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. 2015 Mar;26(3):636-46.
doi: 10.1681/ASN.2014020210. Epub 2014 Jul 28.

Cardiac Myocyte-Derived Follistatin-Like 1 Prevents Renal Injury in a Subtotal Nephrectomy Model

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Free PMC article

Cardiac Myocyte-Derived Follistatin-Like 1 Prevents Renal Injury in a Subtotal Nephrectomy Model

Satoko Hayakawa et al. J Am Soc Nephrol. .
Free PMC article

Abstract

Heart disease contributes to the progression of CKD. Heart tissue produces a number of secreted proteins, also known as cardiokines, which participate in intercellular and intertissue communication. We recently reported that follistatin-like 1 (Fstl1) functions as a cardiokine with cardioprotective properties. Here, we investigated the role of cardiac Fstl1 in renal injury after subtotal nephrectomy. Cardiac-specific Fstl1-deficient (cFstl1-KO) mice and wild-type mice were subjected to subtotal (5/6) nephrectomy. cFstl1-KO mice showed exacerbation of urinary albumin excretion, glomerular hypertrophy, and tubulointerstitial fibrosis after subtotal renal ablation compared with wild-type mice. cFstl1-KO mice also exhibited increased mRNA levels of proinflammatory cytokines, including TNF-α and IL-6, NADPH oxidase components, and fibrotic mediators, in the remnant kidney. Conversely, systemic administration of adenoviral vectors expressing Fstl1 (Ad-Fstl1) to wild-type mice with subtotal nephrectomy led to amelioration of albuminuria, glomerular hypertrophy, and tubulointerstitial fibrosis, accompanied by reduced expression of proinflammatory mediators, NADPH oxidase components, and fibrotic markers in the remnant kidney. In cultured human mesangial cells, treatment with recombinant FSTL1 attenuated TNF-α-stimulated expression of proinflammatory cytokines. Treatment of mesangial cells with FSTL1 augmented the phosphorylation of AMP-activated protein kinase (AMPK), and inhibition of AMPK activation abrogated the anti-inflammatory effects of FSTL1. These data suggest that Fstl1 functions in cardiorenal communication and that the lack of Fstl1 production by myocytes promotes glomerular and tubulointerstitial damage in the kidney.

Keywords: albuminuria; cardiovascular; cell signaling; chronic inflammation; mesangial cells; renal injury.

