Abstract
Whether metabolites derived from injured renal tubular epithelial cells
(TECs) participate in renal fibrosis is poorly explored. After TEC
injury, various metabolites are released and among the most potent is
adenosine triphosphate (ATP), which is released via ATP-permeable
channels. In these hemichannels, connexin 43 (Cx43) is the most common
member. However, its role in renal interstitial fibrosis (RIF) has not
been fully examined. We analyzed renal samples from patients with
obstructive nephropathy and mice with unilateral ureteral obstruction
(UUO). Cx43-KSP mice were generated to deplete Cx43 in TECs. Through
transcriptomics, metabolomics, and single-cell sequencing multi-omics
analysis, the relationship among tubular Cx43, ATP, and macrophages in
renal fibrosis was explored. The expression of Cx43 in TECs was
upregulated in both patients and mice with obstructive nephropathy.
Knockdown of Cx43 in TECs or using Cx43-specific inhibitors reduced
UUO-induced inflammation and fibrosis in mice. Single-cell RNA
sequencing showed that ATP specific receptors, including P2rx4 and
P2rx7, were distributed mainly on macrophages. We found that P2rx4- or
P2rx7-positive macrophages underwent pyroptosis after UUO, and in vitro
ATP directly induced pyroptosis by macrophages. The administration of
P2 receptor or P2X7 receptor blockers to UUO mice inhibited macrophage
pyroptosis and demonstrated a similar degree of renoprotection as Cx43
genetic depletion. Further, we found that GAP 26 (a Cx43 hemichannel
inhibitor) and A-839977 (an inhibitor of the pyroptosis receptor)
alleviated UUO-induced fibrosis, while BzATP (the agonist of pyroptosis
receptor) exacerbated fibrosis. Single-cell sequencing demonstrated
that the pyroptotic macrophages upregulated the release of CXCL10,
which activated intrarenal fibroblasts. Cx43 mediates the release of
ATP from TECs during renal injury, inducing peritubular macrophage
pyroptosis, which subsequently leads to the release of CXCL10 and
activation of intrarenal fibroblasts and acceleration of renal
fibrosis.
Subject terms: Extracellular signalling molecules, Cell death
Introduction
Renal interstitial fibrosis (RIF) is a pathological disorder
characterized by the excessive deposition of extracellular matrix
material and the proliferation of fibroblasts in the renal interstitium
[[68]1, [69]2]. RIF has been identified as the common route by which
all forms of chronic kidney disease (CKD) progress to end-stage renal
disease [[70]3]. Once RIF occurs, it is irreversible [[71]4].
Therefore, exploring the mechanism(s) underlying RIF development may
lead to effective therapies to prevent CKD progression, which is an
urgent clinical need.
It has long been accepted that renal tubule injury is the ultimate
endpoint of kidney disease [[72]5]. Moreover, increasing evidence has
shown that renal functional decline is more tightly correlated with
tubular injury than with glomerular injury [[73]6, [74]7]. However,
renal tubular epithelial cells (TECs) are now considered not only the
sites of injury, but also as key pro-inflammatory and fibrogenic cells
that promote the progression from acute to chronic kidney disease
[[75]8, [76]9]. After injury, TECs release pro-inflammatory factors and
recruit inflammatory cells, aggravating kidney damage [[77]9–[78]11].
Thus, preventing such functions by TECs is a potential avenue to delay
RIF.
Gap junction proteins (GJs) are a type of cellular junction channels,
which act as a hemichannel to transmit signals and death information to
neighboring cells [[79]12–[80]14]. GJs are upregulated after kidney
injury in humans and rodents [[81]15–[82]18]. Although GJs have been
reported to be involved in renal disease, our understanding of the
involvement of GJs in renal pathophysiology is limited [[83]19–[84]24].
Among these GJs, connexin 43 (Cx43) is the most common member [[85]25,
[86]26]. It has been identified that the development of cardiac
fibrosis, liver fibrosis, and the acute phase of lung injury in
patients are correlated with Cx43 expression [[87]27–[88]33]. Heqing
Huang et al. demonstrated that the expression of Cx43 prevented the
progression of diabetic kidney disease [[89]34]. However,
Chadjichristos et al. determined that the progression of CKD is delayed
after Cx43 is systemically blocked [[90]15]. So, the role of Cx43 in
renal fibrosis has not been fully identified. Cx43 is widely
distributed in various types of kidney cells. As TECs are essential in
the maintenance of kidney function, the role of Cx43 in TECs attracted
our attention [[91]16, [92]19].
Cx43 can facilitate ATP release in various tissues under physiological
and pathological conditions [[93]35–[94]38]. In this study, we
genetically depleted and pharmacologically inhibited Cx43 to explore
its role in RIF. We found that Cx43 promoted the release of ATP from
injured TECs, which in turn induces the pyroptosis of macrophages
surrounding these epithelial cells, thus activating an inflammatory
reaction that promotes renal fibrosis.
Results
The expression of Cx43 is positively correlated with the severity of renal
injury
Numerous studies have shown that GJs control the flow of metabolites
across the epithelial layer, and thus they play an indispensable role
in renal physiological and pathological process [[95]39–[96]42]. In
this study, we measured the expression of Cx43 in kidneys after UUO and
BIR injuries at different time points (day 1 (D1), day 2, 3, 7, 14 and
28) (Fig. [97]1A). We observed exacerbated tubular injury over time as
well as continuously increased expression of Cx43 located in TECs (Fig.
[98]1B, [99]C). Flow cytometry gate strategies (Supplementary Fig.
[100]S1A, [101]B) and analysis revealed that both the percentage and
count of Cx43-positive cells increased in kidneys after UUO and BIR
(Fig. [102]1D), with a similar tendency as that in immunofluorescence
staining (Fig. [103]1C). This finding was further confirmed by RT-qPCR
and WB (Supplementary Fig. [104]S1C, [105]D, and [106]E).
Fig. 1. The expression of Cx43 correlates with the severity of kidney injury.
[107]Fig. 1
[108]Open in a new tab
A Scheme. time-related comparison after UUO and BIR. B PAS staining.
