2.4.11) into their respective channels (autofluorescence,
DAPI, Alexa Fluor 488, and Cy5).
Tissue Lipid Extraction and Analysis
A transverse kidney piece was homogenized in methanol
and extracted overnight in methanol and chloroform (CAS
No. 67-66-3) at a 2:1 ratio. Samples were then centrifuged,
and the supernatant was decanted and evaporated to yield
an oily residue of extracted lipids. Lipids were resuspended
by sonication in methanol and water (CAS No. 7732-18-5) at a
1:1 ratio immediately before injection.
Relative S1P levels were quantified using high-performance
liquid chromatography-tandem mass spectrometry as previ-
ously described (70). Data were analyzed in Analyst (v1.6.2), and
the relative abundance of S1P was normalized to tissue weight.
Statistical Analyses
RNA-sequencing data were analyzed with BioJupies and
R programming software (64). Kendall rank correlations
were calculated with Free Statistics Software (v1.2.1) (71). All
other statistical analyses were performed with GraphPad
Figure 6. Biological processes related to neutrophils are increased in adenine mice. Principal component analysis (PCA) and Gene Ontology (GO)
enrichment analysis were performed on RNA-sequencing data from PMID 33779314. A description of the experiment is in Supplemental Table S1. A:
PCA reduces dimensionality of the dataset, and each datum represents one mouse. Band C: the adjusted Pvalues (B), gene ratios (B), and fold
increases (C) are shown for the 250 most upregulated genes in adenine mice compared with control.
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Prism (San Diego, CA). Statistical tests are described in the
respective figures. For ANOVA post hoc tests, the decision
to compare only the following groups was made a priori: 1)
control þvehicle versus control þdrug, 2) control þvehi-
cle versus disease þvehicle, 3) disease þvehicle versus
disease þdrug, and 4) control þvehicle versus disease þ
drug. “Disease”refers to adenine mice or Alport mice.
“Drug”refers to OCA or myriocin.
RESULTS
FXR Activity Is Decreased in Adenine and Alport Mice
We first authenticated two FXR antibodies, key biological
resources used in this study (Fig. 1 and Supplemental Fig. S1).
Renal FXR expression and activity were then quantified in
models of CKD analogous to APRT deficiency (adenine mice)
and Alport syndrome (Alport mice) (Fig. 2Aand Fig. 3Aand
Supplemental Tables S4 and S5). Expression of Nr1h4,the
gene for FXR, was decreased in adenine mice but unchanged
in Alport mice (Fig. 2Band Fig. 3B). Renal FXR protein expres-
sion was unchanged in either model (Fig. 2, Cand D,andFig.
3, Cand D). However, these results should be interpreted with
caution because mRNA and protein levels of nuclear recep-
tors do not necessarily correlate with receptor activity.
Instead, nuclear receptor activity is assessed by quantifying
the expression of their specific target genes. In both adenine
and Alport mice, expression of the canonical FXR target genes
Nr0b2,Ddah1,Slc51a,andSlc51b was decreased, thus imply-
ing reduced FXR activity (Fig. 2Eand Fig. 3, Eand F).
Figure 7. Biological processes related to neutrophils are increased in Alport mice. Principal component analysis (PCA) and Gene Ontology (GO) enrich-
ment analysis were performed on RNA-sequencing data from PMID 35203245. A description of the experiment is in Supplemental Table S1. A:PCA
reduces dimensionality of the dataset, and each datum represents one mouse. Band C: the adjusted Pvalues (B), gene ratios (B), and fold increases (C)
are shown for the 250 most upregulated genes in Alport mice compared with control.
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FXR Agonism Reduces Chronic Kidney Disease in
Adenine Mice
Because renal FXR activity is decreased in the adenine
model of CKD (Fig. 2), we hypothesized that FXR agonism
would reduce disease severity (Fig. 4A). Adenine mice rap-
idly lost weight compared with control mice, despite no
overt and consistent difference in food intake (Supplemental
Fig. S2 and Supplemental Table S6). Adenine mice had
increased BUN and plasma creatinine compared with con-
trol mice, and OCA treatment reduced plasma creatinine
(Table 1).
