Pro-cachectic factors link experimental and human chronic kidney disease to skeletal muscle wasting programs

Abstract

Skeletal muscle wasting is commonly associated with chronic kidney disease (CKD), resulting in increased morbidity and mortality. However, the link between kidney and muscle function remains poorly understood. Here, we took a complementary interorgan approach to investigate skeletal muscle wasting in CKD. We identified increased production and elevated blood levels of soluble pro-cachectic factors, including activin A, directly linking experimental and human CKD to skeletal muscle wasting programs. Single-cell sequencing data identified the expression of activin A in specific kidney cell populations of fibroblasts and cells of the juxtaglomerular apparatus. We propose that persistent and increased kidney production of pro-cachectic factors, combined with a lack of kidney clearance, facilitates a vicious kidney/muscle signaling cycle, leading to exacerbated blood accumulation and, thereby, skeletal muscle wasting. Systemic pharmacological blockade of activin A using soluble activin receptor type IIB ligand trap as well as muscle-specific adeno-associated virus-mediated downregulation of its receptor ACVR2A/B prevented muscle wasting in different mouse models of experimental CKD, suggesting that activin A is a key factor in CKD-induced cachexia. In summary, we uncovered a crosstalk between kidney and muscle and propose modulation of activin signaling as a potential therapeutic strategy for skeletal muscle wasting in CKD.

Trial registration: ClinicalTrials.gov NCT01209000.

Keywords: Chronic kidney disease; Muscle; Muscle Biology; Nephrology.

Conflict of interest statement

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

Figures

Figure 1. Experimental CKD induces loss of muscle mass and function.

(A) Schematic representation of cystic mouse model. (B) Kidney weight/body weight expressed as a percentage and a representative image of the 6-week kidney of WT and Kif3aΔTub (n = 5 mice). (C) Blood urea nitrogen (BUN) measurement (n = 5 mice). Body weight (D) (n = 10 mice) and muscle tissue weights (E) were measured in WT and Kif3aΔTub mice (n = 14 mice). (F) Representative image of the 6-week GC muscle of WT and Kif3aΔTub mice. (G) Frequency histogram showing the distribution of cross-sectional areas (μm2) in TA muscle of WT and Kif3aΔTub mice (n = 3 mice). (H) Contractile properties of the EDL muscle (n = 5 mice). Specific force denotes tetanic force normalized to wet muscle mass. Data shown as mean ± SEM of 3 independent experiments. Student’s t test used for statistical significance. *P < 0.05, ***P < 0.001.

Figure 2. Characterization of CKD-related muscle wasting.

(A) Quantification of satellite cell number on freshly isolated EDL fibers (n = 2 mice). (B) Profiling of differentiation (WT n = 20, Kif3aΔTubn = 20). (C) Quantification of puromycin incorporation in GC muscle from 6-week-old WT and Kif3aΔTub mice, using in vivo SUnSET technique (WT n = 20, Kif3aΔTubn = 20). (D) Autophagy flux analysis with colchicine treatment based on immunoblot analysis of p62 and LC3 of protein extracts from of GC muscles from fed and starved 6-week-old WT and Kif3aΔTub mice (n = 3 mice). (E) SDH staining and quantification of EDL muscles of WT and Kif3aΔTub mice (n = 3 mice). (F) Quantification of SS and intermyofibrillar (IMF) mitochondrial density and size. One-way ANOVA followed by Bonferroni’s multiple-comparison tests (SS: WT n = 4, Kif3aΔTub 8; IMS: WT n = 8, Kif3aΔTub 13). (G) Quantitative gene expression of oxidative stress/UPRmt genes in GC muscle from 6-week-old WT and Kif3aΔTub mice (n = 4 mice). Data shown as mean ± SEM. Student’s t test used for statistical significance, unless otherwise stated. Comparisons of more than 2 groups were calculated using 1-way ANOVA with Tukey’s multiple-comparison tests. *P< 0.05; **P < 0.01; ***P < 0.001; ns, not statistically significant.

Figure 3. Experimental CKD leads to renal production of soluble pro-cachectic factors.