PAS staining was executed to evaluate renal tubular injury by
calculating damaged tubules. C Immunofluorescence. Cx43-positive
tubules were counted at different points. D The percentage (left) and
number (right) of Cx43-positive tubules analyses by flow cytometry from
the whole kidneys. E Immunofluorescence. The immunofluorescence
co-staining of NCC (a collecting tubule cells specific marker), KSP (a
renal tubular epithelial cell-specific marker), LTL (a proximal tubule
cells specific marker) or DBA (a distal tubule cells specific marker)
with Cx43. Data are presented as the mean ± SEM, n = 4–7/group,
*P < 0.05, **P < 0.01, ***P < 0.001; Scale bar: 100 μm.
As the Cx43 expression and specificity were higher in UUO on Day 7, we
chose the model and the timepoint for further experiments. We detected
the exact location of Cx43 in UUO kidneys. Immunofluorescence staining
of LTL (a proximal tubule cells specific marker) and DBA (a distal
tubule cells specific marker) along with Cx43 showed no regions of
co-staining. The co-staining of NCC (a collecting tubule cells specific
marker) or KSP (a renal tubular epithelial cell-specific marker) with
Cx43 presented co-localization (Fig. [109]1E), implying that Cx43 is
located in the collecting duct segment.
Pharmacological blockade of Cx43 hemichannel attenuates UUO-induced renal
injury and RIF
To further understand the role of Cx43 in renal injury and fibrosis, we
applied the specific Cx43 hemichannel blockers, GAP26 after UUO surgery
(Fig. [110]2A). RT-qPCR showed that the expression of α-SMA and of
fibronectin in the GAP26-treated group was significantly lower compared
to the untreated UUO group (P < 0.05) (Fig. [111]2B). PAS staining
showed that there were massive renal tubule expansion, along with
epithelial cell brush border loss, in the UUO group, while in the
UUO + GAP26 group the number of injured tubules was lower (P < 0.05).
Immunofluorescence staining of α-SMA, a marker of myofibroblasts and
fibrosis, showed that numerous α-SMA-positive cells appeared in the
renal interstitium in the UUO group, while in the GAP26-treated groups
the number of α-SMA-positive cells was lower (P < 0.05) (Fig. [112]2C).
These results suggest that blocking Cx43 hemichannel prevents
UUO-induced renal injury and RIF.
Fig. 2. Cx43 hemichannel blocker attenuates kidney injury and RIF after UUO.
[113]Fig. 2
[114]Open in a new tab
A Scheme. mice were treated with GAP26 by i.p. injection after UUO
surgery. B RT-qPCR. Levels of mRNA encoding α-SMA, Fibronectin by
RT-qPCR. C PAS and immunofluorescence staining. PAS staining was
performed to evaluate renal tubular injury by calculating tubules about
GAP26. α-SMA is a marker of myofibroblast. Corresponding graphs
indicate the expression of α-SMA for evaluation of myofibroblast
infiltration. Data are presented as the mean ± SEM, n = 4–7/group,
*P < 0.05, **P < 0.01, ***P < 0.001; Scale bar: 100 μm.
Cx43 gene knockdown in TECs prevents UUO-induced renal injury and RIF
Cx43 plays an important role in normal physiology, and thus systemic
depletion of Cx43 expression causes serious complications, such as
arrhythmia [[115]15, [116]43, [117]44]. Therefore, to clarify the role
of Cx43 in renal injury and fibrosis, we generated tubular-specific
Cx43 knockout mice Cx43^fl/fl,Ksp-icre named C-K mice (Supplementary
Fig. [118]S2A, [119]B). The morphology of organs excluding kidney
showed no pathological change between the UUO and C-K UUO groups
(Supplementary Fig. [120]S2C). In vivo, Cx43 expression was knocked
down by injection of tamoxifen, and then these mice were subjected to
UUO surgery (herein, referred to as C-K UUO mice). We isolated primary
TECs (pTECs) from C-K UUO mice and wild type mice injected by tamoxifen
with UUO or not. RT-qPCR found the expression of Cx43 in the C-K UUO
mice was significantly lower compared to the UUO. (Fig. [121]3A).
Transforming growth factor β (TGF-β) has been identified as the main
inducer of epithelial-to-mesenchymal transition and thus fibrosis in
the kidney [[122]45]. In vitro, the pTECs from C-K mice were cultured
with 4-OHT, an active form of tamoxifen, to knockdown Cx43, and
simultaneously treated with TGF-β. We found the most effective
concentration of 4-OHT was 10 μM, in which the expression of Cx43 was
lower compared with the TGF-β group (Fig. [123]3B). These data further
confirmed the successful construction of Cx43^fl/fl,Ksp-icre mice. PAS
staining showed that in the C-K UUO group the number of injured tubules
and the tubular damage score per high-power filed (HPF) were lower.
Sirius Red (SR) staining showed the percentage of the deposition of
fibrotic collagen in the whole field was lower in the C-K UUO group
(4.14% HPF) compared to the UUO group (8.67% HPF) (Fig. [124]3C). After
intraperitoneal injection of tamoxifen, the expression of Cx43 showed
about a 72.67% ((25-6.83)/25%) reduction in the C-K UUO group compared
with the UUO group, implying successful knockdown of Cx43 expression in
TECs (Fig. [125]3D and Supplementary Fig. [126]S2D). Next, we stained
for α-SMA and found an 8.86% α-SMA-positive area in the UUO group
compared to 5.16% in the C-K UUO group. Fibronectin staining showed a
similar tendency, in which the percentage of the fibronectin-positive
area declined from 7.26% in the UUO group to 4.76% in the C-K UUO group
(Fig. [127]3D). RT-qPCR confirmed the down-regulated expression of
renal fibrosis-related genes, including PDGFR-β, α-SMA, and Collagen
Iα1, in the C-K UUO group compared to the UUO group (Fig. [128]3E).
These data suggest that Cx43 gene knockdown in TECs prevents
UUO-induced renal injury and renal fibrosis.
Fig. 3. Knockout the Cx43 gene of TECs prevents UUO-induced renal injury and
RIF.
[129]Fig. 3
[130]Open in a new tab
A, B Experiment schematic and Levels of mRNA encoding Cx43 by RT-qPCR.
C PAS staining was performed to evaluate renal tubular injury by
calculating damaged tubules with dilation swelling or epithelial cell
brush border loss. The red area in SR staining indicates extracellular
fibrotic collagen deposition in renal interstitium. D
Immunofluorescence. Representative photomicrographs for
immunofluorescence labeled Cx43 (green), α-SMA (red), and FN (green).
