Scanning ion conductance microscopy of live human glomerulus

Abstract Podocyte damage is a hallmark of glomerular diseases, such as focal segmental glomerulosclerosis, typically associated with marked albuminuria and progression of renal pathology. Podocyte structural abnormalities and loss are also linked to minimal change disease and more common diabetic kidney disease. Here we applied the first‐time scanning ion conductance microscopy (SICM) technique to assess the freshly isolated human glomerulus's topology. SICM provides a unique opportunity to evaluate glomerulus podocytes as well as other nephron structural segments with electron microscopy resolution but in live samples. Shown here is the application of the SICM method in the live human glomerulus, which provides proof of principle for future dynamic analysis of membrane morphology and various functional parameters in living cells.


| INTRODUC TI ON
Glomerulus represents a tuft of capillaries located at the entry portion of the nephron, where blood is selectively filtered across the glomerular filtration barrier consisting of endothelial cells, glomerular basement membrane, and podocytes. Primary and secondary processes of adjacent podocytes intertwine, creating a slit diaphragm that tightly covers glomeruli capillaries. This diaphragm has a complex morphology and forms the final barrier for blood filtration contributing to size selectivity and permitting permeability to molecules smaller than albumin. A reduction in podocyte number and foot process effacement was reported in focal segmental glomerulosclerosis, minimal change disease and diabetic nephropathy. [1][2][3] Moreover, podocytes' loss and modification of their architecture can be evident at the very early stages of kidney diseases that may underlie/exacerbate their progression. High-resolution imaging of cell morphology is becoming an essential and useful tool for studying cellular structures and the role they play in cell function and disease progression. 4 Scanning electron microscopy (SEM) is a widely used method for conducting such studies. Still, it requires multiple complex procedures for sample preparation, making it impossible to analyse a living sample. 5 Therefore, there is a critical need

Abstract
Podocyte damage is a hallmark of glomerular diseases, such as focal segmental glomerulosclerosis, typically associated with marked albuminuria and progression of renal pathology. Podocyte structural abnormalities and loss are also linked to minimal change disease and more common diabetic kidney disease. Here we applied the firsttime scanning ion conductance microscopy (SICM) technique to assess the freshly isolated human glomerulus's topology. SICM provides a unique opportunity to evaluate glomerulus podocytes as well as other nephron structural segments with electron microscopy resolution but in live samples. Shown here is the application of the SICM method in the live human glomerulus, which provides proof of principle for future dynamic analysis of membrane morphology and various functional parameters in living cells.
to develop new tools to study alterations of podocyte morphology in live samples. Hopping Probe mode of Scanning Ion Conductance Microscopy (SICM) is a technique that enables high-resolution, nonoptical imaging of living cell surfaces with complex morphology. [6][7][8] The major advantage of this method is a close to several nanometres spatial resolution and a long Z (vertical) range, which can be applied to live samples with convoluted morphology under physiologically relevant conditions. SICM is a multimodal imaging technique that can be combined with other established techniques, concurrent and dynamic analysis of membrane morphology and various functional parameters, such as cell volume, membrane potentials, single ion-channel currents and even the dynamics of membrane protein complexes in living cells. [9][10][11] Here, we provide an example of the application of SICM to analyse the morphology of live human glomerulus structure.

| MATERIAL S AND ME THODS
For the experiment, we used a part of the cortical area from the discarded transplant human kidney dissected and stored in Wisconsin preservation solution followed by the incubation in an oxygenated physiological saline solution (PSS). 12 Renal glomeruli were isolated using a vibrodissociation technique as previously described. 13 This approach allows the rapid isolation of well-preserved renal glomeruli from human kidneys. To perform SICM imaging, freshly iso-

| RE SULTS AND D ISCUSS I ON
SICM image quality is comparable to SEM and allows precise examination of glomerulus surface structure, including blood vessels and podocytes, and the architecture of the secondary foot processes. 4 In our previous study, we used SICM imaging to estimate the pathological structural changes in podocyte foot processes in type 2 diabetic nephropathy (T2DN) rats. 15 We conducted a comparative analysis of the three-dimensional architecture of the podocyte filtration barrier in non-diabetic and T2DN rats. Our data revealed a significant loss of podocyte foot processes in T2DN glomeruli providing an accurate evaluation for histopathological changes occurred in diabetic nephropathy that underlie nephrinuria and albuminuria in these rats. 15 Here, for the first time, we demonstrated a successful approach in assessing the three-dimensional surface structure of freshly isolated,  In summary, the method described here has excellent potential for the studies of glomeruli and other freshly isolated nephron segments. Furthermore, it shows that it is possible to use human kidney samples without elaborate preparation to evaluate morphological changes in the study of pathologies.

ACK N OWLED G EM ENTS
Research in the authors' laboratory was funded by the Department of Veteran Affairs I01 BX004024, the National Heart, Lung, and Blood Institute R35 HL135749 and P01 HL116264, the National Institute of Diabetes and Digestive and Kidney Diseases R01 DK126720, and the American Physiological Society Research Career Enhancement Award.

CO N FLI C T O F I NTE R E S T
Dr Andrew Shevchuk is a shareholder and receives the consulting fees from the ICAPPIC Ltd. All other authors declared no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.