Abstract
Recent studies have indicated that the development of acute and chronic kidney disease including renal fibrosis is associated with endoplasmic reticulum (ER) stress. S100 calcium-binding protein 16 (S100A16) as a novel member of the S100 family is involved in kidney disease; however, few studies have examined fibrotic kidneys for a relationship between S100A16 and ER stress. In our previous study, we identified GRP78 as a protein partner of S100A16 in HK-2 cells. Here, we confirmed a physical interaction between GRP78 and S100A16 in HK-2 cells and a markedly increased expression of GRP78 in the kidneys of unilateral ureteral occlusion mice. S100A16 overexpression in HK-2 cells by infection with Lenti-S100A16 also induced upregulation of ER stress markers, including GRP78, p-IRE1α, and XBP1s. Immunofluorescence staining demonstrated that the interaction between S100A16 and GRP78 predominantly occurred in the ER of control HK-2 cells. By contrast, HK-2 cells overexpressing S100A16 showed colocalization of S100A16 and GRP78 mainly in the cytoplasm. Pretreatment with BAPTA-AM, a calcium chelator, blunted the upregulation of renal fibrosis genes and ER stress markers induced by S100A16 overexpression in HK-2 cells and suppressed the cytoplasmic colocalization of GRP78 and S100A16. Co-immunoprecipitation studies suggested a competitive binding between S100A16 and IRE1α with GRP78 in HK-2 cells. Taken together, our findings demonstrate a significant increase in S100A16 expression in the cytoplasm following renal injury. GRP78 then moves into the cytoplasm and binds with S100A16 to promote the release of IRE1α. The subsequent phosphorylation of IRE1α then leads to XBP1 splicing that activates ER stress.
Highlights
The main pathological features of renal fibrosis are the proliferation of renal interstitial fibroblast and the accumulation of extracellular matrix (ECM)
We found that the protein expression of S100 calcium-binding protein 16 (S100A16) is significantly increased in the kidneys of unilateral ureteral occlusion (UUO) mice, an animal model of kidney fibrosis, and that the characteristic pathological changes of renal tubulointerstitial fibrosis appeared in the conditions and was further supported by IP experiments using Glucose-regulated protein 78 kD (GRP78) antibodies and blotting with S100A16 antibodies (Fig. 3A)
Overexpression in Human kidney-2 (HK-2) cells dramatically promoted the upregula-. These findings suggested that S100A16 regulates the epithelial-mesenchymal transition (EMT) tion of fibronectin, collagen I, and α-smooth muscle actin (α-SMA) compared with Lentiprocess of renal tubular cells by promoting the reorganization scramble infected HK-2 cells, in agreement with our previous of the cytoskeleton-associated with renal tubulointerstitial fibrosis [14]
Summary
The main pathological features of renal fibrosis are the proliferation of renal interstitial fibroblast and the accumulation of extracellular matrix (ECM). ER stress signaling, called the unfolded protein response (UPR), is a cellular adaptive mechanism that occurs in response to the disruption of ER homeostasis. GRP78 binds to the three ER stress sensors including protein kinase RNA-like ER kinase (PERK), inositolrequiring endoribonuclease 1α (IRE1α), and activating transcription factor 6 (ATF6). The PERK autophosphorylation activated the PERK-signaling pathway, and phosphorylates eukaryotic initiation factor-2 α (eIF2α). In order to prevent unfolded protein accumulation, cells suffered from ER stress will initiate the UPR. It can Received: 30 May 2021 Revised: 25 September 2021 Accepted: 1 October 2021
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