Membrane raft redox signalling contributes to renal tubular cell epithelial–mesenchymal transition and fibrosis and inflammation of the kidneys

Chronic renal disease is often associated with tubulointerstitial fibrosis, which can contribute to organ insufficiency and failure. Renal tubulointerstitial fibrosis is characterized by epithelial–mesenchymal transition (EMT), during which epithelial cells transform into myofibroblasts. Reduced cell–cell adhesion, lowered E-cadherin expression, increased synthesis of α-smooth muscle actin (α-SMA), actin reorganization, collagen and fibronectin deposition, and tubular basement membrane disruption are all associated with EMT. Membrane rafts (MRs), defined as sterol, sphingolipid and glycosphingolipid domains on cellular membranes, are closely associated with NADPH oxidase redox activity. NADPH oxidase contributes to EMT in response to several stimuli, including transforming growth factor-β1 (TGF-β1), angiotensin II (AngII) and glucose. However, the exact role of the MR redox signalling pathway in TGF-β1-induced EMT and AngII-induced inflammation and fibrosis remains unknown. In a recent study published in The Journal of Physiology, Han et al. (2018) manipulated the MR redox signalling pathway using small hairpin RNA (shRNA) for translocated lysosomal V-type proton ATPase subunit E2 (Atp6v1e2) and sphingomyelin phosphodiesterase 1 (SMPD1). Atp6v1e2 contributes to a local microenvironment that facilitates SMPD1 activation, which leads to MR clustering and activation. By knocking down these proteins, and thereby inhibiting the MR redox signalling pathway, the researchers were able to study how MR redox signalling is implicated in renal EMT, inflammation and fibrosis. The authors began their study by using labelled ganglioside GM1 to show that TGF-β1-induced MR clustering could be significantly inhibited with Atp6v1e2 and SMPD1 shRNA in renal tubular cells. Furthermore, TGF-β1-treated renal tubular cells that were transfected with Atp6v1e2 and SMPD1 shRNA showed decreased expression of mesenchymal markers, including α-SMA and fibroblast-specific protein-1, as well as increased expression of E-cadherin, compared to scrambled shRNA-transfected, TGF-β1-treated controls. Collectively, these data demonstrate that TGF-β1 activates the MR redox signalling pathway, which contributes to EMT in renal tubular cells. Next, the authors used immunoblotting to show a time-dependent increase in extracellular signal-regulated kinases 1 and 2 (Erk1/2) phosphorylation following TGF-β1 treatment in renal tubular cells. Moreover, they were able to show attenuated Erk1/2 phosphorylation in TGF-β1-treated renal tubular cells that were transfected with Atp6v1e2 and SMPD1 shRNA. It is already well established that Erk1/2 phosphorylation, downstream of reactive oxygen species (ROS) formation, is critical for EMT. Importantly, the authors reason that TGF-β1-induced EMT in renal tubular cells is partially mediated through ROS, derived from the MR redox signalling pathway and subsequent Erk1/2 phosphorylation. Thereafter, using immunoblotting, Han et al. (2018) demonstrated that simvastatin treatment significantly inhibited TGF-β1-induced ROS production, EMT and Erk1/2 phosphorylation in renal tubular cells. Previously, statins have been shown to play a protective role against ROS-mediated renal EMT and fibrosis. Consistent with these previous findings, the authors showed for the first time that ROS, derived from the MR redox signalling pathway, play a critical role in TGF-β1-induced EMT by acting through Erk1/2 phosphorylation in renal tubular cells. In addition to their in vitro studies, Han et al. (2018) also investigated the in vivo role of the MR redox signalling pathway. Chronic AngII infusion was used to induce fibrosis and inflammation in adult rats, thereby allowing the researchers to examine whether MR redox signalling is involved in the development of renal pathology. First, the authors found that chronic AngII infusion increased the systolic blood pressure of the rats; however, increases in blood pressure did not differ between scramble control, Atp6v1e2 and SMPD1 shRNA-transfected rats. Moreover, none of the rats exhibited changes in body weight and renal blood flow following chronic AngII infusion. These results suggest that the effects observed in Atp6v1e2 and SMPD1 knock-down mice are not a result of changes to systolic blood pressure and renal blood flow. Rather, the authors speculated that any potential effects of Atp6v1e2 and SMPD1 shRNAs in transfected rats would be a result of the inhibition of the MR redox signalling pathway. To further investigate the effects of MR redox signalling in adult rats, renal tubular cells were isolated with Percoll density gradient centrifugation, and a three-layer flotation assay was used to assess MR