Corresponding graphs indicate the expression of Cx43 (green), α-SMA
(red) and FN (green). E RT-qPCR. Levels of mRNA encoding α-SMA,
Fibronectin, Collagen Iα1 by RT-qPCR. Data are presented as the
mean ± SEM, n = 3–6/group, *P < 0.05, **P < 0.01, ***P < 0.001; Scale
bar: 100 μm.
Knockdown of Cx43 expression influences the purine metabolism pathway in TECs
To explore the mechanism by which Cx43 promotes renal injury and
fibrosis, we extracted pTECs from Sham, UUO and C-K UUO mice for
transcriptome RNA sequencing (RNA-seq). The whole RNA-seq analysis
showed that pTECs from the C-K UUO group had 3604 down-regulated genes
compared to the UUO group (Fig. [131]4A). These genes were mostly
involved in purine metabolism by KEGG-pathway enrichment analysis (Fig.
[132]4B). We further analyzed the metabolome of urine samples and also
found that the differential metabolites between the UUO group and C-K
UUO group were mostly related to purine metabolism (Fig. [133]4C–F).
Furthermore, Cx43 knockdown reduced the levels of purine metabolites in
the urine, suggesting Cx43 participates in the regulation of purine
metabolism in TECs. As a hemichannel, Cx43 responds to various external
stimuli by mediating the release of adenosine triphosphate (ATP),
glutamate, NAD+ and prostaglandin E2, in which ATP is the major source
of purine-related metabolites [[134]18, [135]23, [136]46, [137]47].
Therefore, as Cx43 expression is associated with worse UUO-induced
renal fibrosis and the main metabolite released from the injured TECs
is ATP, we hypothesized that this release is a major mechanism by which
Cx43 promotes RIF.
Fig. 4. Transcriptomics and Metabolomics of C-K UUO mice revealed that
knockout of Cx43 gene mainly affects the accumulation of purine substances in
TECs.
[138]Fig. 4
[139]Open in a new tab
A Differentially expression genes. B KEGG-pathway enrichment analysis
of C-K UUO/UUO downregulated DEGs. C Principal component analysis (PCA)
score plot. D Network analysis of differential products between C-K UUO
and UUO group. E Heatmap of selected enriched terms (FDR ≤ 0.01) from
KEGG-pathway analysis of downregulated DEGs in C-K UUO group and
upregulated DEGs in UUO group. F The bubble plot shows that the main
difference between UUO and C-KUUO group is purine metabolites.
The Cx43 hemichannel on TECs regulates ATP outflow for binding to P2X7
receptor participates in renal fibrosis
To explore this possibility further, we extracted pTECs from the UUO,
C-K UUO and UUO + GAP26 groups and then measured their intracellular
ATP concentrations (Fig. [140]5A). We found that compared to the Sham
group, the intracellular ATP concentration was lower in the UUO group,
while the concentration was higher in the C-K UUO group compared to UUO
group, implying knockdown of Cx43 in TECs prevented the outflow of ATP
to the extracellular space. A similar change was observed after using
GAP26 (Fig. [141]5B), further suggesting the outflow of ATP in TECs is
dependent on Cx43.
Fig. 5. The Cx43 hemichannel of TECs mediate ATP outflow and ATP receptor
analysis.
[142]Fig. 5
[143]Open in a new tab
A, B Detection of intracellular ATP content in TECs. C scRNAseq
identified clusters of cells in the Sham and UUO kidneys after surgery
at day 7. UMAP plot representation of 30,788 kidney cells, including
20,590 cells from UUO kidney and 10,198 cells from Sham kidney. DotPlot
(D, E), UMAP (F) and violin plots (G) depicted genes related to ATP
receptor and pyroptosis. Data are presented as the mean ± SEM,
n = 4–7/group, ***P < 0.001.
Previous reports showed that ATP mainly binds to purinergic receptors
on the surface of target cells to mediate cell proliferation,
migration, differentiation and death [[144]48–[145]51]. According to
the scRNA-seq cell clustering and marker genes, we divided all cells
into six groups: TECs, macrophage & dendritic cells, lymphocytes,
endothelial cells, neutrophils, and stromal cells (Fig. [146]5C). ATP
receptors include P1 and P2 (P2X and P2Y). The P2X receptor is an
ATP-specific receptor, including P2X1 to P2X7 [[147]52]. Among P2X1 to
P2X7, we observed that P2X4 and P2X7 are the ones most highly expressed
in the kidney, mainly in renal macrophages (Fig. [148]5D). As the
activation of P2 receptors is related to the activation of
inflammasomes and pyroptosis, we analyzed pyroptosis-related genes and
found that they were mainly located in macrophages (Fig. [149]5E).
Jeffrey John Bajramovic et al. suggested that ATP-induced IL-1β
secretion in BMDM was fully dependent on P2X7 signaling [[150]53]. From
UMAP and VlnPlot plots, we also found that the P2X7 receptor gene was
specifically upregulated in macrophages after UUO (Fig. [151]5F,
[152]G). Thus, we assumed that ATP-associated macrophage pyroptosis
might play an important role in renal fibrosis via ATP outflow from
TECs.
To explore the effect of tubular ATP outflow and its receptor
activation on renal injury and fibrosis, we used a P2 receptor
inhibitor (suramin) [[153]54] or a P2X7 receptor inhibitor (A-839977)
[[154]55] (Supplementary Fig. [155]S3A). PAS and SR staining showed
that compared with the UUO group, the tubular damage score was lower
and fibrotic area was larger in the UUO + A839977 or +suramin group
(Supplementary Fig. [156]S3B, C). In addition, we found that treatment
with a P2X7 receptor agonist (BzATP) [[157]56] induced worse
UUO-induced renal injury compared with the untreated UUO group
(Supplementary Fig. [158]S3D–F). These data indicate that the P2X7
receptor participates in UUO-induced renal fibrosis.
