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. 2022 May 31;13(5):511.
doi: 10.1038/s41419-022-04910-w.

Blocking connexin 43 and its promotion of ATP release from renal tubular epithelial cells ameliorates renal fibrosis

Huzi Xu #  1 , Meng Wang #  1 , Yinzheng Li  1 , Mengxia Shi  1 , Zheng Wang  1 , Chujin Cao  1 , Yu Hong  1 , Bin Hu  1 , Han Zhu  1 , Zhi Zhao  1 , Xiaoxin Chu  1 , Fan Zhu  1 , Xuan Deng  1 , Jianliang Wu  1 , Fenfei Zhao  2   3 , Jing Guo  2   3 , Yuxi Wang  1 , Guangchang Pei  1 , Fengming Zhu  1 , Xiaoyan Wang  4 , Juan Yang  1 , Ying Yao  5   6 , Rui Zeng  7
Affiliations

Blocking connexin 43 and its promotion of ATP release from renal tubular epithelial cells ameliorates renal fibrosis

Huzi Xu et al. Cell Death Dis. .

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. The expression of Cx43 correlates with the severity of kidney injury.
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.
Fig. 2
Fig. 2. Cx43 hemichannel blocker attenuates kidney injury and RIF after UUO.
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.
Fig. 3
Fig. 3. Knockout the Cx43 gene of TECs prevents UUO-induced renal injury and RIF.
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.
Fig. 4
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.
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.
Fig. 5
Fig. 5. The Cx43 hemichannel of TECs mediate ATP outflow and ATP receptor analysis.
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.
Fig. 6
Fig. 6. The ATP outflow from TECs increase macrophage pyroptosis via P2 receptor.
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.
Fig. 7
Fig. 7. The expression of Cx43 and GSDMD genes was associated with renal function in human obstructive nephropathy.
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.
Fig. 8
Fig. 8. CXCL10 can aggravate the progression of obstructive nephropathy.
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.
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