Histopathological staining showed that adenine mice had
increased renal fibrosis compared with control mice, and it
was reduced by OCA (Fig. 4, Band D, and Supplemental Fig.
S3A). Oil red O staining revealed that adenine mice had
increased kidney lipid accumulation, and this was prevented
by FXR agonism (Fig. 4, Cand E, and Supplemental Fig.
S3B). Renal 2,8-DHA crystals were quantified, but there was
no difference between vehicle or treatment groups, suggest-
ing that the treatment benefit is not secondary to reduced
crystal deposition (Fig. 4, Cand F, and Supplemental Fig.
S3C). When taken together, these data provide unequivocal
support for the nephroprotective effect of FXR agonism in
the adenine diet model of APRT deficiency.
FXR Agonism Reduces Chronic Kidney Disease in Alport
Mice
Because renal FXR activity is decreased in the Alport
model of CKD (Fig. 3), we hypothesized that FXR agonism
would reduce disease severity (Fig. 5Aand Supplemental
Table S7). Within the diseased group, OCA reduced BUN,
plasma creatinine (trend, P¼0.0557), and the urinary
Figure 8. Neutrophilic infiltrate is abundant in adenine mice. A: immunohistochemistry for CD45 shows that adenine mice have extensive immune cell
infiltration compared with control mice. Band C: immunohistochemistry for Ly-6G (B) and MPO (C), both neutrophil-specific markers, shows that a subset
of the CD45-positive cells are neutrophils. The identical staining patterns in(B)and(C) clearly demonstrates that the cells are neutrophils. Scale bars rep-
resent 1 mm in the slide scans (both control and adenine) and 100 lmintheinset images. Samples in A,B,andCare serial sections, 5 lm each. Images
are representative of four control and four adenine mice. MPO, myeloperoxidase.
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albumin-to-creatinine ratio (ACR) (Fig. 5B). As described
in MATERIALS AND METHODS, urinary ACR was quantified
for all available Alport samples. Immunostaining for syn-
aptopodin, a podocyte-specific marker, was done to con-
firm the urinary ACR results. Glomerular synaptopodin
density was reduced in Alport mice and restored by OCA
(Supplemental Fig. S4A). OCA also reduced renal fibrosis
and renal cortical fibronectin expression (Fig. 5, Cand D,
and Supplemental Fig. S4B). These data demonstrate the
nephroprotective effects of FXR agonism in this model of
Alport syndrome.
Neutrophils Are Abundant in Adenine and Alport Mice
NETosis has been reported in models of CKD arising
from other etiologies, but it has not yet been reported in ad-
enine or Alport mice. Therefore, we examined these models
to determine if neutrophils are an important component
of the immune infiltrate. We analyzed published RNA-
sequencing datasets from four independent experiments,
two from each model (Supplemental Table S1) (66–69). In
all four experiments, gene ontology biological processes
related to neutrophils were among the most significantly
increased in adenine and Alport mice (Figs. 6 and 7and
Supplemental Figs. S5 and S6). Next, we stained serial sec-
tions from both models with CD45, Ly-6G, and MPO. The
staining patterns for Ly-6G and MPO (membranous and
cytoplasmic markers of neutrophils, respectively) were
identical, and they represented a subset of the CD45-posi-
tive cells (Figs. 8 and 9). Altogether, these data represent
very strong evidence that neutrophils are a prominent
component of the immune infiltrate in adenine and Alport
mice.
Figure 9. Neutrophilic infiltrate is abundant in Alport mice. A: immunohistochemistry for CD45 shows that Alport mice (8-wk-old, 129S1/SvImJ) have
extensive immune cell infiltration compared with control mice. Band C: immunohistochemistry for Ly-6G (B)andMPO(C), both neutrophil-specific
markers, shows that a subset of the CD45-positive cells are neutrophils. The identical staining patterns in (B)and(C) clearly demonstrates that the cells
are neutrophils. Scale bars represent 1 mm in the slide scans and 50 lmintheinset images. Samples in A,B,andCare serial sections, 3 lmeach.
Images are representative of five control and three Alport mice. MPO, myeloperoxidase.