The ATP outflow from TECs increases macrophage pyroptosis
A recent study reported that ATP activates inflammasomes after binding
to P2X7 receptors [[159]57]. In addition, it has been suggested that
the activation of inflammasomes is closely related to immune cell
pyroptosis [[160]58]. In Fig. [161]5E, we observed the genes related to
the inflammasome (Nlrc4, Nlrp3, Aim2, Casp1) and pyroptosis (Gsdmd,
Il18) were mainly expressed in macrophages. To investigate the specific
effects of ATP on the macrophage pyroptosis, we induced cellular
pyroptosis in bone marrow-derived macrophages (BMDMs) by stimulating
them with LPS and ATP (Fig. [162]6A). Pyroptosis was observed by
scanning electronic microscopy, as marked by characteristic swelling,
nuclei concentration and the emergence of vesicle-like pyroptotic
bodies, in LPS + ATP-treated cells (Fig. [163]6B). WB analysis showed
that NLRP3, Caspase-1, GSDMD-N and GSDMD-FL (GSDMD-full length) were
markedly activated after LPS + ATP stimulation. However, these proteins
were not significant changes when BMDM cells were stimulated with LPS
or ATP alone (Fig. [164]6C). Immunofluorescence staining demonstrated
that LPS + ATP induced the translocation of GSDMD-N towards the plasma
membranes, forming pyroptotic bodies and activated cell pyroptosis
(Fig. [165]6D).
Fig. 6. The ATP outflow from TECs increase macrophage pyroptosis via P2
receptor.
[166]Fig. 6
[167]Open in a new tab
A Scheme. BMDMs were primed with or without LPS (500 ng/ml) for 4 h,
followed by stimulation with ATP (5 mM) for 6 h. B Representative
scanning electronic microscopy (SEM) images of BMDM cells treated with
LPS and ATP. Yellow arrow points to bubbling of pyroptotic cells. Scale
bar, 10 µm. C LPS with or without ATP induction upregulated the protein
expression of NLRP3, caspase1. LPS with ATP upregulated the protein
expression of GSDMD-N/GSDMD-(FL + N). Corresponding graphs indicate the
expression of NLRP3, caspase1, GSDMD-N/GSDMD-(FL + N). D–F
Immunofluorescence. BMDMs Representative photomicrographs for
immunofluorescence labeled GSDMD and DAPI of BMDMs indicated that LPS
with ATP could active macrophage pyroptosis (D) and the pyroptotic
cells reduced after knockout the Cx43 gene by immunofluorescence
labeled GSDMD, F4/80 and DAPI (E). The pyroptotic macrophages were
mainly concentrated around Cx43-positive TECs (F). Data are presented
as the mean ± SEM, n = 3–6/group, *P < 0.05, **P < 0.01, ***P < 0.001;
Scale bar: 100 μm.
Next, we extracted pTECs from mice in the Sham group and the C-K group,
and co-cultured them with BMDMs. The extracellular ATP concentrations
of BMDM were regulated by an ATP analog (ATPγS) or an ATP-depleting
agent (Apyrase). RT-qPCR revealed that the expression of the pyroptosis
related gene IL-1β in the BMDMs was higher when the extracellular ATP
concentrations increased and lower as the extracellular ATP
concentrations dropped (Supplementary Fig. [168]S4). Immunofluorescence
co-staining of Cx43 or F4/80 with GSDMD showed that GSDMD distributed
mainly around Cx43-positive tubules in the UUO kidneys, and the F4/80-
and GSDMD-double positive cells were lower in the C-K UUO kidney,
implying that Cx43 depletion in TECs reduced peritubular macrophage
pyroptosis (Fig. [169]6E, [170]F). WB analysis showed that NLRP3,
caspase-1, ASC-1 and GSDMD-N were markedly reduced in the C-K UUO
kidneys (Supplementary Fig. [171]S5). Therefore, the outflow of ATP
from TECs induced the activation of macrophage inflammasomes and
triggered macrophage pyroptosis after UUO.
The expression of Cx43 and GSDMD are associated with renal function decline
in human obstructive nephropathy
We detected the expression of Cx43 and GSDMD in human kidney diseases.
Immunohistochemical staining showed greater expression of tubular Cx43
in renal biopsy specimens from individuals diagnosed with obstructive
nephropathy and lupus nephritis (Fig. [172]7A). GSDMD was observed to
be expressed in renal interstitial cells with obstructive nephropathy.
Neither Cx43 nor GSDMD were found in normal kidney specimens. Further,
we divided individuals into two groups: those with an eGFR
<90 ml/min/1.73 m^2 and those with an eGFR ≥90 ml/min/1.73 m^2 group.
Peritubular GSDMD high cell was defined as the number of GSDMD-positive
cells ≥50th percentiles. Individuals in the eGFR <90 ml/min 1.73 m^2
group showed higher levels of Cx43 (P < 0.015) and GSDMD (P < 0.041) at
the time of biopsy than the other group (Fig. [173]7B). Univariate and
multivariate binary logistic regression indicated that appearance of
Cx43 in TECs and GSDMD in interstitial cells were independent risk
factors for the decline of renal function in subjects with obstructive
nephropathy (Tables [174]1, [175]2).
Fig. 7. The expression of Cx43 and GSDMD genes was associated with renal
function in human obstructive nephropathy.
[176]Fig. 7
[177]Open in a new tab
A The representative image of Cx43 immunohistochemistry in TECs in
human renal obstruction disease and lupus nephritis. B The
representative image of GSDMD immunohistochemistry in GSDMD low group
and GSDMD high group. Data are presented as the mean ± SEM, n.s. no
significance, ***P < 0.001; Scale bar: 100 μm.
Table 1.
Obstructive nephropathy patients in the Cx43 positive group and GSDMD
high group showed a decline of kidney function.
