Calponin 2 harnesses metabolic reprogramming to determine kidney fibrosis

Abstract

Objective: In the fibrotic kidneys, the extent of a formed deleterious microenvironment is determined by cellular mechanical forces. This process requires metabolism for energy. However, how cellular mechanics and metabolism are connected remains unclear.

Methods: A multi-disciplinary approach was employed: the fibrotic kidney disease models were induced by renal ischemia-reperfusion injury and unilateral ureteral obstruction in Calponin 2 (CNN2) knockdown mice. Proteomics, bioinformatics, and in vivo and in vitro molecular experimental pathology studies were performed.

Result: Our proteomics revealed that actin filament binding and cell metabolism are the two most dysregulated events in the fibrotic kidneys. As a prominent actin stabilizer, CNN2 was predominantly expressed in fibroblasts and pericytes. In CKD patients, CNN2 levels was markedly induced in blood. In mice, CNN2 knockdown preserves kidney function and alleviates fibrosis. Global proteomics profiled that CNN2 knockdown enhanced the activities of the key rate-limiting enzymes and regulators of fatty acid oxidation (FAO) in the diseased kidneys. Inhibiting carnitine palmitoyltransferase 1α in the FAO pathway resulted in lipid accumulation and extracellular matrix deposition in the fibrotic kidneys, which were restored after CNN2 knockdown. Bioinformatics and chromatin immunoprecipitation showed that CNN2 interactor, estrogen receptor 2 (ESR2), binds peroxisome proliferator-activated receptor-α (PPARα) to transcriptionally regulate FAO downstream target genes expression amid kidney fibrosis. In vitro, ESR2 knockdown repressed the mRNA levels of PPARα and the key genes in the FAO pathway. Conversely, activation of PPARα reduced CNN2-induced matrix inductions.

Conclusions: Our results suggest that balancing cell mechanics and metabolism is crucial to develop therapeutic strategies to halt kidney fibrosis.

Keywords: Calponin 2; Chronic kidney disease; ESR2; Fatty acid oxidation; Proteomics.

Figure 1

The landscape of proteomes in CKD. (A) Principal component analysis of proteomes in control and IRI-induced CKD kidneys. (B) Correlation of kidney proteome profiles between control and IRI-induced CKD kidneys. The color scale represents R² values. (C) Violin plot of ANOVA significant proteins (Permutation FDR 0.05) among control and IRI-induced CKD kidneys. Label-free quantitation (LFQ) intensities of represented proteins were z-scored and plotted according to the color bar. (D) Hierarchical clustering of the intensity (plotted as z-score) of the proteins identified in the control and ischemic kidneys by quantitative proteomic analysis. Two clusters of proteins with different patterns of abundance profiles were highlighted in the heatmap. (E) KEGG pathway enrichment analyses revealed the top 20 activated pathways in the ischemic kidneys after CKD.

Figure 2

Calponin 2 is a key actin filament-associated regulatory protein identified in the fibrotic kidneys. (A) Gene Ontology (GO) enrichment analysis revealed that actin filament binding is listed as one of the top 3 significantly upregulated events under molecular function terms. (B) Heatmap of the differentially expressed actin filament-associated regulatory proteins between controls and CKD induced by IRI. (C) Volcano plot showed calponin 2 (CNN2) is upregulated in the ischemic kidneys after CKD. (D) Quantitative RT-PCR analysis revealed that CNN2 mRNA levels were upregulated in the fibrotic kidneys induced by IRI, UUO, and ADR nephropathy, respectively. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 5–6). (E) Western blot assays demonstrated the expression of CNN2 protein in the fibrotic kidneys induced by IRI, UUO, and ADR nephropathy. Numbers indicate individual animals within each group. (F) Single nucleus RNA sequencing showed CNN2 mainly expressed by fibroblasts (Fib) and pericytes (Per) after IRI. (Data were extracted from the online database provided by Dr. Benjamin Humphrey's laboratory at the Washington University in St. Louis, http://humphreyslab.com/SingleCell/displaycharts.php). (G) Immunohistochemical staining showed CNN2 distributions in the fibrotic kidneys after IRI, UUO, and ADR nephropathy, respectively. (H) Immunofluorescence staining demonstrated the co-staining of CNN2 (red) and platelet-derived growth factor receptor β (green) in the ischemic kidneys. (I) Representative immunohistochemical staining images showed CNN2 expression in the non-tumor normal human kidneys and the kidney biopsy specimens from CKD patients diagnosed with IgA nephropathy (IgAN), focal segmental glomerulosclerosis (FSGS), and membrane nephropathy (MN). Boxed areas are zoomed. (J) Serum CNN2 levels in healthy adults (n = 75) and non-diabetic CKD patients (n = 84). Graphs are presented as means ± SEM. Arrows indicate positive staining. Scale bar, 50 μm. IRI, ischemia reperfusion injury; UUO, unilateral ureteral obstruction; ADR, Adriamycin.

