Abstract
Indoxyl sulfate (IS) accumulation occurs early during chronic kidney disease (CKD) progression and contributes to renal dysfunction by inducing fibrosis, inflammation, oxidative stress, and tissue remodeling. Renal toxicity of high IS concentrations (250 μM) has been widely explored, particularly in resident tubular and glomerular cells, while the effect of a moderate IS increase on kidneys is still mostly unknown. To define the effects of IS accumulation on renal fibroblasts, we first analyzed kidneys of C57BL/6 mice receiving IS (0.1%) in drinking water for 12 weeks. As a next step, we treated renal fibroblasts (NRK-49F) with IS (20 μM) with or without the HSP90 inhibitor 17-AAG (1 μM). In mouse kidneys, IS increased the collagen deposition and HSP90 and α-SMA expression (immunohistochemistry) in interstitial fibroblasts and caused tubular necrosis (histological H&E and picrosirius red staining). In NRK-49F cells, IS induced MCP1, TGF-β, collagen I, α-SMA, and HSP90 gene/protein expression and Smad2/3 pathway activation. IS had no effects on fibroblast proliferation and ROS production. 17-AAG counteracted IS-induced MCP1, TGF-β, collagen I, and α-SMA expression and Smad2/3 phosphorylation. Our study demonstrates that the IS increase promotes renal fibroblast activation by a HSP90-dependent pathway and indicates HSP90 inhibition as a potential strategy to restrain IS-induced kidney inflammation and fibrosis in CKD.
Highlights
In patients with chronic kidney disease (CKD), the progressive decline of the glomerular filtration rate (GFR) and kidney metabolic function hinders the removal of several endogenous toxins which are normally cleared by the kidney
Kidneys of C57BL/6 mice receiving continuous supplementation of 0.1% Indoxyl sulfate (IS) in drinking water displayed signs of tubular necrosis (Figure 1(a)) and interstitial fibrosis whose severity was evaluated by picrosirius red staining (Figure 1(b))
We found a significant increase in α-SMA protein in interstitial fibroblasts (2-fold vs. control mice, p < 0 05) (Figure 1(c)) and upregulation of HSP90 in the tubular-interstitial compartment (1.4-fold increase vs. control mice, p < 0 05) (Figure 1(d))
Summary
In patients with chronic kidney disease (CKD), the progressive decline of the glomerular filtration rate (GFR) and kidney metabolic function hinders the removal of several endogenous toxins which are normally cleared by the kidney. Previous in vitro observations reveal that even a moderate increase in IS affects cell homeostasis and induces tissue remodeling [5], and several clinical studies point out that IS levels predict the progression of CKD [6]. Renal toxicity of high IS concentrations (exceeding 250 μM) has been widely explored, in particular in resident tubular and glomerular cells. Both in proximal tubule cells and in podocytes [7], IS has profibrotic [8], prooxidant [9], and proinflammatory [10] action, while the effect of a moderate increase in circulating IS levels in kidneys is far to be Oxidative Medicine and Cellular Longevity defined. Renal fibrosis is a common adaptive response to a variety of pathological triggers, and fibroblast activation in the kidney contributes to tissue remodeling by collagen production and release of profibrotic factors [11], being involved in the activation of multiple pathways which include the TGF-β and the Smad downward signaling [12]
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