Na/K-ATPase/src complex mediates regulation of CD40 in renal parenchyma

Na/K-ATPase/src complex mediates regulation of CD40 in renal parenchyma Jeffrey X. Xie1, Shungang Zhang1, Xiaoyu Cui2, Jue Zhang3, Hui Yu3, Fatimah K. Khalaf1, Deepak Malhotra1, David J. Kennedy1, Joseph I. Shapiro2, Jiang Tian1 and Steven T. Haller1 1 Department of Medicine, University of Toledo College of Medicine and Life Sciences, Toledo, OH, USA, 2Marshall Institute for Interdisciplinary Research, Marshall University, Huntington, WV, USA and 3Department of Pediatrics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Correspondence and offprint requests to: Steven T. Haller; E-Mail: ABSTRACT INTRODUCTION Background. Recent studies have highlighted a critical role for CD40 in the pathogenesis of renal injury and fibrosis. However, little is currently understood about the regulation of CD40 in this setting. Methods. We use novel Na/K-ATPase cell lines and inhibitors in order to demonstrate the regulatory function of Na/KATPase with regards to CD40 expression and function. We utilize 5/6 partial nephrectomy as well as direct infusion of a Na/K-ATPase ligand to demonstrate this mechanism exists in vivo. Results. We demonstrate that knockdown of the a1 isoform of Na/K-ATPase causes a reduction in CD40 while rescue of the a1 but not the a2 isoform restores CD40 expression in renal epithelial cells. Second, because the major functional difference between a1 and a2 is the ability of a1 to form a functional signaling complex with Src, we examined whether the Na/KATPase/Src complex is important for CD40 expression. We show that a gain-of-Src binding a2 mutant restores CD40 expression while loss-of-Src binding a1 reduces CD40 expression. Furthermore, loss of a functional Na/K-ATPase/Src complex also disrupts CD40 signaling. Importantly, we show that use of a specific Na/K-ATPase/Src complex antagonist, pNaKtide, can attenuate cardiotonic steroid (CTS)-induced induction of CD40 expression in vitro. Conclusions. Because the Na/K-ATPase/Src complex is also a key player in the pathogenesis of renal injury and fibrosis, our new findings suggest that Na/K-ATPase and CD40 may comprise a pro-fibrotic feed-forward loop in the kidney and that pharmacological inhibition of this loop may be useful in the treatment of renal fibrosis. Na/K-ATPase is a P-type ATPase capable of transporting sodium and potassium against their concentration gradients [1]. It has been established that four isoforms of the a subunit of Na/K-ATPase exist—of note, the a1 isoform is expressed ubiquitously, while the a2 isoform is predominately found in skeletal muscle [2]. Interestingly, studies from our group and others have demonstrated that in addition to its role as an ion transporter, the a1 isoform can form a functional signaling complex with Src, a non-receptor tyrosine kinase, which is known to be involved in numerous signaling cascades [3]. This Na/K-ATPase/Src signaling complex can be activated by the binding of cardiotonic steroids (CTSs) [4–6]. However, some controversy still exists regarding whether Na/K-ATPase and Src directly interact within this complex [7]. CD40, a type I transmembrane receptor of the toll-like receptor superfamily, was initially discovered as a mediator of humoral immune reactions [8]. However, it is now understood that CD40 is widely expressed, including within renal proximal tubular epithelial cells, and may serve broader functions [9, 10]. Interestingly, we have previously shown that circulating levels of the ligand for CD40, soluble CD40 ligand (sCD40L), are significantly increased in subjects with renal artery stenosis [11]. Follow-up studies have identified circulating CD40 mediators as predictive of changes in renal function in subjects with renal artery stenosis [12] and chronic kidney disease [13]. Furthermore, we have recently demonstrated that functional deletion of CD40 in a strain of rat prone to kidney disease improved renal function and attenuated renal fibrosis [14]. However, little is currently understood about the regulation of CD40 in the setting of renal disease. Recent studies have established a critical role of the Na/K-ATPase/Src signaling complex in the regulation of Keywords: CD40/CD40L, inflammation, Na/K-ATPase, renal fibrosis, Src C The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. V 1138 ORIGINAL ARTICLE Nephrol Dial Transplant (2018) 33: 1138–1149 doi: 10.1093/ndt/gfx334 Advance Access publication 22 December 2017 cellular signaling pathways important for reactive oxygen species (ROS) generation, inflammation and fibrosis in the kidney [15–17]. Given that both Na/K-ATPase and CD40 play central roles in mediating renal fibrosis and are known to reside in caveolae [18, 19], we speculate that Na/K-ATPase may crosstalk and be critically involved in the regulation of CD40 within renal parenchyma. Demonstration of a relationship between Na/KATPase and CD40 would reveal the existence of a profibrotic feed-forward signaling loop and identify a new pharmacological target for the treatment of renal fibrosis. MATERIALS AND METHODS Materials Cell culture LLC-PK1 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA; CL-101). For a detailed description of the generation of stable mutant Na/K-ATPase cell lines, please see relevant publications and Table 2 [20–23]. Cells were cultured in DMEM supplemented with 10% FBS with 100 U/mL penicillin and streptomycin. Animals Male Sprague Dawley rats were used for all of the studies. All of the animal experimentation described in the article was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals using protocols approved by the University of Toledo, Health Science Campus, Institutional Animal Use and Care Committee. Rats weighing between 250 and 300 g (n  8 per group) were Na/K-ATPase mediated regulation of CD40 Immunoblot analysis (in vivo) Renal cortex tissue was isolated from kidneys and homogenized in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (pH 7.0) (Santa Cruz Biotechnology; sc-24948). The proteins, obtained from tissue homogenates, were resolved on an SDS-PAGE using Precast Ready Gels 4–15% Tris-HCl, purchased from Bio-Rad. In all, 60 lg of protein per sample were loaded into each well. The proteins from the gel were electrotransferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in 20 mmol/L Tris-HCl (pH 7.5, 150 mmol/L NaCl and 0.1% Tween 20). Mouse antiCD40 antibody (Abcam) was used to probe for CD40 and secondary anti-mouse antibody was purchased from Invitrogen (Grand Island, NY, USA). Immunoblot analysis (in vitro) Cells were washed with cold phosphate-buffered saline (PBS) and then lysed into radioimmunoprecipitation buffer (0.25% deoxycholate, 1% Nonidet P-40, 1 mM EDTA) with 1 Halt Protease and Phosphatase Inhibitor (Thermo Fisher, #78440). Cell lysates wer (...truncated)