Variables All (n = 48) eGFR ≥ 90 ml/min 1.73 m^2 (n = 16)
eGFR < 90 ml/min 1.73 m^2 (n = 32) P
Gender male, n% 23/48 (47.9%) 5/23 (21.7%) 18/23 (78.3%) 0.102^c
Age, years 48.12 ± 15.11 40.63 ± 13.35 52.75 ± 13.05 0.004^a
WBC, 10^9/L 9.84 ± 3.24 10.16 ± 3.49 9.71 ± 3.21 0.658^a
N, 10^9/L 7.90 ± 3.27 8.15 ± 3.55 7.84 ± 3.18 0.759^a
L, 10^9/L 1.17 (0.74-1.53) 1.01 (0.70-1.77) 1.21 (0.7-1.52) 0.702^b
M, 10^9/L 0.63 (0.5-0.8) 0.68 (0.59-0.87) 0.57 (0.41-0.78) 0.137^b
Hb, g/L 121.44 ± 16.02 117.31 ± 17.91 123.53 ± 14.45 0.201^a
Platelets, 10^9/L 191 (157-229) 195.5 (152.75-258.5) 189 (157-220.75)
0.519^b
Na^+, mmol/L 139.3 ± 2.60 138.53 ± 2.24 139.71 ± 2.75 0.144^a
K^+, mmol/L 4.06 ± 0.39 3.96 ± 0.27 4.12 ± 0.44 0.186^a
Cl^-, mmol/L 103.84 ± 2.98 103.53 ± 3.37 103.97 ± 2.86 0.642^a
Ca^2+, mmol/L 2.18 ± 0.13 2.21 ± 0.11 2.16 ± 0.13 0.227^a
P, mmol/L 1.05 ± 0.23 1.09 ± 0.19 1.03 ± 0.25 0.507^a
ALT, U/L 11 (8–21) 10 (6–16) 12 (9-23.50) 0.130^b
AST, U/L 16 (13.5-21) 15 (12.5-16) 18.5 (15-21.5) 0.023^b
TP, g/L 62.87 ± 5.81 61.63 ± 4.52 63.09 ± 6.02 0.396^a
ALB, g/L 36.54 ± 3.90 35.89 ± 3.56 36.53 ± 3.69 0.569^a
GLB, g/L 26.40 ± 3.41 25.96 ± 2.50 26.56 ± 3.83 0.573^a
Chol, mmol/L 3.54 (3.11-4.12) 3.96 (3.33-4.47) 3.46 (3.08-3.96) 0.068^b
UA, μmol/L 302.20 ± 82.357 273.73 ± 76.20 316.25 ± 84.08 0.096^a
Proteinuria positive, n% 13/48 (27%) 3/13 (23.1%) 10/13 (76.9%) 0.358^c
Hematuria positive, n% 12/48 (25%) 4/12 (33.3%) 8/12 (66.7%) 1.000^c
pyuria positive, n% 8/48 (16.7%) 1/8 (12.5%) 7/8 (87.5%) 0.171^c
Cx43 positive, n% 37/48 (77.1%) 9/37 (24.3%) 28/37 (75.7%) 0.015^c
GSDMD high, n% 25/48 (52.1%) 5/25 (20%) 20/25 (80%) 0.041^c
[178]Open in a new tab
The bold values indicate P < 0.05.
Data are presented as mean ± SD or median (25–75th percentiles) or a
percentage.
Peritubular GSDMD high cell was defined as the number of GSDMD positive
cells ≥50th percentiles.
WBC White blood cell, N Neutrophils, L Lymphocytes, M Monocyte, TP
Total protein, ALB Albumin, GLB Globulin, Chol Cholesterol, UA Uric
acid.
^at-test, ^bMann–Whitney U test, ^cPearson’s chi-squared test.
Table 2.
Risk factors associated with eGFR <90 ml/min/1.73 m^2 during follow-up
periods.
Variables Univariate analysis Multivariate analysis
OR 95%CI P OR 95%CI P
Gender male 2.829 (0.797–10.042) 0.108
Age, years 1.07 (1.017–1.126) 0.009 1.104 (1.027–1.186) 0.007
Cx43 in TECs (positive vs negative) (n = 48) 5.444 (1.290–22.976) 0.021
10.388 (1.439–74.997) 0.020
GSDMD in interstitial cells (high vs low)^a (n = 48) 3.667
(1.023–13.143) 0.046 6.929 (1.189–40.374) 0.031
[179]Open in a new tab
The bold values indicate P < 0.05.
^aGSDMD (GSDMD high was defined as the number of GSDMD positive cells
≥50th percentiles, 1, GSDMD positive cells ≥50th percentiles; 0, GSDMD
positive cells <50th percentiles).
Pyroptotic macrophage-derived CXCL10 aggravates the progression of
UUO-induced renal fibrosis
To explore the mechanism by which macrophage pyroptosis induces renal
fibrosis, we divided macrophages into two groups: the GSDMD-negative
and the GSDMD-positive groups, and analyzed them by scRNA-seq. The
fibrosis-related chemokine CXCL10 ranked among the top three of
differential genes (Fig. [180]8A). It has been reported that CXCL10
plays a key role in wound healing, pulmonary fibrosis, and liver
fibrosis [[181]59–[182]61]. During kidney fibrosis, the
transdifferentiation of fibroblasts into myofibroblasts is an
indispensable process. In order to confirm whether the GSDMD-positive
macrophages were more likely to communicate with fibroblast. We
calculated the attraction strengths of ligand-receptor pairs in our
scRNA-seq dataset by using a simulation analysis similar to previous
methods [[183]62, [184]63]. Of the ligand receptor pairs pertaining to
macrophage and stromal cell (including fibroblast), CXCL10-CXCR3 was
significantly enriched in GSDMD-positive macrophages compared to
GSDMD-negative macrophages in UUO kidneys (Fig. [185]8B), implicating a
potential role of GSDMD-positive macrophage in recruiting or activating
fibroblasts. Thus, we cultured NIH3T3 cells with CXCL10 in vitro and
found that CXCL10 aggravated fibrosis. This result implies that CXCL10
from GSDMD-positive macrophages participates in the activation of
fibroblasts (Fig. [186]8C,[187]D).
Fig. 8. CXCL10 can aggravate the progression of obstructive nephropathy.
[188]Fig. 8
[189]Open in a new tab
A One of the top 3 differential genes between the GSDMD positive
macrophage and GSDMD-negative macrophage was CXCL10 by scRNA-seq. B
Bubble heatmap showing the mean attraction strength for selected
ligand-receptor pairs between the GSDMD-positive/negative macrophage
with Stromal cell. Dot size indicates P-value generated by permutation
test, colored by attraction strength levels. C Scheme. D Levels of mRNA
encoding Fibronectin, collagen I1α by RT-qPCR. E Representative overall
photo and photomicrographs of PAS staining. F Immunofluorescence.
Representative photomicrographs for immunofluorescence labeled α-SMA
(red) and FN (green). Corresponding graphs indicate the expression of
α-SMA (red) and FN (green). Data are presented as the mean ± SEM,
n = 5/group, **P < 0.01, ***P < 0.001; Scale bar: 100 μm.