Figures

Figure 1.
Figure 1.
cFstl1-KO mice show exacerbation of albuminuria, glomerular hypertrophy, and fibrosis after renal ablation. (A) Plasma and cardiac Fstl1 levels of control and cFstl1-KO mice after nephrectomy or sham operation. Left panels show representative bands of Fstl1 by Western blot analysis. Right panel shows quantitative analysis of plasma Fstl1 protein levels evaluated by ImageJ program. n=4 in each group. (B) Urine and plasma parameters of renal function in control and cFstl1-KO mice after nephrectomy or sham operation. Left panel shows urinary albumin excretion normalized to urinary creatinine. Center and right panels show plasma concentration of urea nitrogen and creatinine. n=5–8 in each group. (C) Histologic evaluation of glomerular hypertrophy. Upper panels show representative photos of remnant kidneys from control and cFstl1-KO mice as determined by PAS staining. Lower panels show quantitative analyses of glomerular cross-sectional area (left) and intraglomerular cell number (right). n=4 in each group. Scale bars represent 50 μm. (D) Evaluation of interstitial fibrosis in injured kidneys. Upper panels show representative photos of remnant kidney from control and cFstl1-KO mice as stained with Masson’s Trichrome. Lower panel shows quantitative analysis of fibrosis area as measured by WinROOF program. UN, urea nitrogen. All data are presented as mean±SEM. n=4 in each group. Scale bars represent 50 μm.
Figure 1.
Figure 1.
cFstl1-KO mice show exacerbation of albuminuria, glomerular hypertrophy, and fibrosis after renal ablation. (A) Plasma and cardiac Fstl1 levels of control and cFstl1-KO mice after nephrectomy or sham operation. Left panels show representative bands of Fstl1 by Western blot analysis. Right panel shows quantitative analysis of plasma Fstl1 protein levels evaluated by ImageJ program. n=4 in each group. (B) Urine and plasma parameters of renal function in control and cFstl1-KO mice after nephrectomy or sham operation. Left panel shows urinary albumin excretion normalized to urinary creatinine. Center and right panels show plasma concentration of urea nitrogen and creatinine. n=5–8 in each group. (C) Histologic evaluation of glomerular hypertrophy. Upper panels show representative photos of remnant kidneys from control and cFstl1-KO mice as determined by PAS staining. Lower panels show quantitative analyses of glomerular cross-sectional area (left) and intraglomerular cell number (right). n=4 in each group. Scale bars represent 50 μm. (D) Evaluation of interstitial fibrosis in injured kidneys. Upper panels show representative photos of remnant kidney from control and cFstl1-KO mice as stained with Masson’s Trichrome. Lower panel shows quantitative analysis of fibrosis area as measured by WinROOF program. UN, urea nitrogen. All data are presented as mean±SEM. n=4 in each group. Scale bars represent 50 μm.
Figure 2.
Figure 2.
cFstl1-KO mice show elevated expressions of proinflammatory cytokines, NADPH oxidase components, and fibrosis markers in remnant kidney. (A) mRNA levels of TNF-α, IL-6, IL-1β, and MCP-1 (control: black bars, cFstl1-KO: white bars). n=8 in each group. (B) mRNA levels of P40phox, P67phox, P47phox, and P22phox (control: black bars, cFstl1-KO: white bars). n=8 in each group. (C) mRNA levels of collagen I, collagen III, TGF-β1, and AMPK (control: black bars, cFstl1-KO: white bars). n=8 in each group. mRNA levels were measured by quantitative RT-PCR method. All results are normalized to 36B4. CTGF, connective tissue growth factor. All data are presented as mean±SEM. *P<0.05 for control group.
Figure 3.
Figure 3.
Systemic delivery of Fstl1 ameliorates renal injury in wild-type mice after subtotal nephrectomy. (A) Plasma Fstl1 levels in wild-type mice at 4 weeks after intravenous administration of Ad-β-gal (1×109 plaque-forming units) or Ad-Fstl1 (1×109 plaque-forming units). Lower panel shows quantitative analysis of plasma Fstl1 levels evaluated by ImageJ program. n=6 in each group. (B) Quantitative analysis of urinary albumin excretion normalized to urinary creatinine in sham-operated and subtotal nephrectomy–operated wild-type mice at 4 weeks after intravenous administration of Ad-β-gal or Ad-Fstl1. n=4 in each sham-operated group. n=8 in each nephrectomy-operated group. (C) Histologic assessment of renal hypertrophy by PAS staining. Upper panels show representative photos of remnant kidneys from Ad-β-gal–treated or Ad-Fstl1–treated wild-type mice. Lower panels show quantitative analyses of glomerular cross-sectional area (left) and intraglomerular cell number (right). n=6 in each group. Scale bars represent 50 μm. (D) Evaluation of interstitial fibrosis by Masson’s Trichrome staining. Upper panels show representative photos of remnant kidney from Ad-β-gal–treated or Ad-Fstl1–treated wild-type mice. Lower panel shows quantitative analysis of fibrosis area evaluated by a WinROOF program. n=6 in each group. Scale bars represent 50 μm. (E) Relative mRNA levels of proinflammatory mediators (TNF-α, IL-6, and MCP-1), NADPH oxidase components (P40phox, P47phox, and P22phox), and fibrosis parameters (collagen I, collagen III, TGF-β1, and CTGF) in remnant kidneys in wild-type mice treated with Ad-β-gal (black bars) or Ad-Fstl1 (white bars). mRNA levels were measured by quantitative RT-PCR method. All samples are normalized to 36B4. n=8 in each group. CTGF, connective tissue growth factor. All data are presented as mean±SEM. *P<0.05 for Ad-β-gal group.
Figure 4.
Figure 4.
FSTL1 protein attenuates inflammatory response to TNF-α in cultured human mesangial cells. These cells were pretreated with FSTL1 protein, at 100 or 250 ng/ml, or vehicle for 1 hour, followed by stimulation with or without TNF-α (10 ng/ml). mRNA levels of IL-6 (left panel) and TNF-α (right panel) were determined by quantitative RT-PCR method. Baseline in the absence of FSTL1 and TNF-α means vehicle-treated control. All data are presented as mean±SEM. n=4 in each group. All samples are normalized to 36B4.
Figure 5.
Figure 5.
Fstl1 activates AMPK signaling in cultured mesangial cells and remnant kidney. (A) Time course analysis of phospho-ACC (pACC), ACC, phospho-AMPK (pAMPK), AMPK, and α-tubulin (tubulin) by Western blot analysis. Mesangial cells were treated with FSTL1 protein (250 ng/ml) for the indicated length of time (0, 5, 15, 30, and 60 minutes). (B) Immunostaining of phosphorylated AMPK and DAPI in remnant kidney from control and cFstl1-KO mice after renal ablation. Original magnification, ×200. (C) Immunostaining of phosphorylated AMPK and DAPI in remnant kidney from Ad-β-gal–treated and Ad-Fstl1–treated wild-type mice after renal ablation. Original magnification, ×200. (D) Protein levels of pAMPK, AMPK, and α-tubulin in the remnant kidneys from control and cFstl1-KO mice after sham or subtotal nephrectomy surgery. Right panel shows quantitative analysis of pAMPK/α-tubulin as evaluated by ImageJ program. All data are presented as mean±SEM. n=4 in each group. DAPI, 4,6-diamidino-2-phenylindole.
Figure 6.
Figure 6.
FSTL1 suppresses the expression of proinflammatory cytokines in cultured mesangial cells via activation of AMPK. (A) Protein levels of phosphorylated ACC (P-ACC), ACC, C-Myc, and α-tubulin by Western blot analysis. Human mesangial cells were treated with adenovirus expressing dominant negative mutant form of AMPK tagged with c-myc (Ad-dn-AMPK, 100 moi) or β-galactosidase (Ad-β-gal, 100 moi) for 24 hours, followed by 5-minute treatment with FSTL1 at 250 ng/ml or vehicle. (B) Relative mRNA levels of IL-6 and TNF-α in mesangial cells treated with FSTL1 and/or TNF-α after inhibition of AMPK activation. Cells were transduced with Ad-β-gal or Ad-dn-AMPK for 24 hours and treated with FSTL1 at 250 ng/ml or vehicle for 1 hour, followed by 4-hour stimulation with or without TNF-α (10 ng/ml). All data are presented as mean±SEM. n=4 in each group. All samples are normalized to 36B4.

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