Figure 3

Knockdown of CNN2 alleviates kidney fibrosis. (A) Experiment design. The first dose of ShCNN2 plasmid was administrated in mice 1 day (d) before unilateral IRI (UIRI) surgery. At d10 after IRI (1d before unilateral nephrectomy, UNX), the second dose of ShCNN2 plasmid was injected. The mice were sacrificed on d18. (B) Quantitative RT-PCR (qPCR) analysis showed the changes of CNN2 mRNA levels in the kidneys of ShNC and ShCNN2 mice after IRI. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 6). (C) Western blot assay demonstrated CNN2 protein expression in the kidneys of ShNC and ShCNN2 mice after IRI. Numbers indicate individual animals within each group. (D) Immunohistochemical staining and immunofluorescence co-staining of CNN2 (red) and platelet-derived growth factor receptor β (green) showed CNN2 expression and distribution in the kidneys of ShNC and ShCNN2 mice after IRI. Scale bar, 50 μm. (E, F) Blood urea nitrogen (BUN) and serum creatinine (Scr) levels in ShNC and ShCNN2 mice after IRI. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 6–8). (G) qPCR analyses revealed the mRNA abundance of FN, α1 type I collagen (Col1α1), and α1 type III collagen (Col3α1) in the kidneys of ShNC and ShCNN2 mice after IRI. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 6). (H) Western blot assay demonstrated Col1α1, FN, α-smooth muscle actin (α-SMA), and platelet-derived growth factor receptor β (PDGFRβ) protein expression in the kidneys of ShNC and ShCNN2 mice after IRI. Numbers indicate individual animals within each group. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 6). (I) Representative micrographs for α-SMA, Col1α1, FN staining, Masson Trichrome Staining, CD45, and CD3 in the fibrotic kidneys of ShNC and ShCNN2 mice after IRI-induced CKD. Arrows indicate positive staining. Scale bar, 50 μm. (J) qPCR analyses revealed the mRNA abundance of monocyte chemoattractant protein-1 (MCP1), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) in the kidneys of ShNC and ShCNN2 mice after IRI. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 6).

Figure 4

Global proteomics reveals fatty acid oxidation is a key pathway in mediating kidney fibrosis after knockdown of CNN2. (A) Experimental workflow of the global proteomic analysis. In each group, kidney samples from six mice were used for mass spectrometry. (B) Principal component analysis of global proteomes from ShNC and ShCNN2 fibrotic kidneys after IRI. (C) Heatmap of t-test significant proteins (Permutation FDR 0.05). Two clusters of proteins with different patterns of abundance profiles are highlighted in the heatmap. (D) Volcano plot showed the differential proteins (Permutation FDR 0.05) between ShNC and ShCNN2 fibrotic kidneys. Up- and down-regulated proteins (fold-change, FC) are colored in red and blue, respectively. (E, F) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis highlighted upregulated metabolic pathways in fibrotic kidneys from ShNC and ShCNN2 mice after IRI. (G) Gene Ontology (GO) biological process terms in each cluster of proteins are plotted with their names and significance. The fatty acid metabolic process is highlighted to indicate the group with the largest difference in upregulated proteins. (H) Heatmap of the differentially expressed proteins in the fatty acid oxidation pathway in ShNC and ShCNN2 mice fibrotic kidneys after IRI.