(A) Volcano plot of genes encoding for secreted proteins (red, genes encoding pro-cachectic factors) (n = 4 mice). (B) Quantitative reverse transcription PCR (RT-PCR) analysis of Inhba in different organs (n = 4 mice). (C) t-Distributed stochastic neighbor embedding (t-SNE) projection of 6779 cells from the 5-week Kif3aΔTub kidney demonstrating 17 cell types. (D) t-SNE plot showing the normalized/average expression level of Inhba in Kif3aΔTub kidney cell types. (E) Dot plot showing the expression of Inhba in the Kif3aΔTub and control kidney. (F) Violin plot showing cluster-specific gene expression of Inhba (n = 1 mouse). (G) Blood levels of activin A in WT and Kif3aΔTub mice determined by ELISA (WT n = 3, KO n = 5). Data shown as mean ± SEM. Student’s t test used for statistical significance, unless otherwise stated. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. Increased production of pro-cachectic factors in patients with CKD.

(A) Activin A blood level in healthy controls and in patients with CKD (n = 75 patients). (B) Spearman’s correlation analysis of serum activin A and eGFR. **P < 0.01. (C) Spearman’s correlation analysis of IF/TA% (renal interstitial fibrosis and tubular atrophy) and eGFR. (D) Spearman’s correlation analysis of serum activin A and IF/TA% (renal interstitial fibrosis and tubular atrophy). (E) Log fold change of quantitative gene expression of genes encoding pro-cachectic factors in glomeruli of manually microdissected biopsies from patients with different CKD stages (Glomerular: CKD1: n = 55; CKD2: n = 52; CKD3: n = 44; CKD4: n = 26; CKD5: n = 10. Tubular: CKD1: n = 56; CKD2: n = 46; CKD3: n = 37; CKD4: n = 26; CKD5: n = 10. Live donor: n = 42). A q value below 5% was considered statistically significant. Nonsignificantly changed genes are denoted as ns and genes below background cutoff as BC. GDF15, growth differentiation factor 15. (F) Spearman’s correlation analysis for glomerular activin A mRNA expression with eGFR. (G) Spearman’s correlation analysis of patients with focal segmental glomerulosclerosis (FSGS) of INHBA mRNA expression with log2eGFR (n = 66 patients). (H) Dot plot shows the relative average mRNA expression of INHBA across the 31 clusters identified from the combined analysis of the 24 adult human kidney scRNA-Seq data sets.

Figure 5. Skeletal muscle atrophy and accumulation of activin A in the blood are common features of different models of kidney fibrosis.

(A) Schematic representation of 2,8-DHA nephropathy model (AN): mice are fed for 21 days with adenine-enriched diet. (B) BUN measurement (n = 5 mice). (C) Body weight (D) and lean mass curve in WT and AN mice (n = 5 mice). (E) GC muscle weights (n = 5 mice). (F) Frequency histogram showing the distribution of cross-sectional areas (μm2) in TA of WT and AN mice (n = 4 mice). (G) Electron micrographs of EDL muscles of WT and AN mice. Scale bar: 2 μm. (H) Quantification of SS and IMF mitochondrial density and size. One-way ANOVA followed by Bonferroni’s multiple-comparison tests. (I) Quantitative RT-PCR analysis of Inhba in different organs (n = 5 mice). (J) In situ hybridization was performed with an RNAscope probe targeting Inhba mRNA. Representative images of WT and AN kidney. Inhba mRNA shown in green and nuclei counterstained with DAPI (blue). Scale bars: 10 μm. (K) Blood levels of activin A in WT and AN mice determined by ELISA (n = 5 mice). Values are mean ± SEM. Student’s t test used for statistical significance, unless otherwise stated. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6. Pharmacological inhibition of activin A prevents muscle wasting in experimental CKD.