Next, we applied CXCL10 to UUO mice. We found that this treatment
resulted in more serious tubular injury and fibrosis compared to the
untreated UUO group (Fig. [190]8E, F). These data demonstrate that
macrophage-derived CXCL10 after pyroptosis accelerates UUO-induced
renal fibrosis.
Discussion
The expression and distribution of GJs have been reported to have a
significant impact on various diseases [[191]20, [192]22, [193]25,
[194]64]. In this study, we demonstrated that Cx43-positive TECs
communicate with macrophages through release of ATP and promotion of
renal fibrosis, while inhibiting or blocking Cx43 improved renal
structure and function.
The expression and distribution of Cx43 has been reported in various
inflammatory diseases regulating the progression of inflammatory injury
[[195]20, [196]65–[197]67]. Renal tubular epithelial cell injury is one
of the key causes of renal fibrosis. Injured TECs aggravate renal
fibrosis by releasing inflammatory factors, chemokines, and exosomes
[[198]9]. In addition to exosomes and exocytosis, some metabolites are
directly released through specific channels, such as GJ channels. Among
these, Cx43 is the most widely expressed and the best studied [[199]25,
[200]26]. Cx43 expression has a strong correlation with myocardial
fibrosis, liver fibrosis, and pulmonary fibrosis [[201]32,
[202]68–[203]71], but it has been less studied in renal fibrosis.
Heqing Huang et al. demonstrated that the expression of Cx43 prevented
the progression of diabetic RIF [[204]34]. However, Chadjichristos et
al. determined that the progression of CKD is delayed after Cx43 is
systemically blocked [[205]15]. Therefore, the role of Cx43 on the
kidney is controversial and worthy of study. In the kidney, Cx43 is
distributed on various types of kidney cells [[206]16, [207]19,
[208]72, [209]73]. Here, we showed that Cx43 expression is higher on
collecting duct cells after renal injuries. To investigate whether Cx43
mediated renal fibrosis, we took advantage of gene editing technology
to construct Cx43-KSP mice. We found that knockout of Cx43 on TECs
alleviates renal injury and fibrosis. Through transcriptome and
metabolomics analysis, we confirmed that Cx43 aggravates renal fibrosis
by mediating ATP outflow. It has been demonstrated that extracellular
ATP acting as a damage associated molecular pattern (DAMP) in various
diseases is sensed by P2X receptors where its activation mainly
promotes the formation of inflammasomes and the initiation of
pyroptosis [[210]74–[211]76]. Our previous findings showed that
pyroptosis after kidney injury mainly occurs in macrophages [[212]77].
By scRNA-seq, we found that the P2X receptor in the injured kidney is
mainly distributed on macrophages and participates in macrophage
pyroptosis. This result is consistent with previously reported results
[[213]57]. We further confirmed here that Cx43 activates renal
macrophage pyroptosis by facilitating the release of ATP from
neighboring TECs, which in turn binds to the P2X receptor on
macrophages.
Previous studies have shown that macrophage pyroptosis mainly promotes
inflammation. By using scRNA-seq, we found that CXCL10 is one of the
top three differentially expressed genes between GSDMD-positive and
GSDMD-negative macrophages after UUO. Previous reports have shown that
CXCL10 is strongly positively correlated with the progression of organ
fibrosis, such as skin, liver, and lung [[214]59–[215]61]. Our study
confirmed that CXCL10 directly activates the proliferation of
fibroblasts. Thus, the present study provides new insight into the
mechanism of renal fibrosis.
While pharmacological agents that specifically target the hemichannel
function of on TECs is not available, by using nanotechnology to embed
Cx43 hemichannel blockers into nanomaterials with renal tubular
specific targeting it is possible that such a future therapy may be
developed. In spite of this current limitation, our study provides
important new insight as it provides evidence for the protective effect
of targeting tubular Cx43 on renal fibrosis via inhibiting ATP outflow
and the resulting macrophage pyroptosis. Together, this insight
provides a novel mechanism and a potential therapeutic strategy to
prevent renal fibrosis.
Methods
Animal model
All mice were kept in well-controlled animal housing facilities with a
12 h day and night cycle, had free access to tap water and pellet food
in accordance with the Experimental Animal Ethics Committee of Huazhong
University of Science and Technology. Male C57BL/6 mice (8–10 weeks
old, weighing 22–25 g, Vital River Laboratory, Beijing, China) were
anaesthetized with 1% sodium pentobarbital solution (0.009 ml/g, Sigma,
USA) by intraperitoneal injection. The animal genotype was not known
when the animal model was constructed and the medicine was
administered. Finally, it was judged by the mouse ear mark.
Unilateral ureteral obstruction (UUO) model: The left ureter was
ligated with 4-0 silk at 2 points, close to the renal pelvis. Then the
muscle layer and skin were closed with 4-0 silk. Sham animal models
were subjected to a similar surgical procedure without ligating the
left ureter.
Bilateral ischemia-reperfusion injury (BIR) model: both renal pedicles
were clamped with an atraumatic vascular clip for 30 min (Roboz
Surgical Instrument Co, Germany). The technical success of
ischemia-reperfusion was checked by observing the kidney color after
clamping and after removing the clamps. The body temperature was
controlled at 36.6–37.2 °C by a sensitive rectal probe throughout the
procedure (FHC, USA). Then, the muscle layer and skin were closed with
4-0 silk. Sham operations were performed with exposure of the left
kidney, but without induction of ischemia.
All animals were housed in the specific pathogenfree laboratory animal
center of Huazhong University of science and technology ([2017] IACUC
Number: 2471).
Cx43-KSP gene mice and chemical
Cx43-loxp mice were purchased from Jackson Laboratory, USA. KSP-icreERT
mice were developed in cooperation with Beijing Biocytogen company
Corporation using CRISPR/Cas9 technology. Cx43-KSP gene mice were
generated by crossing Cx43-loxp gene mice and KSP-icreERT mice to
deplete the Cx43 gene of renal tubular epithelial cells. In vivo
experiment, we used different concentrations of 4-OHT to induce Cx43
knockout and found 10 μM was the best dose. The control mice were for
Cx43-loxp or KSP-icreERT mice. A rang doses of 0.01 μg/μl, 0.02 μg/μl,
0.04 μg/μl were injected into the mice with GAP26 for 8 days.
