Application of Scanning Ion Conductance Microscopy in the Studies of Podocytes Morphological Changes

Abstract

High‐resolution imaging of glomerular podocytes morphology is becoming an essential and helpful tool for studying their role in renal disease progression. Electron microscopy (EM) is commonly used to analyze the effacement of the podocytes’ cell foot processes and the glomerular filtration barrier integrity loss. However, EM does not allow studying living samples and requires multiple complex procedures for sample preparation. Here, we propose and demonstrate the successful application of Scanning Ion Conductance Microscopy (SICM) for studying morphological changes of cultured podocytes and freshly isolated glomeruli.SICM provides a high‐resolution (comparable to EM) non‐optical imaging of cell topography using hopping probe microscopy principles and new dynamic reconstruction software. This technique can be used for analysis of cell membrane morphology and different functional characteristics, such as cell volume, membrane potentials, single ion‐channel currents, and other parameters. The major advantage of the current method is that it can be applied to live samples with complex morphology, such as intact glomeruli, under physiologically relevant liquid conditions.We have established and demonstrated a successful application of the SICM method in the live human glomerulus and provided a proof of principle for future dynamic analysis of membrane morphology and varied functional parameters in the living glomerulus. As a proof of principle, we applied this method for estimation the pathological structural changes in podocyte foot processes in type 2 diabetic nephropathy (T2DN) rats. Using topographical quantification of the length and irregular shape of the podocyte foot processes, we showed an absence of interdigitated foot processes on the selected region of the glomerular slit diaphragm in isolated T2DN glomeruli.In addition to the application of SICM with freshly isolated glomeruli, we used this approach to visualize and quantify foot process dynamics in cultured human podocytes during pharmacological applications. Thus, we showed that destroying the F‐actin with cytochalasin D completely stopped the migration of the podocyte filopodium through the remodeling of the actin cytoskeleton. Here, we further applied SICM to detect morphological changes in human podocytes after activation of Protease‐activated receptor‐1 (PAR1) by acute application of agonist TFLLR‐NH2. A series of experiments revealed that PAR1 activation led to retraction of the lamellipodium and a significant decrease in cell surface area (+51±46 vs. ‐15±8 µm2, 30 min after application of vehicle or TFLLR, respectively; n=5, p<0.05).In summary, SICM has numerous advantages and has excellent potential for precise quantitative analyses of the morphological changes in the glomerulus and cultured podocytes. Moreover, it opens the possibility of performing patch‐clamp recordings on the specific parts of the podocyte cell body and advances our understanding of the function and mechanism of the glomerulus filtration barrier.