clustering in the kidney. The results showed that cells isolated from AngII-infused rats exhibited higher ROS expression, increased SMPD1 activity, and translocation of NADPH oxidase subunit 4 (Nox4) from the non-MR domain to the MR domain, all of which are indicative of MR redox signalling pathway activation. These data collectively demonstrate that chronic AngII infusion activates the MR redox signalling pathway in adult rat renal tubular cells. Finally, Han et al. (2018) used immunoblotting and histology to demonstrate that chronic AngII infusion induced fibrosis in rat kidneys. The researchers observed characteristic features of fibrosis, including elevated collagen deposition and increased activation of fibroblasts in the interstitium and renal tubule. Likewise, rats with chronic AngII infusion exhibited higher expression of classic fibrotic markers including fibronectin, α-SMA and collagen type 1. The transfection of Atp6v1e2 and SMPD1 shRNA into adult rats significantly reduced AngII-induced increases in collagen deposition, myofibroblast numbers and profibrotic markers. As well, the authors demonstrated a decreased expression of inflammatory markers, including monocyte chemoattractant protein 1 (MCP-1), tumour necrosis factor α (TNF-α) and intercellular adhesion molecule 1 (ICAM-1), in AngII-infused, Atp6v1e2 and SMPD1 knock-down rats. Together, these data show that the MR redox signalling pathway contributes significantly to AngII-induced renal fibrosis and inflammation in rats. For the first time, Han et al. (2018) provide evidence, both in vitro and in vivo, of the role that MR redox signalling plays in renal EMT, inflammation and fibrosis. Their results demonstrate that TGF-β1, which serves to activate the MR redox signalling pathway, induces EMT through ROS production via Nox4, Erk1/2 phosphorylation and MR clustering in renal tubular cells. Likewise, the authors demonstrate that AngII-induced inflammation and fibrosis can be attenuated in rats that are deficient of Atp6v1e2 and SMPD1 proteins, which are critical for MR redox signalling in vivo. Several studies have implicated EMT in the progression of various fibrotic diseases, including cardiac, pulmonary and renal fibrosis (Lamouille et al. 2014). Han et al. (2018) have successfully shown that the inhibition of the MR redox signalling pathway minimizes EMT in vitro. Further, the group also showed that preventing MR redox signalling in vivo attenuates fibrosis. However, the researchers did not investigate whether the attenuation of AngII-induced fibrosis was mediated through the inhibition of EMT in vivo. In the future, researchers can investigate whether inhibition of the MR redox signalling pathway prevents EMT in vivo, thereby attenuating the development of fibrosis. Moreover, Han et al. (2018) were able to show that inflammation, as assessed by the production of the cytokines MCP-1, ICAM-1 and TNF-α, was minimized in AngII-infused rats following inhibition of the MR redox signalling pathway. Previously, studies have shown that inflammation plays a critical role in mediating the progression of various pathologies, including renal fibrosis, by acting through several complex and interconnected pathways (Eddy, 2000). Given the critical role of inflammation in disease progression, researchers should investigate the inflammatory pathways associated with or downstream of MR redox signalling. Furthermore, a better understanding of the associations between MR redox signalling and inflammation may reveal novel therapeutic targets for pathologies characterized by fibrosis and inflammation. Irrespective of the affected organ, fibrosis is characterized by pathological tissue remodelling, including extracellular matrix deposition and EMT, which can ultimately lead to organ failure and death (Wynn et al. 2012). Han et al. (2018) demonstrated features characteristic of fibrosis in their AngII-induced model of renal tubulointerstitial fibrosis. However, their studies provide an additional insight into how the MR redox signalling pathway contributes to the development of fibrosis. In the future, researchers can investigate whether the MR redox signalling pathway plays a similar role in other fibrotic diseases, like cardiac fibrosis or pulmonary fibrosis. Overall, Han et al. (2018) have shown that the MR redox signalling pathway plays a critical role in mediating renal tubular cell EMT in vitro, and fibrosis and inflammation of the kidneys in vivo. Although the authors have made great progress in characterizing MR redox signalling, there remains great potential for further research in this area. None. Both H.S. and A.H. designed, drafted and provided critical revisions to the manuscript. Both authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. None.

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