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. 2023 Nov 8;8(21):e170521.
doi: 10.1172/jci.insight.170521.

Calponin 2 regulates ketogenesis to mitigate acute kidney injury

Yuan Gui  1 , Zachary Palanza  1 , Priya Gupta  1 , Hanwen Li  2 , Yuchen Pan  3 , Yuanyuan Wang  1 , Geneva Hargis  4 , Donald L Kreutzer  5 , Yanlin Wang  1 , Sheldon I Bastacky  6 , Yansheng Liu  7   8 , Silvia Liu  6 , Dong Zhou  1
Affiliations

Calponin 2 regulates ketogenesis to mitigate acute kidney injury

Yuan Gui et al. JCI Insight. .

Abstract

Calponin 2 (CNN2) is a prominent actin stabilizer. It regulates fatty acid oxidation (FAO) by interacting with estrogen receptor 2 (ESR2) to determine kidney fibrosis. However, whether CNN2 is actively involved in acute kidney injury (AKI) remains unclear. Here, we report that CNN2 was induced in human and animal kidneys after AKI. Knockdown of CNN2 preserved kidney function, mitigated tubular cell death and inflammation, and promoted cell proliferation. Distinct from kidney fibrosis, proteomics showed that the key elements in the FAO pathway had few changes during AKI, but we identified that 3-hydroxymethylglutaryl-CoA synthase 2 (Hmgcs2), a rate-limiting enzyme of endogenous ketogenesis that promotes cell self-renewal, was markedly increased in CNN2-knockdown kidneys. The production of ketone body β-hydroxybutyrate and ATP was increased in CNN2-knockdown mice. Mechanistically, CNN2 interacted with ESR2 to negatively regulate the activities of mitochondrial sirtuin 5. Activated sirtuin 5 subsequently desuccinylated Hmgcs2 to produce energy for mitigating AKI. Understanding CNN2-mediated discrete fine-tuning of protein posttranslational modification is critical to optimize organ performance after AKI.