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FXR Agonism Reduces Renal Neutrophilic Infiltrate and
NETosis in Adenine-Treated Mice
FXR agonism prevents kidney inflammation, but the mech-
anistic basis underlying this has not been fully elucidated (18,
20,22,72). Given that renal calculi stimulate NETosis in
humans (40) and S1P promotes neutrophil infiltration and
NETosis, we hypothesized that 2,8-DHA crystals would stimu-
late NETosis and that FXR agonism would abrogate this via
reduced Sphk1 expression.
Adenine mice had extensive NETosis, quantified by co-
localization of MPO (neutrophil marker) and citrullinated his-
tone H3 (Cit-H3, decondensed DNA) in the absence of CD68
(macrophage marker) to rule out macrophage extracellular
traps (Fig. 10A,arrows).2,8-DHAcrystalsstainednon-specifi-
cally, and they were excluded during quantitation (Fig. 10A,
arrowheads, and Supplemental Fig. S7). NETosis occurring on
the periphery of intact 2,8-DHA crystals was included during
quantitation. Remarkably, OCA treatment promoted the reso-
lution of neutrophilic inflammation and decreased NETosis
in adenine mice (Fig. 10B). The characteristic web-like pat-
tern of colocalized MPO and Cit-H3 can be clearly seen
with high magnification confocal microscopy (Fig. 10C).
The arrows annotate areas of unambiguous extracellular
MPO and Cit-H3 colocalization, confirming that this is
trueNETosis,notjustmildcitrullinationofhistoneH3
Figure 10. FXR agonism reduces neutrophilic inflammation and NETosis in adenine mice. Kidneys from surviving vehicle- and OCA-treated adenine mice
were stained for NETosis, and they were compared with three unremarkable control kidneys. A: adenine mice have extensive NETosis (arrows) as shown by
colocalization (yellow) of myeloperoxidase (MPO, green) and citrullinated histone H3 (Cit-H3, red). 2,8-DHA crystals bind antibodies non-specifically (arrow-
heads in A), and this signal was excluded during quantification. B:quantification of Ashows that OCA reduced both MPO-positive neutrophilic inflammation
and NETosis in adenine mice. C:highmagnification (63) confocal microscopy of a vehicle-treated adenine mouse shows characteristic “web-like”NETosis
morphology, strongly supporting the conclusion that these lesions are NETs. D:thissubfigure is best viewed on a computer. Slide scan (20) showing the re-
markable severity of NETosis in adenine mice. DAPI and CD68 single-channel images are in Supplemental Fig. S8A. Scale bars represent 100 lm(A), 20 lm
(C), and 1 mm (D). Significance was determined by one-way ANOVA with the Holm–
Sídák correction for multiple comparisons, and exact Pvalues are shown.
Data are expressed as the means ± SD, and each datum represents one mouse. FXR, farnesoid X receptor; OCA, obeticholic acid.
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within MPO-positive immune cells. A fluorescence slide scan
(best viewed on a computer) showed the remarkable severity
of NETosis in adenine mice (Fig. 10Dand Supplemental Fig.
S8A).
FXR Agonism Reduces Renal Neutrophilic Inflammation
and NETosis in Alport Mice
We then investigated Alport mice to see if FXR agonism also
modulated NETosis in that mouse model. Alport mice had
extensive neutrophilic infiltrate and NETosis, and both were
reduced by FXR agonism (Fig. 11, Aand B). A fluorescence slide
scan (best viewed on a computer) showed that Alport mice also
have extensive NETosis (Fig. 11Cand Supplemental Fig. S8B).
Neither Adenine nor Alport Mice Have Macrophage
Extracellular Traps
Macrophage extracellular traps (METs) have been
reported in rhabdomyolysis-induced acute kidney injury (73,
74). We did not observe METs in any of our adenine or
Alport mice, although we cannot exclude very rare occur-
rences. Notably, macrophages could be adjacent to, and
even phagocytose shards of, 2,8-DHA crystals without under-
going METosis (Supplemental Fig. S9).
