Quantifying cellular mechanics and adhesion in renal tubular injury using single cell force spectroscopy

Abstract

Tubulointerstitial fibrosis represents the major underlying pathology of diabetic nephropathy where loss of cell-to-cell adhesion is a critical step. To date, research has predominantly focussed on the loss of cell surface molecular binding events that include altered protein ligation. In the current study, atomic force microscopy single cell force spectroscopy (AFM-SCFS) was used to quantify changes in cellular stiffness and cell adhesion in TGF-β1 treated kidney cells of the human proximal tubule (HK2). AFM indentation of TGF-β1 treated HK2 cells showed a significant increase (42%) in the Elastic modulus (stiffness) compared to control. Fluorescence microscopy confirmed that increased cell stiffness is accompanied by reorganization of the cytoskeleton. The corresponding changes in stiffness, due to F-actin rearrangement, affected the work of detachment by changing the separation distance between two adherent cells. Overall, our novel data quantitatively demonstrate a correlation between cellular elasticity, adhesion and early morphologic/phenotypic changes associated with tubular injury.

Connexin‐mediated cell communication in the kidney: A potential therapeutic target for future intervention of diabetic kidney disease?

“…Whilst the structural and functional disturbances discussed above are synonymous with early stages of diabetic nephropathy, tubulointerstitial fibrosis (TIF), represents the final common pathway of chronic renal failure and exhibits a positive correlation to the onset of end‐stage renal disease and entry onto the renal transplantation programme (Hodgkins & Schnaper, ). Recent findings from our laboratory confirm that tubular CX43 and CX26 expression is increased in biopsy material from people with diabetic nephropathy (Siamantouras, Hills, Squires, & Liu, ), and work by Abed et al ., reported that patients with chronic kidney disease (CKD) exhibit elevated levels of CX43. Using a mouse model of hypertension‐induced CKD, they demonstrated decreased expression of cell adhesion markers, reduced monocyte infiltration and interstitial renal fibrosis when CX43 expression was reduced by 50%.…”

“…How aberrant CX‐mediated communication impacts on cell function forms the basis of our ongoing studies. In early TIF, exposure to glucose and downstream pro‐fibrotic cytokines initiates a series of events beginning with altered expression of the epithelial adhesion protein E‐cadherin (Hills et al., ; Siamantouras et al., ). Cadherins help form the multi‐protein adherens junction that links cell‐to‐cell contact to the actin cytoskeleton and associated signalling molecules.…”

“…It is therefore unsurprising that intercellular adhesion is a pre‐requisite for connexin oligomerisation and gap junction formation. The loss of cell adhesion is the driving force behind morphological and phenotypic changes associated with early tubular injury (Siamantouras et al., ), and has been linked to glucose‐evoked changes in connexin expression and GJIC (Siamantouras et al., ). Although more than a dozen fibrogenic factors affect renal function, it is widely recognised that TGF‐β1 and downstream Smad signalling represent the key pathway orchestrating renal fibrosis in diabetic nephropathy (reviewed in Hills & Squires, ; Rauchman & Griggs, ).…”

Transforming Growth Factor Beta 1 Drives a Switch in Connexin Mediated Cell-to-Cell Communication in Tubular Cells of the Diabetic Kidney

“…Human kidney (HK2) cells (passage 18-30) were maintained in DMEM/Hams F12 medium, supplemented with 10% FCS wt/vol, glutamine (2mmol/l) and EGF (5ng/ml), and cultured at 37°C in a humidified atmosphere with 5% CO 2 . Prior to treatment, cells were transferred to DMEM/F12 low glucose (5mmol/l) for 48h as described previously [26]. Cells were serum-starved overnight before incubating in TGF-β1 (2–10ng/ml), glucose (25mmol/l), conditioned media or L-glucose (25mmol/l) for 48h.…”

Identification of Transcription Factors related to Diabetic Tubulointerstitial Injury

“…Besides, particular molecules derived from extracellular exosomes have been suggested to serve as potential diagnostic biomarkers in DN including AQP2(22), AQP5(22) and let-7c-5p(23). The loss of molecularbinding events between cell surfaces is also involved in diabetic tubulointerstitial fibrosis(24). KEGG pathway analysis of targeted DEGs showed that these DEGs were primarily mapped to complement and coagulation cascades, tight junction and cell adhesion molecules.…”