In vitro-based model
In vitro, the pTECs from C-K mice were cultured with 4-OHT, an active
form of tamoxifen, to knockdown Cx43, we used a rang doses of 0.1 μM,
1 μM, 10 μM, and simultaneously treated with TGF-β (10 ng/ml) for 12 h.
Cellular pyroptosis model: By stimulating the BMDM cells with 5 mM ATP
alone for 8 h, or with 500 ng/ml LPS alone for 4 h and then with 5 mM
ATP alone for 8 h [[216]78].
Histology and immunofluorescence
Periodic acid-schiff (PAS) staining was used to evaluate kidney
pathological injury, and Sirius Red (SR) staining were carried out to
estimate the extent of tubular interstitial fibrosis.
Immunofluorescence (IF) renal sections were dewaxed in a constant
temperature oven and subjected to heat antigen retrieval in a microwave
oven. The nonspecific antigens were blocked with serum for 30 min at
room temperature. The slides were then incubated with specific primary
antibodies against Cx43 (1:100, Sigma, USA), KIM-1 (1:1000, R&D system,
USA), LTL (1:50, Vector Laboratories, USA), NCC (1:100, Millipore,
USA), DBA (1:300, Vector Laboratories, USA), KSP (1:100, Proteintech,
China), α-SMA (1:100, Abcam, UK), Fibronectin (1:100, R&D system, USA),
GSDMD (1:300, Santa cruz, USA) and F4/80 (1:100, Abcam, UK) at 4 °C for
24 h. Then labeling fluorescent secondary antibodies for IF. Nuclei
were stained with DAPI. Staining was carefully quantified in each slide
by capturing ten randomly visions in a blind manner by two experienced
renal pathologists and the data was analyzed by Image Pro Plus software
(Media Cybernetics, Rockville, MD, USA).
Western blotting
Renal tissues were lysed in RIPA lysis buffer (Promoter, Wuhan, China)
containing protease inhibitors (Promoter, Wuhan, China). Equal amounts
of proteins (40 μg) were loaded and separated by SDS-PAGE. The gel was
transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The
membranes were blocked with 5% skimmed milk in TBST for 1 h at 37 °C
and were then incubated with primary antibodies against Cx43 (1:1000,
sigma, USA), GAPDH (1:5000, Abclonal, China), NLRP3 (1:1000, CST, USA),
ASC-1 (1:1000, CST, USA), Caspase-1 (1:1000, CST, USA) and GSDMD
(1:1000, Santa cruz, USA) at 4 °C overnight. The PVDF membranes were
incubated with HRP-conjugated secondary antibodies for 1 h at 37 °C and
were visualized by enhanced chemiluminescence (ECL, Biosharp, China).
The signal intensity of the targeted band was quantified using Image J
(NIH, USA).
Quantitative real time-PCR
Total RNA was extracted from renal tissues using Trizol reagent
according to the manufacturer’s instructions (Invitrogen, USA). Reverse
transcribed into first strand cDNA using the reverse transcription
system (Vazyme, Nanjing, China). Quantitative PCR was conducted using
the SYBR mastermix (Vazyme, Nanjing, China) on the ABI Step-One.
Relative mRNA expression levels were calculated using the 2^−ΔΔCt
method and normalized to the expression levels of GAPDH. The primers
used are listed in the table below.
Gene name Forward Reverse
GAPDH 5′-TGACCTCAACTACATGGTCTACA-3′ 5′-CTTCCCATTCTCGGCCTTG-3′
Cx43 5′-ACAAGGTCCAAGCCTACTCCA-3′ 5′-CCGGGTTGTTGAGTGTTACAG-3′
α-SMA 5′-CCCAGACATCAGGGAGTAATGG-3′ 5′-TCTATCGGATACTTCAGCGTCA-3′
Fibronectin 5′-GCTCAGCAAATCGTGCAGC-3′ 5′-CTAGGTAGGTCCGTTCCCACT-3′
PDGFR-β 5′-AGGAGTGATACCAGCTTTAGTCC-3′ 5′-CCGAGCAGGTCAGAACAAAGG-3′
Collagen Ia1 5′-GCTCCTCTTAGGGGCCACT-3′ 5′-CCACGTCTCACCATTGGGG-3′
IL-1β 5′-GTGGCTGTGGAGAAGCTGTG-3′ 5′-GAAGGTCCACGGGAAAGACAC-3′
[217]Open in a new tab
Relative mRNA expression levels were quantified according to the
2^−ΔΔCt method and were normalized to the expression levels of GAPDH.
Flow cytometry
Mouse kidney tissue was chopped and digested with Collagenase-IV
(Promoter, Wuhan, China) at 37 °C for 1 h. The suspension was filtered
through a 200 mesh and a 400 mesh filter cloth to generate a
Single-cell suspension after lysis of red blood cells (BD, USA). The
cells were then incubated with the following fluorescent antibodies for
30 min shielded from light at room temperature: FITC-conjugated
anti-CD45 (Biolegend, USA), APC-conjugated anti-KSP (Novus, USA),
PE-conjugated anti-Cx43 (Santa Cruz, USA), Zombie Violet™ Fixable
Viability Kit (Biolegend, USA). The cells were sorted using a Beckman
CytoFLEX and the data were analysed using the CytoExpert for DxFLEX
software. We added 10 µL of precision-count beads to each sample before
sorting cells. The absolute cell count was calculated by using the
following formula: Absolute Cell Count =
[MATH: CellsA^μL=CellCountPrecision−CountBeadCount :MATH]
x Precision-Count Bead Concentration
[MATH: (BeadsA^μL)
:MATH]
.
Transcriptome sequencing and bioinformatics analysis
Total RNA was obtained from TECs using Trizol reagent (Invitrogen,
USA). The total RNAs were subjected to cDNA synthesis, fragmentation,
adapter ligation, and amplification. Sequencing was performed on
Illumina HiSeq platform. The sequencing reads were further processed
with determination of quality using the SOAPnuke tool. We mapped clean
reads to mouse mm9 genome using HISAT (Hierarchical Indexing for
Spliced Alignment of Transcripts). The fragments per kilobase million
(FPKM) and differential expression genes (DEGs) were obtained using
RSEM and DEGseq software, respectively (Fold Change ≥2 and Adjusted P
value ≤ 0.001). R package was used for generation of GO and KEGG
analysis.