NETosis Occurs in Kidney Biopsies From Humans With
Alport Syndrome
After confirming the relevance of neutrophils and NETosis in
the adenine and Alport models, we then sought to visualize
NETosis in human kidney biopsies. NETs were present in 13 of
15 human Alport kidney biopsies, and the degree of NETosis var-
ied between mild, moderate, and severe (Fig. 12, A–E,Table 2,
and Supplemental Figs. S10 and S11). Kendall rank correlation
revealed that NETosis grade was positively correlated with se-
rum creatinine, interstitial fibrosis and tubular atrophy (IFTA),
and glomerulosclerosis (GS) (Fig. 12, F–H). There was no correla-
tion between NETosis and proteinuria; however, interpretation
of this result is limited because only five of the biopsies had
unambiguously labeled 24-h proteinuria data available (Fig. 12I).
Figure 11. FXR agonism reduces neutrophilic inflammation and NETosis in Alport mice. A: kidneys from one-half of the vehicle- and OCA-treated Alport
mice were randomly chosen for NETosis staining. Alport mice on the fast-progressing 129S1/SvImJ background have extensive NETosis (arrows) as shown
by colocalization (yellow) of myeloperoxidase (MPO, green) and citrullinated histone H3 (Cit-H3, red). B:quantification of Ashows that OCA reduced MPO-
positive neutrophilic inflammation and NETosis in Alport mice. C:thissubfigure is best viewed on a computer. Slide scan (20)showingtheremarkablese-
verity of NETosis in Alport mice. DAPI and CD68 single-channel images are in Supplemental Fig. S8B. Scale bars represent 100 lm(A)and1mm(C).
Significance was determined by one-way ANOVA with the Holm–
Sídák correction for multiple comparisons, and exact Pvalues are shown. Data are
expressed as the means ± SD, and each datum represents one mouse. FXR, farnesoid X receptor; OCA, obeticholic acid.
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Semiquantitative values and measurements with ambigu-
ous units were disregarded in these analyses. Also, the 18-yr-
old male biopsy was disregarded as an outlier because it was
only a fragment of tissue. It was 10 times smaller than the
next smallest biopsy and 20 times smaller than the largest
one. Healthy control biopsies were not investigated and are
assumed to be unremarkable. We were unable to acquire
biopsies from patients with APRT deficiency.
After verifying that renal NETosis occurs in patients with
Alport syndrome, we then investigated if reduced kidney S1P
signaling is a mechanism underlying FXR-mediated inhibi-
tion of neutrophilic inflammation and NETosis.
Genetic Deletion of FXR Increases Sphk1 Expression in
the Kidney
To determine if FXR modulates inflammation via S1P
signaling, we firstsoughttoidentifyadirectlinkbetween
reduced FXR activity and increased renal Sphk1 expres-
sion. We quantified Sphk1 in otherwise healthy FXR-null
mice, and genetic deletion of FXR increased kidney Sphk1
Figure 12. Renal NETosis occurs in humans with Alport syndrome and correlates with the severity of kidney disease. This figure is best viewed on a computer. A–
C: kidney biopsies from humans with Alport syndrome were stained for NETosis, identified by colocalization (yellow) of myeloperoxidase (MPO, green) and citrulli-
nated histone H3 (Cit-H3, red). Representative multispectral images (20) are shown from biopsies with mild (A), moderate (B), and severe (C) NETosis. Only two of
the 15 kidney biopsies did not have any NETosis (data not shown). Arrows in Cindicate areas of significant NETosis that were not chosen for inset images. Isolated
NETting neutrophils are scattered throughout Cand are unlabeled. D: multispectral image (20) showing NETs from inset in C. Additional inset images from Care
provided in Supplemental Fig. S10. E: multispectral image (40) showing NETs from inset in D.F–H: NETosis grade is positively correlated with serum creatinine (F),
interstitial fibrosis and tubular atrophy (IFTA) (G), and glomerulosclerosis (GS) (H). I: NETosis grade is not correlated with proteinuria. Scale bars represent 1 mm in the
large images and 100 lmintheinset images. All images in this figure were spectrally unmixed to remove autofluorescence, provided in Supplemental Fig. S11.
Kendall rank correlation was performed, and exact Pvalues are shown. Each datum represents one biopsy. NET, Neutrophil extracellular trap.
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expression (Fig. 13A). We then quantified Sphk1 expression
in our models.
FXR Agonism Prevents Sphk1 Expression and S1P
Production in the Kidney
Kidney Sphk1 expression was increased in both adenine
and Alport mice, and FXR agonism prevented this increase
(Fig. 13, Band C). Spearman’s rank correlation revealed a
positive association between Sphk1 expression and NETosis
in adenine (trend, P¼0.0968) and Alport (significant, P¼
0.0172) mice (Fig. 13, Dand E).