Keywords: Apoptosis; Mouse models; Nephrology; Pericytes.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. CNN2 inductions in the AKI kidneys.
(A) The percentage of cells expressing CNN2 in multiple organs based on a single-cell transcriptomic atlas of humans. (B) Bulk RNA sequencing revealed CNN2 was induced in the mouse kidneys after renal IRI at 1 and 3 days. Left panel: principal component analysis (PCA) colored by different time points. Right panel: normalized CNN2 expression at 0, 1, and 3 days. (C) Single-nucleus RNA sequencing showed CNN2 is predominantly expressed by fibroblasts and pericytes after IRI. Costaining for CNN2 (red) and the marker for fibroblasts/pericytes, PDGFR-β (green). Scale bar, 25 μm. Arrows indicate positive staining. (D) Representative immunohistochemical staining images showed CNN2 expression in nontumor normal human kidney and kidney biopsy specimens from patients with AKI. Boxed areas are zoomed (original magnification, 20×). Arrows indicate positive staining. Scale bar, 50 μm. (E) Quantitative real-time PCR analysis revealed the levels of CNN2 mRNA in the diseased kidneys after IRI and cisplatin injection, respectively. *P < 0.05 (n = 6). (F) Western blot assays show CNN2 protein expression in the diseased kidneys after IRI (left panel) and cisplatin (right panel), respectively. Numbers indicate individual animals within each group. (G) Immunohistochemical staining showed the distribution of CNN2 in mouse kidneys after IRI and cisplatin. Original magnification, 40×. Graphs are presented as means ± SEM. Differences between groups were analyzed using unpaired t tests or ANOVA followed by the Student-Newman-Keuls test. IRI, ischemia/reperfusion injury; PDGFR-β, PDGF receptor-β.
Figure 2
Figure 2. Knockdown of CNN2 mitigates ischemic AKI.
(A) Experiment design. ShCNN2 plasmid was administrated in mice 7 days (d) and 1 d before IRI, respectively. The mice were sacrificed at 1 d after IRI. (B) Quantitative real-time PCR analysis showed the changes of CNN2 mRNA levels in kidneys of ShNC and ShCNN2 mice after IRI. *P < 0.05 (n = 6). (C and D) Western blot assay demonstrated CNN2 protein expression in kidneys of ShNC and ShCNN2 mice after IRI (C), and quantified data were presented (D). Numbers indicate individual animals within each group. *P < 0.05 (n = 5). (E) Immunohistochemical staining showed CNN2 expression and distribution in kidneys of ShNC and ShCNN2 mice after IRI. Scale bar, 25 μm. Arrows indicate positive staining. (F) Costaining for CNN2 (red) and PDGFR-β (green) in the kidneys demonstrated CNN2 induction was largely abolished in fibroblasts/pericytes. Scale bar, 50 μm. Arrows indicate positive staining. (G) Serum creatinine (Scr) and blood urea nitrogen (BUN) levels in ShNC and ShCNN2 mice at 1 d after IRI or 3 d after cisplatin injection. *P < 0.05 (n = 7–9). (H–J) Representative Western blots (H and I) and quantified data (J) of NGAL protein expression in ShNC and ShCNN2 kidneys at 1 day after IRI or 3 days after cisplatin injection. Numbers indicate individual animals within each group. *P < 0.05 (n = 6). (K) The changes of kidney histology as shown by periodic acid–Schiff (PAS) staining in ShNC and ShCNN2 mice at 1 d after IRI or 3 d after cisplatin injection. Scale bar, 50 μm. Blue asterisks indicate injured tubules. Graphs are presented as means ± SEM. Differences between groups were analyzed using unpaired t tests.
Figure 3
Figure 3. Knockdown of CNN2 attenuates tubular cell death and kidney inflammation in AKI.
(A and B) Representative micrographs (A) and quantitative data (B) of TUNEL staining in ShNC and ShCNN2 mouse kidneys 1 day after IRI or 3 days after cisplatin injection. Arrows indicate apoptotic cells. DAPI is a nuclear counterstain. Scale bar, 50 µm. *P < 0.05 (n = 5). (C) Western blot assay demonstrated the expression of AIF, FADD, Bax, and ARC in ShNC and ShCNN2 mouse kidneys at 1 day after IRI (n = 6). (D) Western blot assay demonstrated p-MLKL, MLKL, and GPX4 expression in ShNC and ShCNN2 mouse kidneys at 1 day after IRI. (E) Representative immunohistochemical staining micrographs for p-MLKL, GPX4, CD45, Ki67, and PCNA in ShNC and ShCNN2 mouse kidneys 1 day after IRI or 3 days after cisplatin injection. Scale bar, 50 µm. Arrows indicate positive staining. (F) Quantitative real-time PCR analyses revealed the mRNA abundance of Rantes, IL-6, and TNF-α in ShNC and ShCNN2 mouse kidneys 1 day after IRI or 3 days after cisplatin injection. *P < 0.05 (n = 6). (G) Immunofluorescence staining showed F4/80+ macrophages in ShNC and ShCNN2 mouse kidneys 1 day after IRI. Scale bar, 50 µm. Arrows indicate positive staining. (H) Western blot assays demonstrated PCNA expression in ShNC and ShCNN2 mouse kidneys at 1 day after IRI or 3 days after cisplatin injection. (I) Western blot assays demonstrated PDGFR-β and α-SMA expression in the kidneys from ShNC and ShCNN2 mice 1 day after IRI. For all Western blot panels, numbers indicate individual animals within each group. Graphs are presented as means ± SEM. Differences between groups were analyzed using unpaired t tests or ANOVA followed by the Student-Newman-Keuls test. AIF, apoptosis inducing factor; ARC, apoptosis repressor with caspase recruitment domain; FADD, Fas-associated protein with death domain; p-MLKL, phosphorylated mixed lineage kinase domain-like protein; GPX4, glutathione peroxidase 4; α-SMA, α–smooth muscle actin.