Figure 5

CNN2 modifies lipid accumulation and fatty acid oxidation pathway amid kidney fibrosis. (A, B) The enzyme-linked immunosorbent assay (ELISA) showed triglyceride contents in serum (A) and kidney (B) collected from ShNC and ShCNN2 mice after IRI. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 6). (C) Representative micrographs of Oil Red-O staining in the fibrotic kidneys from ShNC and ShCNN2 mice after IRI. Arrows indicate lipid accumulation in tubules. Scale bar, 50 μm. (D) Western blot assay demonstrated perilipin 2 (Plin2) protein expression in ShNC and ShCNN2 mice fibrotic kidneys after IRI. Numbers indicate individual animals within each group. (E) Immunohistochemical staining showed Plin2 distributions in the fibrotic kidneys of ShNC and ShCNN2 mice after IRI. Arrows indicate positive staining. Scale bar, 50 μm. (F) ELISA showed ATP levels in the total kidneys collected from ShNC and ShCNN2 mice after IRI. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 6). (G) Quantitative RT-PCR analyses revealed the mRNA abundance of PPARα, CPT1α, ACOX1-3, Acsm1, Acsm2, Acsm5, Acadm, and Acadl in fibrotic kidneys from ShNC and ShCNN2 mice after IRI. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 6). (H) Western blot assays demonstrated PPARα, PGC1α, CPT1α, ACOX1, Acsm5, and Acadm protein expression in ShNC and ShCNN2 mice fibrotic kidneys after IRI. Numbers indicate individual animals within each group. (I) Representative micrographs of PPARα, CPT1α, ACOX1, and Acadm staining in the fibrotic kidneys from ShNC and ShCNN2 mice after IRI. Boxed areas are enlarged. Arrows indicate positive staining. Scale bar, 50 μm.

Figure 6

Knockdown of CNN2 enhances CPT1α activity to alleviate kidney fibrosis (A) Experiment design. CPT1α inhibitor, Etomoxir (Eto), was administrated in mice 2 days before unilateral IRI (UIRI) and nephrectomy (UNX), respectively. ShNC and ShCNN2 plasmids were injected at day (d) −1 and 10 through the tail vein. The mice were sacrificed on d18. (B) Quantitative RT-PCR (qPCR) analysis showed the changes of CPT1α mRNA levels in the fibrotic kidneys of ShNC mice, ShNC mice that received Etomoxir, and ShCNN2 mice that received Etomoxir after IRI. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 5). (C) Western blot assay demonstrated CPT1α protein expression in the fibrotic kidneys of ShNC mice, ShNC mice that received Etomoxir, and ShCNN2 mice that received Etomoxir after IRI. Numbers indicate individual animals within each group. (D, E) Blood urea nitrogen (BUN) and serum creatinine (Scr) levels in ShNC mice, ShNC mice that received Etomoxir, and ShCNN2 that received Etomoxir after IRI. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 5). (F) Representative micrographs for Oil Red O Staining and perilipin 2 (Plin2) immunohistochemical staining showed lipid accumulations in fibrotic kidneys of ShNC mice, ShNC mice that received Etomoxir, and ShCNN2 mice that received Etomoxir after IRI. Arrows indicate positive staining. Scale bar, 50 μm. (G) qPCR analyses revealed the mRNA abundance of FN, α1 type I collagen, and α1 type III collagen in the fibrotic kidneys of ShNC mice, ShNC mice that received Etomoxir, and ShCNN2 mice that received Etomoxir after IRI. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 5). (H) Western blot assay demonstrated FN and α-SMA proteins expression in the fibrotic kidneys of ShNC mice, ShNC mice that received Etomoxir, and ShCNN2 mice that received Etomoxir after IRI. Numbers indicate individual animals within each group. (I) Representative micrographs for immunostaining of α-SMA, Col1α1, fibronectin (FN), and Masson Trichrome Staining (MTS) showed myofibroblast activation and collagen deposition in the fibrotic kidneys of ShNC mice, ShNC mice that received Etomoxir, and ShCNN2 mice that received Etomoxir after IRI. Arrows indicate positive staining. Scale bar, 50 μm.