Next, we validated that FXR agonism reduced kidney S1P
levels, not just Sphk1 expression. High-performance liquid
chromatography-tandem mass spectrometry performed on
Table 2. Clinical metadata associated with human Alport kidney biopsies
Sex
Age,
yr
NETosis
Grade
Serum Cr,
mg/dL Proteinuria Histology IFTA, % GS, %
Interstitial
Foam Cells Misc. Info.
M 4 1 0.3 500 mg/day Thin GBM 0 0 n.d. n.d.
M 9 2 n.d. n.d. GBM remodeling 20 9 Positive n.d.
M 13 1 0.7 Present GBM remodeling mild 5 n.d. n.d.
M 18 0 n.d. Nephrotic GBM remodeling 40 66 n.d. n.d.
M 38 1 n.d. n.d. GBM remodeling 0 20 n.d. Transplant
M 52 3 1.59 UPCR 2.2 g/g GBM remodeling, FSGS 15 33 Positive n.d.
F 7 1 0.6 150 mg/24h GBM remodeling 10 5 n.d. n.d.
F 9 1 n.d. 2 g/24 h Remodeled GBM, FSGS 0 3 Positive Possible immune
deposits
F 16 1 n.d. n.d. GBM remodeling 0 0 n.d. n.d.
F 23 0 0.6 3.6 g Remodeled GBM, FSGS 10 16 Positive Interstitial
eosinophils
F 28 1 n.d. n.d. Thin GBM 10 0 n.d. n.d.
F 34 2 0.83 4.5 g/24 h Thin GBM, FSGS with hyalinosis 10 33 n.d. n.d.
F 39 3 1.26 2.8 g/24 h Thin GBM with abnormal Col IV
staining; FSGS
20 29 n.d. n.d.
F 41 3 n.d. n.d. FSGS with hyalinosis mild 30 n.d. n.d.
F 48 1 0.5 Trace GBM remodeling 10 21 n.d. n.d.
NETosis was graded as none (0), mild (1), moderate (2), and severe (3). Cr, creatinine; F, female; FSGS, focal segmental glomerulosclerosis;
GBM, glomerular basement membrane; GS, glomerulosclerosis; IFTA, interstitial fibrosis and tubular atrophy; M, male; n.d., no data.
Figure 13. FXR prevents kidney Sphk1 expression and reduces S1P levels. A:Sphk1 expression is increased upon genetic deletion of FXR. B:Sphk1 expression
is increased in adenine mice and reduced by FXR agonism. C:Sphk1 expression is increased in Alport mice and reduced by FXR agonism. D:Sphk1 expression
is positively correlated (trend) with NETosis in adenine mice. E:Sphk1 expression is positively correlated with NETosis in Alport mice. F:analysisofextractedlip-
ids shows that relative kidney S1P levels are increased in Alport mice and reduced by FXR agonism. Significance was determined by Student’sttest (A), one-
way ANOVA with the Holm–
Sídák correction for multiple comparisons (B,C,andF), and Spearman’s rank correlation (Dand E). Exact Pvalues are shown. Data
are expressed as the means ± SD, and each datum represents one mouse. FXR, farnesoid X receptor; S1P, sphingosine-1-phosphate.
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extracted kidney lipids revealed that renal S1P in Alport
mice was over twice that of control mice, and FXR agonism
abrogated this increase (Fig. 13F, Supplemental Fig. S12, and
Supplemental Table S8).
Inhibition of De Novo Sphingosine Synthesis Reduces
Renal Neutrophilic Infiltrate and NETosis
We then sought to establish a causal link between inhibiting
S1P signaling and reducing renal neutrophilic infiltrate and
NETosis in CKD. To this end, slow-progressing C57BL/6J
Alport mice with established kidney disease were treated with
a short-term course of myriocin to block de novo sphingosine
synthesis (Fig. 14A). In agreement with our data from the fast-
progressing 129S1/SvImJ Alport mice, renal S1P in the slow-pro-
gressing C57BL/6J Alport mice was twice that of control mice.