(A) Schematic representation of experimental design: treatment of WT and Kif3aΔTub mice twice weekly by intraperitoneal (i.p.) injections of vehicle treatment or 10 mg/kg sActRIIB starting at 2 weeks of age for 4 weeks. (B) Left panel: representative image of 6-week-old body size of WT, Kif3aΔTub, and Kif3aΔTub sActRIIB mice. Right panel: growth curve of WT, Kif3aΔTub, and Kif3aΔTub sActRIIB mice (n = 5 mice). (C) GC weight of WT, Kif3aΔTub, and Kif3aΔTub sActRIIB mice (n = 13 mice). (D) Analysis of the number of fibers per muscle in WT and Kif3aΔTub mice and Kif3aΔTub sActRIIB mice (n = 3 mice). (E) Frequency histogram showing the distribution of cross-sectional areas (μm2) in TA of WT, Kif3aΔTub, and Kif3aΔTub sActRIIB mice (n = 3 mice). (F) Quantification of satellite cell number on freshly isolated EDL fibers in WT and Kif3aΔTub mice and Kif3aΔTub sActRIIB mice (n = 2 mice). Data of WT and Kif3aΔTub have already been presented in Figure 2B. (G) Tetanic specific force measurement in EDL muscle of WT and Kif3aΔTub mice and Kif3aΔTub sActRIIB mice. Data of WT and Kif3aΔTub have already been presented in Figure 1H (n = 5 mice). (H) Quantification of SS and IMF mitochondrial density and size. One-way ANOVA followed by Bonferroni’s multiple-comparison tests. Data of WT and Kif3aΔTub have already been presented in Figure 2F (n = 3 mice). (I) Quantitative gene expression of UPRmt genes in GC muscle from 6-week-old WT, Kif3aΔTub, and Kif3aΔTub sActRIIB mice (n = 4 mice). Data shown as mean ± SEM of 3 independent experiments. Comparisons of more than 2 groups were calculated using 1-way ANOVA with Tukey’s multiple-comparison tests: *when comparing WT vs. Kif3aΔTub, §when comparing WT vs. Kif3aΔTub sActRIIB,#when comparing Kif3aΔTub vs. Kif3aΔTub sActRIIB. *,§,#P < 0.05, **,§§P < 0.01, ***,§§§,###P < 0.001.

Figure 7. Pharmacological inhibition of activin restores protein synthesis while inhibiting protein breakdown.

(A and B) Activin A, IL-6, and corticosterone blood level determined by ELISA in WT, Kif3aΔTub, and Kif3aΔTub sActRIIB mice. A: WT n = 3, Kif3aΔTubn = 5, Kif3aΔTub sActRIIB n = 5.B left panel: WT n = 4, Kif3aΔTubn = 4, Kif3aΔTub sActRIIB n = 4.B right panel: WT n = 10, Kif3aΔTubn = 8, Kif3aΔTub sActRIIB n = 10. (C and D) Total protein extracts from muscle of 6-week-old WT, Kif3aΔTub, and Kif3aΔTub sActRIIB mice were immunoblotted with the indicated antibodies (n = 3 mice). (E) Quantification of puromycin incorporation in GC muscle from 6-week-old WT Kif3aΔTub and Kif3aΔTub sActRIIB mice, using in vivo SUnSET technique (n = 3 mice). (F and G) Total protein extracts from muscle of 6-week-old WT, Kif3aΔTub, and Kif3aΔTub sActRIIB mice were immunoblotted with the indicated antibodies (n = 3 mice). (H) Autophagy flux analysis with colchicine treatment. Immunoblot analysis of p62 and LC3 of protein extracts from GC muscles from fed and starved 6-week-old WT, Kif3aΔTub, and Kif3aΔTub sActRIIB mice (n = 3 mice). Values are mean ± SEM. Comparisons of more than 2 groups were calculated using 1-way ANOVA with Tukey’s multiple-comparison tests. *P < 0.05, **P < 0.01, ***P < 0.001. In H, only statistically significant comparisons are shown.

Figure 8. Muscle-specific downregulation of activin pathway protects from CKD-induced skeletal muscle atrophy.

(A) Schematic representation of experimental design: mice were fed with adenine-enriched diet to induce 2,8-DHA nephropathy (AN), and TA muscles were transfected with (AAV-)Acvr2a/b-shRNA or (AAV-)Scramble-shRNA. Muscles were examined 3 weeks later. (B) Representative images of the renal histology of WT and AN kidney. Scale bars represent 60 μm. PAS, periodic acid–Schiff. (C) Quantitative RT-PCR analysis of Inhba in kidneys of WT and AN mice (n = 4 mice). (D) Blood levels of activin A in WT and AN mice determined by ELISA (n = 4 mice). (E) Body weights were measured in WT and AN mice (n = 7 mice). (F) The weight of (AAV-)Acvr2a/b-shRNA– and (AAV-)Scramble-shRNA–infected muscles of WT and AN mice were measured (n = 4 mice). (G) Average fiber cross-sectional area of TA muscle of WT and AN mice infected with (AAV-)Acvr2a/b-shRNA or (AAV-)Scramble-shRNA (n = 3 mice). Data shown as mean ± SEM. Student’s t test used for statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 9. Schematic overview of proposed crosstalk between kidney and muscle during CKD.

We propose a new kidney/muscle axis where the soluble pro-cachectic factor activin A is produced in injured kidney, accumulates in the blood, and destroys muscle homeostasis and induces muscle wasting.