CST3 and GDF15 ameliorate renal fibrosis by inhibiting fibroblast growth and activation

https://doi.org/10.1016/j.bbrc.2018.04.061Get rights and content

Highlights

  • CST3 and GDF15 inhibit survival and collagen production of kidney fibroblasts.

  • CST3 inactivates fibroblasts by antagonizing the TGF-β-SMAD signaling pathway.

  • GDF15 inhibits the N-Myc induction in activated kidney fibroblasts.

  • Recombinant CST3 and GDF15 ameliorate kidney fibrosis in vivo.

Abstract

The final strategies to care patients with end-stage renal fibrosis rely on dialysis and kidney transplantation. Because such treatments are invasive and cause health problems eventually, it is necessary to develop new therapeutic strategies for delaying the disease progress. We here searched for cytokines showing an anti-fibrotic activity in cell-based experiments. Cystatin C (CST3) and Growth differentiation factor 15 (GDF15) were identified to have anti-fibrotic activities in a cytokine array screening. In primary fibroblasts isolated from the mouse kidneys subjected to ureteral obstruction-induced fibrosis, each cytokine induced apoptotic cell death and reduced collagen production. These anti-fibrotic effects were further augmented by co-administration of both cytokines. Mechanistically, CST3 and GDF15 were found to block the TGF-β receptor and the N-Myc signaling pathways, respectively. In mice with unilateral ureter obstruction, each cytokine and the combination of two cytokines effectively reduced the fibrotic burden in the subjected kidneys. Therefore, we propose that CST3 and GDF15 could be potential candidates for biopharmaceutics to ameliorate renal fibrosis.

Introduction

Chronic kidney disease (CKD), which includes all degrees of failure in renal function, evokes public health problem in worldwide [1,2]. Renal fibrosis is a representative pathogenesis in progressive CKD [3]. Renal fibrosis is defined as glomerulosclerosis, tubulointerstitial fibrosis, inflammatory infiltration, and parenchymal loss with tubular atrophy, capillary loss, and podocyte depletion [4]. It is associated with aberrant wound repair processes that stimulate the excessive deposition of extracellular matrix proteins, such as collagen, fibronectin, and laminin, between tubules and peritubular capillaries [5].

Renal fibrosis is initially involved in abnormal angiogenesis, capillary obliteration, and stimulation of pericytes and perivascular cells, followed by further stimulation of kidney resident cells through pro-inflammatory cytokines. Next, mesangial cells, fibroblasts, and tubular epithelial cells produce and deposit a large amount of extracellular matrix components in the interstitial space [4,6], which eventually cause chronic renal failure. Of diverse resident and infiltrating cells, activated fibroblast mainly contributes to extracellular matrix (ECM) deposition in the kidney. In wound healing process, fibroblast is activated through various injury signals such as TGF-β1, PDGF, and FGF-2, which is also escalated under inflammatory microenvironmental factors such as hypoxia, hyperglycemia, immune cell infiltration, and altered ECM composition [7]. Therefore, the clearance of activated fibroblasts is considered as a reasonable strategy for preventing or delaying the progression of renal fibrosis.

To overcome renal fibrosis, many drugs have been tested in clinical trials. Immunosuppressive agents, including corticosteroids and cyclosporine A, are considered as conventional therapeutics to delay the fibrogenic process. However, the long-term usage of such agents is not recommended because of serious metabolic disorders and kidney injury. Some inhibitors of angiotensin-converting enzyme or antagonists of angiotensin II receptor have been also suggested as useful regimens to preserve the glomerular function in renal fibrosis, but these agents also evoke severe side effects such as hypotension, decreased GFR and hyperkalaemia [8]. Recently, new anti-fibrotic strategies to specifically target fibroblasts have been developed, which include antibodies neutralizing PDGFs and TGF-β, ALK5 receptor kinase inhibitors, Ras inhibitors, or a fibroblast inactivator pirfenidone [9]. Despite of the advances of treatment strategy of renal fibrosis, translation to clinical trials has not shown successful result.

At the terminal stage of wound healing, fibroblast growth and activation naturally subside, which is reorganized by repaired epithelium. There is a growing consensus of opinion on the anti-fibrotic roles of the epithelium-derived cytokines. Accordingly, we tried to search for anti-fibrotic cytokines in epithelial cell conditioned media. In the present study, we identified and characterized Cystatin C (CST3) and Growth differentiation factor 15 (GDF15) as the inhibitors of activated renal fibroblasts. We also verified the effects of the cytokines against renal fibrosis in mice subjected to unilateral ureter obstruction (UUO).

Section snippets

Unilateral ureter obstruction (UUO)

Ten week-old c57BL/B6 mice were purchased from Central Laboratory Animal Inc. (Seoul, Korea). The left ureter was tied off at two points for UUO or not for Sham operation [10,11]. CST3/GDF15 peptides (50 μg/kg each) or PBS were injected intraperitoneally into mice every other day after the surgery. Recombinant peptides of active CST3 (aa. 27–146, NM_000099) and GDF15 (aa. 195–308, NM_004864) were purchased from Abcam (Cambridge, MA) and Sino Biological Inc. (Beijing, China), respectively. On

CST3 and GDF15 inhibit survival and collagen production of kidney fibroblasts

We tested the effects of various CMs on fibroblast growth. Of the CMs, HaCaT CM alone significantly inhibited the growth of MEF (Supplementary Fig. 1A), and it also induced cell death (Supplementary Fig. 1B). To examine whether peptide-based factors are responsible for the fibroblast growth inhibition, CMs were incubated at 95 °C for 30 min to denature peptides. The heat-inactivated HaCaT CM lose its inhibitory effect to fibroblast growth (Supplementary Fig. 1C), suggesting that some peptides

Discussion

Inactivation of fibroblasts must be one of the most compelling strategy to treat renal fibrosis. Although a few anti-fibrotic agents like Fresolimumab and Pirfenidone were tested in clinical studies, the outcomes of these trials were evaluated to be unsatisfactory in terms of renal failure treatment [16] [17]. Therefore, new agents should be developed to preserve renal function in patients with renal fibrosis-associated CKD. In this study, we propose that recombinant CST3 and GDF15 be potential

Competing financial interests

None of the authors have any competing interests.

Conflicts of interest

The authors declare no conflict of interest that might be construed to influence the results or interpretation of their manuscript.

Acknowledgements

This work was supported by the grant (2016R1A2A1A05005082) funded by the National Research Foundation of Korea.

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