As expected, myriocin treatment restored S1P to baseline levels
(Fig. 14B, Supplemental Fig. S13, and Supplemental Table S9).
The rapid reduction in kidney S1P after inhibiting de novo
sphingosine synthesis is a good biological positive control that
validates our protocol. After only 12 days of treatment, there
was a dramatic reduction of kidney neutrophilic inflammation
and NETosis in the myriocin-treated mice compared with vehi-
cle-treated littermate controls (Fig. 14, Cand D). This demon-
strates that inhibition of de novo sphingosine synthesis
reverses neutrophilic inflammation and NETosis in the kidney.
Altogether, our results show that FXR agonism represses
kidney Sphk1 expression and thus S1P production. In turn,
this protects the kidney by reducing both renal neutrophilic
inflammation and renal NETosis (Fig. 15).
DISCUSSION
We have made several noteworthy advancements presented
herein. We are the first to report NETosis in mouse models of
APRT deficiency and Alport syndrome as well as in kidney
biopsies from patients with Alport syndrome. Additionally, we
are the first to show the benefits of FXR agonism in these
mouse models. Finally, we report a novel mechanism whereby
FXR agonism reduces renal Sphk1 expression and thus
NETosis (Fig. 15).
NETosis is emerging as a critical player involved in the
pathogenesis of kidney diseases, including lupus nephri-
tis, hypertensive nephropathy, diabetic nephropathy, re-
nal vasculitis, renal calculi, and acute kidney injury (33–
41). We show the role of neutrophils and NETosis in mouse
models of APRT deficiency and Alport syndrome. Notably,
these models have the most severe NETosis that we have
observed. We also demonstrate that NETosis is present in
kidney biopsies from patients with Alport syndrome and
that NETosis severity is positively correlated with clinical
markers of kidney disease.
As of this writing, OCA has not been approved for the treat-
ment of kidney disease, but the nephroprotective role of FXR
in preclinical studies is becoming increasingly apparent. We
are the first to demonstrate that FXR agonism protects the
kidney in mouse models of APRT deficiency and Alport syn-
drome, and our results are consistent with many other studies
showing that OCA protects the kidney (20–24,75–77).
FXR agonism is known to decrease renal inflammation, but
the mechanism underlying this effect is poorly understood.
Figure 14. Inhibition of de novo sphingosine synthesis reverses NETosis and promotes resolution of neutrophilic inflammation. A: experimental design:
Alport mice on the slow-progressing C57BL/6J background were treated with or without myriocin for 2 wk. B: analysis of extracted lipids shows that rela-
tive kidney S1P levels are increased in Alport mice and reduced by myriocin. Cand D: Alport mice had moderate NETosis (arrows) which was reversed
by short-term inhibition of de novo sphingosine synthesis. Scale bars represent 100 μm. Significance was determined by one-way ANOVA with the
Holm–
Sídák correction for multiple comparisons, and exact Pvalues are shown. Data are expressed as the means ± SD, and each datum represents
one mouse. S1P, sphingosine-1-phosphate.
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We investigated S1P signaling as a potential mediator of cross-
talk between FXR-expressing renal parenchymal cells and
neutrophils which do not express detectable levels of FXR on
RNA sequencing (78). In both adenine and Alport mice, Sphk1
expression was increased with kidney disease and reduced by
FXR agonism. Furthermore, Sphk1 expression was positively
correlated with the severity of NETosis. Finally, kidney Sphk1
expression was elevated in otherwise healthy FXR-null mice,
confirming a mechanistic link between increased FXR func-
tion and reduced Sphk1 expression.
Our final key advancement was to establish a causal link
between FXR agonism, reduced S1P signaling, and the preven-
tion of neutrophilic inflammation and NETosis. To this end,
we blocked de novo sphingosine synthesis in Alport mice
with established kidney disease. Myriocin-treated mice had
both less neutrophilic infiltrate and less NETosis than vehi-
cle-treated mice, showing that reducing S1P prevents NETosis
and promotes the resolution of neutrophilic inflammation.
Our results are consistent with recent reports where inhibition
of S1P signaling reduced NETosis and promoted the resolu-
tion of neutrophilic inflammation in the liver (48,50,52).