Metabolomics
Metabolomics project data analysis based on Ultra High Performance
Liquid Tandem Chromatography Quadrupole Time of Flight Mass
Spectrometry, UHPLC-QTOFMS is mainly divided into three parts: basic
data analysis, advanced data analysis and optional data analysis. Basic
data analysis is to carry out univariate statistical analysis and
multivariate statistical analysis of the qualitative and quantitative
results of the metabolome, and to screen the metabolites with
significant differences; optional data analysis is based on the basic
data analysis to conduct significant differences in metabolites Series
of bioinformatics analysis. The R packages include bubble plot: ggplot2
ggrepel, heatmap: pheatmap, PCA: ggplot2, network: igraph ggraph.
Commercial databases including KEGG ([218]http://www.genome.jp/kegg/)
and MetaboAnalyst ([219]http://www.metaboanalyst.ca/) were used for
pathway enrichment analysis.
10× Genomics
Single cells in nanoliter-scale oil droplets by Chromium Controller and
to generate Gel Bead-In-EMulsions (GEMs). Full length cDNA libraries
were prepared by incubation of GEMs in a thermocycler machine. GEMs
containing cDNAs were crushed and all single-cell cDNA libraries were
collected together, cleaned using DynaBeads MyOne Silane beads (PN
37002D; Fisher). The final constructed single-cell libraries were
sequenced by Illumina Novaseq6000 machine with total reads per cell
targeted, for a minimum of 50,000.
Analysis of single-cell RNA-seq (scRNA-seq) data
Dimension reduction
Principal component analysis was then performed on significantly
variable genes. The variable genes were selected based on dispersion of
binned variance to mean expression ratios using Find Variable Genes
function of Seurat package and removed unwanted cells. Then the
appropriate number principal components were selected as input for
clustering. We performed dimension reduction using gene expression data
for a subset of variable genes.
Determining cluster markers
Differential gene expression testing was performed using the Find
Markers function in Seurat with parameter “test.use = wilcox” by
default and the Benjamini-Hochberg methods was used to estimate the
false discovery rate (FDR). DEGs were filtered using a minimum natural
log (fold change) of 0.25 and a maximum FDR value of 0.05.
Basic information of clinical obstructive nephropathy patients collected from
Tongji Hospital
A total of 48 patients were included in the study who were diagnosis
obstructive nephropathy by ultrasonography and relieve the obstruction
through surgery. We obtained the related data from electronic medical
records and the paraffin section of obstruction nephrology patients
from ward of department of urology, Tongji Hospital of Tongji Medical
college, Huazhong University of Science and Technology in Wuhan, China.
The investigations were conducted in accordance with the principles of
the Declaration of Helsinki and were approved by the Institutional
Review Board at Tongji Medical College, Huazhong University of Science
and Technology (TJ-IRB20210815) and obtaining the informed consent of
the patient.
Statistical analysis
For single cell RNA-seq, the statistical analysis was conducted using
the R machine language, including the graphs made by R packages used:
Seurat/clusterProfiler/tidyverse. We identify a PC threshold by
calculating where the principal components start to elbow by taking the
larger value of: (a) The point where the principal components only
contribute 5% of standard deviation and the principal components
cumulatively contribute 90% of the standard deviation; (b) The point
where the percent change in variation between the consequtive PCs is
less than 0.1%. Here the pc number is 40. Single cell RNAseq
methodology: alignment: STAR software built-in CellRaner software;
mitochondrial DNA thresholds least than 20%; NormalizeData: By default,
the raw counts are normalized using global-scaling normalization by
performing the following: (a) normalizing the gene expression
measurements for each cell by the total expression; (b) multiplying
this by a scale factor (10,000 by default); (c) log-transforming the
result.
Data were prepared using GraphPad Prism software version 6.0 or IBM
SPSS Statistics 23. Data conforming to normal distribution were
presented as mean ± SD, or median and quartiles for non-normal
distribution. Rate comparisons were performed by chi-squared test.
t-Test, Wilcoxon rank-sum test, Wilcoxon signed-rank test, or
Kruskal–Wallis test were used to compare means across groups according
to the number of group and distribution of variable. Preliminary
univariate binary logistic regression was used to select and estimate
the association between Cx43, GSDMD, hematuria, or eGFR and variables
that were clinically relevant on grounds of professional knowledge and
Stepwise multivariate binary logistic regression was used to select the
predictors. Specifically, variables with P < 0.05 in the univariate
analysis were entered into multivariate analysis to select the
predictors (inclusion criterion was P < 0.05 and exclusion criterion
was P ≥ 0.05). Results are presented as odds ratios (ORs) with 95%
confidence intervals (95% CIs) and P values. All the statistical
analyses were two-tailed. n.s. no significance, *P < 0.05, **P < 0.01,
***P < 0.001.
Supplementary information
[220]41419_2022_4910_MOESM1_ESM.docx^ (22.9KB, docx)
Obstructive nephropathy patients in the Cx43 positive group and GSDMD
high group showed a decline of kidney function
[221]41419_2022_4910_MOESM2_ESM.docx^ (18.1KB, docx)
Risk factors associated with eGFR < 90 ml/min/1.73 m^2 during follow-up
periods
[222]41419_2022_4910_MOESM3_ESM.tif^ (1.8MB, tif)
Time-related comparison after UUO and BIR.
[223]41419_2022_4910_MOESM4_ESM.tif^ (8.1MB, tif)
The Cx43 gene knockout mice were constructed
[224]41419_2022_4910_MOESM5_ESM.tif^ (6MB, tif)
P2X7 receptor modulate renal injury and RIF
[225]41419_2022_4910_MOESM6_ESM.tif^ (556KB, tif)
ATP outflow from renal tubular epithelial cells directly reduced BMDM
pyroptosis in vitro
[226]41419_2022_4910_MOESM7_ESM.tif^ (1MB, tif)
Knockout Cx43 gene alleviated kidney pyroptosis
[227]Original Data File^ (144.5KB, pdf)
[228]Reproducibility Checklist^ (1.8MB, pdf)
Acknowledgements