Figure 7

Knockdown of CNN2 promotes ESR2 binding PPARα to enhance fatty acid oxidation (A) Based on the GeneMANIA database, the human protein–protein interaction analysis showed CNN2 interacts with estrogen receptor 2 (ESR2). (B) Quantitative RT-PCR (qPCR) analysis showed the abundance of ESR2 mRNA levels in ShNC and ShCNN2 mice fibrotic kidneys after IRI. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 6). (C) Western blot assay demonstrated ESR2 protein expression in the fibrotic kidneys of ShNC and ShCNN2 mice after IRI. Numbers indicate individual animals within each group. (D) ELISA showed CNN2 levels in the conditioned medium (CM) collected from the cultured fibroblasts after knockdown of CNN2 under TGF-β stress. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 6). (E) Schematic diagram showed CNN2-deprived CM from the cultured fibroblasts or recombinant CNN2 protein were employed to treat human kidney proximal tubular cells (HK-2). (F) qPCR showed ESR2 expression in HK-2 cells after incubation with CNN2-deprived CM or human recombinant CNN2 protein. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 3). (G) Western blot assay demonstrated ESR2 expression in HK-2 cells after incubation with CNN2-deprived CM or human recombinant CNN2 protein. (H) ChIP-qPCR assay showed ESR2 binds to putative sequences in the promoter of PPARα genes. HK-2 cells were treated with ESR2 agonist (LY500307) for 3 h. Chromatin preparations from the cells were immunoprecipitated using an anti-ESR2 antibody, and co-precipitated DNA fragments were amplified using primers specific for the PPARα promoter. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 3). The upper panel showed representative DNA sequence logo representing the binding motif of the ESR2 gene. (I) qPCR analyses revealed the mRNA abundances of PPARα were reduced in HK-2 cells after knockdown of ESR2 under TGFβ1 stress, compared with scramble controls. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 3). (J) Western blot assay demonstrated that knockdown of ESR2 repressed PPARα expression in HK-2 cells under TGFβ1 stress, compared with scramble controls. (K, L) qPCR analyses revealed the mRNA abundances of PPARα were increased in HK-2 cells by CNN2-deprived CM (K) but were repressed by CNN2 recombinant protein (L) under TGFβ1 stress, compared with controls. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 3). (M, N) Western blot assays demonstrated that CNN2-deprived CM enhanced PPARα expression (M) but CNN2 recombinant protein repressed PPARα inductions (N) in HK-2 cells under TGFβ1 stress, compared with controls. (O) qPCR analyses revealed the mRNA abundances of PPARα were induced by CNN2-deprived CM but were repressed after knockdown of ESR2 under TGFβ1 stress. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 3).

Figure 8

Knockdown of CNN2 alleviates defective fatty acid oxidation and ECM deposition in tubular cells. (A) qPCR analyses revealed the mRNA abundances of CPT1α, ACOX1, Acsm1, and Acsm5 were increased in HK-2 cells incubated with CNN2-deprived CM under TGFβ1 stress, compared with controls. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 3). (B) Western blot assay demonstrated that CNN2-deprived CM reduced the inductions of FN and α-SMA in HK-2 cells under TGFβ1 stress, compared with controls. (C) qPCR analyses revealed the mRNA abundances of CPT1α, ACOX1, and Acsm1 were increased by CNN2-deprived CM in HK-2 cells but were repressed after knockdown of ESR2 under TGFβ1 stress, compared with controls. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 3). (D) Western blot assays demonstrated CNN2-deprived CM alleviated FN and α-SMA inductions in HK-2 cells under TGFβ1 stress but they were increased after knockdown of ESR2, compared with controls. (E) Under TGFβ1 stress, HK-2 cells were incubated with CNN2-deprived CM and followed by CPT1α inhibitor Etomoxir (100 μM). Western blot assays showed Etomoxir induced FN and α-SMA expression after incubation with CNN2-deprived CM. (F) qPCR analyses revealed the mRNA abundances of PGC1α, CPT1α, ACOX1, and Acsm5 were reduced in HK-2 cells incubated with CNN2 recombinant protein under TGFβ1 stress. Graphs are presented as means ± SEM. ∗P < 0.05 (n = 3). (G) Western blot assay demonstrated that CNN2 recombinant induced FN and α-SMA expression in HK-2 cells after TGFβ1 stimulations. (H) Under TGFβ1 stress, HK-2 cells were treated with CNN2 recombinant proteins followed by PPARα agonist Fenofibrate (Feno, 80 μM). Western blot assays showed Fenofibrate decreased FN and α-SMA expression after treated with CNN2 recombinant protein. (I) Schematic diagram depicts knockdown of CNN2 promotes ESR2 binding to PPARα to transcriptionally regulate the FAO genes to halt kidney fibrosis.