We acknowledge several limitations to our data. Although our
data from both FXR-null mice and OCA-treated adenine and
Alport mice clearly establish that FXR regulates kidney Sphk1
expression in vivo, they do not fully characterize all the molecu-
lar players involved. A complete mechanistic dissection of this
pathway is outside the scope of the current study. In addition,
we unexpectedly observed that OCA treatment did not reduce
Sphk1 expression in healthy control mice. A possible technical
explanation for this is that decreases in gene expression may be
easier to observe when expression of the gene of interest is
higher, such as in the case of Sphk1 in adenine and Alport mice
compared with their respective controls. A possible biological
explanation for this is that there may be homeostatic mecha-
nisms that prevent decreases in Sphk1 expression to abnormally
low levels. If so, this would explain why the decrease in Sphk1
expression was only observed in adenine and Alport mice, both
of which have abnormally high Sphk1 expression. However,
these explanations are only speculatory, and additional studies
are necessary to identify the underlying causes.
In conclusion, FXR agonism protects the kidney in
mouse models of APRT deficiency and Alport syndrome.
We are the firsttoreportNETsinthesemousemodels,as
well as in kidney biopsies from patients with Alport syn-
drome. Mechanistically, FXR agonism regulates renal inflam-
mation by repressing Sphk1 expression. This inhibits renal
S1P signaling, thereby reducing NETosis and promoting the
resolution of neutrophilic inflammation (Fig. 15).
DATA AVAILABILITY
Data are available from the corresponding author upon reason-
able request.
SUPPLEMENTAL DATA
Supplemental Figs. S1–S13 and Supplemental Tables S1–S9:
https://doi.org/10.6084/m9.figshare.24116088.v1.
ACKNOWLEDGMENTS
We acknowledge Emma Rowland, Carlos Benitez, and Maria
Idalia Cruz for assistance in caring for the mice. Experimental
design subfigures and Fig. 15 were created with BioRender.com.
Figure 15. Proposed mechanism for the FXR-mediated prevention of neutrophilic inflammation and NETosis in the kidney. Under physiological condi-
tions, FXR functions to repress Sphk1 gene expression and maintain kidney S1P at baseline levels. Reduced FXR activity in the setting of chronic kidney
disease drives an increase in Sphk1 gene expression, contributing at least in part to elevated renal S1P. This promotes neutrophilic inflammation and
NETosis in the kidney. Pharmacological FXR agonism, such as with OCA, represses Sphk1 gene expression and thus prevents these changes in the kid-
ney. FXR, farnesoid X receptor; OCA, obeticholic acid; S1P, sphingosine-1-phosphate.
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GRANTS
This work was supported by National Institutes of Health Grants
F30DK129003 (to B.A.J.), R01DK116567 (to M.L.), R01DK127830
(to M.L.), TL1TR001431 (to B.A.J.), P30CA016059, P30CA023074,
and P30CA051008.
DISCLAIMERS
The content is solely the responsibility of the authors and does
not necessarily represent the official views of the National
Institutes of Health.
DISCLOSURES
B.A.J. discloses a financial interest in AllazoHealth (New York,
NY). None of the other authors has any conflicts of interest, finan-
cial or otherwise, to disclose.
AUTHOR CONTRIBUTIONS
B.A.J. and M.L. conceived and designed research; B.A.J., K.M.,
M.G., and J.C.A. performed experiments; B.A.J. and M.G. analyzed
data; B.A.J., L.A.C., A.Z.R., and M.L. interpreted results of experi-
ments; B.A.J. prepared figures; B.A.J. drafted manuscript; B.A.J.,
K.M., M.G., S.D., S.A., I.L.S., I.S., Y.Y., J.C.A., L.A.C., X.X.W., A.Z.R.,
and M.L. edited and revised manuscript; B.A.J., K.M., M.G., S.D.,
S.A., I.L.S, I.S., Y.Y., J.C.A., L.A.C., X.X.W., A.Z.R., and M.L. approved
final version of manuscript.
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F810 AJP-Renal Physiol doi:10.1152/ajprenal.00292.2023 www.ajprenal.org
Downloaded from journals.physiology.org/journal/ajprenal at Virginia Commonwealth Univ (128.172.077.159) on April 18, 2024.
