#1115 TGFβ induced mechanosignaling controls the transcriptome and microenvironmental composition of proximal tubules in chronic kidney disease

Abstract

Abstract Background and Aims Interstitial fibrosis of the renal parenchyma and progressive atrophy of proximal tubules (PT) are hallmark features of chronic kidney disease (CKD). The latter is characterized by pronounced thickening and multilamellation of the tubular basement membrane, whereas fibrosis is promoted by accumulation of extracellular matrix (ECM) translating into increased matrix rigidity. The role of mechanotransduction via the integrin adhesion complex (IAC) as well as the corresponding signaling programs activated in this context remain largely elusive. Here, we aimed to elucidate the functional role of the ILK-Pinch-Parvin (IPP)-complex as an essential part of the IAC in response to PT damage and CKD. Method Morphometric assessment of a CKD patient biopsy cohort as well as ischaemia-reperfusion injury (IRI) mouse models was performed. Transcriptome studies of human proximal tubular epithelial cells (hRPTECs) under pro-fibrotic and rigid matrix conditions were analyzed. An in vitro model, mimicking CKD, was established and functionally characterized. Reanalysis of single-cell RNA sequencing (scRNA-seq) from a human CKD cohort was employed for cross-correlation and validation. Knockout (KO) models for the IPP-complex were generated by CRISPR/Cas9 genome editing and combined with functional assays (e.g. ECM deposition and sensing). Results In IRI mice, we observed a strong recruitment of the ILK-Pinch-Parvin (IPP)-complex and related-IAC proteins to the tubular basement membrane of proximal tubules (PTs) showing features of tubular atrophy. These findings were translated to CKD patients further assessing markers of active mechanosignaling (YAP) as well as tubular damage (SOX9). Rigidity sensing assays in a hRPTEC model were employed to define the underlying mechanisms. Remarkably, extensive transcriptome analysis revealed that only under pro-fibrotic signaling conditions, hRPTECs sensed rigidity alterations in the underlying substratum, and translated these changes into regulation of matrisome- and adhesome-related gene signatures (including members of the IPP-complex). Further filtering and cross-correlation with available scRNA-seq data from CKD-patients highlighted differential expression of secreted signaling proteins including the matricellular molecule CCN1. Subsequent functional studies demonstrated the underlying pivotal role of the IPP-complex as a central signaling node required for active mechanosignaling in damaged hRPTECs. Conclusion In CKD, IPP and other IAC proteins are recruited to the massively thickened tubular basement membrane of atrophic tubules, indicating a critical role in disease progression. Our results demonstrate that hRPTECs do not express IAC proteins in healthy conditions. However, when inflammation processes (TGFβ treatment) occur, PTs undergo a phenotypic switch, resulting in altered IAC expression signatures. Our data indicate that IAC proteins are crucially involved in sensing of the microenvironmental composition of PTs and thereby reciprocally fine-tune auto-/paracrine signaling properties of epithelial cells.