Figure 4
Figure 4. Global proteomics reveals CNN2 knockdown increases Hmgcs2-mediated ketogenesis after AKI.
After IRI at 1 day, (A) principal component analysis of global proteomes from ShNC and ShCNN2 kidneys. (B) Heatmap of t test significant proteins. Two clusters of proteins with different patterns of abundance profiles are highlighted. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis highlighted upregulated pathways in ShNC and ShCNN2 mouse kidneys. (D) Gene Ontology (GO) biological process terms in each cluster of proteins are plotted with their names and significance. (E) Heatmap of the key components in the fatty acid oxidation pathway in ShNC and ShCNN2 mouse kidneys. (F) Volcano plot showed the differential proteins between ShNC and ShCNN2 kidneys. Up- and downregulated proteins (fold-change, FC) are colored in yellow and blue, respectively. (G) Quantitative real-time PCR revealed Hmgcs2 mRNA levels in ShNC and ShCNN2 mouse kidneys. *P < 0.05 (n = 6). (H) Western blot assay demonstrated Hmgcs2 levels in ShNC and ShCNN2 mouse kidneys. (I and J) Immunohistochemical staining (I) showed Hmgcs2 expression in ShNC and ShCNN2 mouse kidneys. Costaining for Hmgcs2 (red) and aquaporin 1 (AQP1, green) in the kidneys (J, upper panel). Immunohistochemical staining showed Hmgcs2 expression in the kidney biopsy specimens from patients with AKI (J, lower panel). Scale bar, 50 µm. Arrows indicate positive staining. (K–M) ELISA showed the levels of β-OHB in blood (K), ATP in total kidney tissue (L), and alanine transaminase (ALT) in blood (M) from ShNC and ShCNN2 mice. *P < 0.05 (n = 7). (N) Western blot assay demonstrated the expression of Hmgcs2 protein in the liver from ShNC and ShCNN2 mice. For Western blot panels, numbers indicate individual animals within each group. Graphs are presented as means ± SEM. Differences between groups were analyzed using unpaired t tests or ANOVA followed by the Student-Newman-Keuls test.
Figure 5
Figure 5. Knockdown of Hmgcs2 aggravates ischemic AKI.
(A) Normal rat kidney proximal tubular cells (NRK-52E) were transfected with Dicer-substrate siHmgcs2, followed by CoCl2 (400 μM) administration. Western blot assay demonstrated that knockdown of Hmgcs2 increased Bax and NGAL in NRK-52E cells, compared with scramble controls. (B) NRK-52E cells were treated with β-OHB at different dosages, followed by CoCl2 (400 µM) administration. Western blot assay showed β-OHB reduced FADD and NGAL induction. (C) Experiment design. (D) Quantitative real-time PCR (qPCR) analysis showed Hmgcs2 mRNA levels in ShNC and ShHmgcs2 mouse kidneys after IRI. *P < 0.05 (n = 6). (E) Serum creatinine (Scr) levels in ShNC and ShHmgcs2 mice after IRI. *P < 0.05 (n = 10). (F) Periodic acid–Schiff (PAS) staining showed morphological changes in ShNC and ShHmgcs2 mice after IRI. Blue asterisks indicate injured tubules. Representative micrographs of TUNEL staining in the kidneys from ShNC and ShHmgcs2 mice after IRI. Arrows indicate apoptotic cells. DAPI is a nuclear counterstain. Scale bar, 50 µm. (G) The quantitative data of apoptotic cells. *P < 0.05 (n = 5). (H and I) Representative Western blots (H) and quantified data (I) of NGAL and Bax expression in ShNC and ShHmgcs2 mouse kidneys after IRI. Numbers indicate individual animals within each group. *P < 0.05 (n = 6). (J) qPCR analyses revealed the mRNA abundance of MCP1, IL-6, and TNF-α in the kidneys from ShNC and ShHmgcs2 mice at 1 day after IRI. *P < 0.05 (n = 6). (K) Representative immunohistochemical staining micrographs for CD45 in ShNC and ShHmgcs2 mouse kidneys after IRI. Scale bar, 50 µm. Arrows indicate positive staining. Graphs are presented as means ± SEM. Differences between groups were analyzed using unpaired t tests or ANOVA followed by the Student-Newman-Keuls test. DsiRNA, Dicer-substrate siRNA; MCP-1, monocyte chemoattractant protein-1; NGAL, neutrophil gelatinase-associated lipocalin.
Figure 6
Figure 6. Knockdown of CNN2 enhances lysine desuccinylation of HMGCS2 in the kidney after AKI.
(A) Schematic diagram. After IRI at 1 day, in ShNC and ShCNN2 mouse kidneys, (B–D) qPCR analyses revealed FOXA2, PPARα, and FGF21 mRNA levels (n = 6). (E and F) Representative Western blots (E) and the quantified data (F) of PPARα (n = 6). (G–I) qPCR analyses revealed sirt2, sirt3, and sirt5 mRNA levels. *P < 0.05 (n = 6). (J and K) Representative Western blots (J) and the quantified data (K) of sirt5. *P < 0.05 (n = 6). (L) Representative micrographs for sirt5 staining. Scale bar, 50 µm. Arrows indicate the positive staining. (M and N) Representative Western blot (M) and quantified data (N) of succinyl-lysine motif (Succ-K). *P < 0.05 (n = 6). (O) Immunoprecipitation of endogenous Hmgcs2 from the kidney lysates of ShNC and ShCNN2 mice. (P) Western blot assay demonstrated that knockdown of sirt5 repressed Hmgcs2 expression in NRK-52E cells under hypoxic stress. (Q) Western blot assay demonstrated that knockdown of CNN2 induced ESR2 after IRI. (R) Western blot assay demonstrated that knockdown of ESR2 repressed sirt5 and Hmgcs2 expression in NRK-52E cells under hypoxic stress. (S) Western blot assay demonstrated that estradiol (100 nM) did not induce Hmgcs2 in NRK-52E cells after knockdown of sirt5, compared with scramble controls. (T) Molecular docking analysis showed the binding sites between CNN2 and ESR2. (U) The strategy of designing a mutant form of CNN2. (V) Western blot assay demonstrated that mutant CNN2 (25 ng/mL) did not affect sirt5 and Hmgcs2 expression in NRK-52E cells, compared with active form of CNN2 human recombinant (rb) protein (25 ng/mL). For Western blot panels, numbers indicate individual animals within each group. Graphs are presented as means ± SEM. Differences between groups were analyzed using unpaired t tests.
Figure 7
Figure 7. Knockdown of CNN2 promotes Hmgcs2 desuccinylation to repress tubular cell death in vitro.
(A) ELISA showed CNN2 levels in the conditioned medium (CM) collected from the cultured fibroblasts after knockdown of CNN2 under hypoxic stress. *P < 0.05 (n = 6). (B) Schematic diagram. (C) Western blot assay demonstrated that CNN2-deprived CM enhanced sirt5 and Hmgcs2 expression in NRK-52E cells stimulated with CoCl2 (400 µM). (D and F) Western blot assay demonstrated CNN2-deprived CM decreased the abundance of succinyl-lysine motif (Succ-K) in NRK-52E cells under hypoxia stress (D), but they were increased after knockdown of sirt5 (F). (E and G) Co-immunoprecipitation of endogenous Hmgcs2 from cell lysates of normal control (NC) CM and CNN2-deprived CM. Immunoprecipitation revealed that lysine succinylation on Hmgcs2 is less in CNN2-deprived CM under hypoxia stress (E) but increased after knockdown of sirt5 (G), compared with controls. (H) Western blot assay demonstrated decreased Bax and NGAL levels after incubation with CNN2-deprived CM under hypoxic stress, compared with vehicles. (I–K) After stimulation with staurosporine (STS, 1 μM) for 3 hours, Western blot assay demonstrated the reduced abundance of cleaved caspase-3 (CCP3) in cultured NRK-52E cells incubated with CNN2-deprived CM (I) and immunofluorescence staining showed fewer CCP3+ tubular cells after treatment with CNN2-deprived CM (J). Quantitative data are presented (K). Scale bar, 25 μm. *P < 0.05 (n = 3). (L) Western blot assay demonstrated that CNN2-deprived CM reduced abundance of CCP3 in cultured NRK-52E cells, but they were increased after knockdown of Hmgcs2, compared with scramble controls. (M) Western blot assay demonstrated CNN2-deprived CM repressed Bax and NGAL inductions in cultured NRK-52E cells under hypoxia stress, but they were increased after knockdown of sirt5. Graphs are presented as means ± SEM. Differences between groups were analyzed using unpaired t tests or ANOVA followed by the Student-Newman-Keuls test.
Figure 8
Figure 8. CNN2 inhibits Hmgcs2 desuccinylation to accelerate tubular cell death in vitro.
(A) Representative Western blots showed that human CNN2 recombinant protein markedly repressed sirt5 and Hmgcs2 expression in NRK-52E cells at different dosages under hypoxic stress. (B) Western blot assay demonstrated CNN2 recombinant protein enhanced the abundance of Succ-K in NRK-52E cells at different dosages under hypoxia stress, compared with controls. (C) Co-immunoprecipitation revealed that lysine succinylation on Hmgcs2 is increased after human CNN2 recombinant protein treatment under hypoxia stress. (D) Western blot assay showed that human CNN2 recombinant protein induced Bax and NGAL expression at different dosages under hypoxic stress, compared with vehicle. (E–G) After stimulation with staurosporine (1 μM) for 3 hours, immunofluorescence staining showed increased CCP3+ tubular cells after being incubated with human CNN2 recombinant protein (E) and quantitative data are presented (F). Scale bar, 25 μm. *P < 0.05 (n = 3). Western blots demonstrated the upregulated abundance of CCP3 in cultured NRK-52E cells after incubation with CNN2 recombinant protein (G). (H) Western blot assay showed β-OHB decreased the abundance of CCP3 after being treated with CNN2 recombinant protein in cultured NRK-52E cells. (I) Schematic diagram depicts knockdown of CNN2 enhanced ketogenesis to mitigate AKI. Graphs are presented as means ± SEM. Differences between groups were analyzed using ANOVA followed by the Student-Newman-Keuls test.
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