HIV infection changes glomerular podocyte cytoskeletal composition and results in distinct cellular mechanical properties

Abstract

In addition to forming the selective filtration barrier for the renal glomerulus, podocytes maintain glomerular capillary architecture by opposing distending hemodynamic forces. To understand the relationship of cytoskeletal properties and the mechanical characteristics of podocytes, we studied filamin expression and distribution and measured cell membrane deformability in conditionally immortalized wild-type (WT) mouse podocytes, and in podocytes derived from a mouse model of HIV-associated nephropathy (HIVAN). In the WT cells, filamin and F-actin were localized at the periphery and in prominent stress fibers. In the HIVAN cells, filamin expression was reduced, and stress fibers were sparse. In a microaspiration assay, HIVAN cells ruptured under minimal negative pressure. Atomic force microscopy demonstrated that the WT cells had a stiffness of 17 kPa, whereas the value for the HIVAN cells was 4 kPa. These results demonstrate that the mechanical properties of WT and HIVAN podocytes are markedly different in a manner that is consistent with differences in the composition and arrangement of their cytoskeletons. The mechanical properties of the WT podocytes suggest that these cells can better maintain capillary integrity than the HIVAN podocytes and implicate pathological assembly of the cytoskeleton as a mechanism of HIVAN.

Biophysical properties of normal and diseased renal glomeruli

“…where fbead is the force on the bead, dcantilever is the deflection of the cantilever measured by the AFM, E is the Young's modulus, v is the Poisson ratio, R is the radius of the bead, and ␦ is the vertical displacement of the cantilever (41). As the external surface of glomeruli is composed primarily of exposed capillaries, the measurements will most likely probe the properties of the external capillary wall.…”

“…AFM probes submicron areas of membrane over a subsecond time scale, and as such, reflects the intrinsic characteristics of a structure since cells and tissues cannot actively remodel their cytoskeletons or membranes over this time scale (41). The external surface of a glomerulus is composed primarily of exposed capillaries, the majority of which are covered by podocyte foot processes as well as some podocyte cell bodies.…”

“…All methods to analyze elasticity involve measuring deformation in response to a mechanical force. Elasticity can be measured by a number of techniques including atomic force microscopy (AFM), a technique that measures local characteristics (subcellular or single cell level), rheometry, or microindentation that measure multicellular or tissue characteristics, or the newly developed capillary micromechanics that measures the complete mechanical characteristics (compressive and shear deformation) of a soft object such as a glomerulus (20,21,26,41). Local and aggregate tissue mechanical properties may have different implications for tissue behavior and response to injury.…”

“…We used AFM to measure the Young's modulus or deformability of the outer surface of glomerular capillaries and the new micromechanics method that involves deformation of whole glomeruli in glass microcapillaries, capillary micromechanics, to measure the full elastic response of whole glomeruli (41,44). To determine whether abnormal glomerular biophysical properties are associated with glomerular disease, we studied glomeruli from two different mouse models of glomerular disease Col4a3…”

Mechanisms of mechanical signaling in development and disease

“…For example, it was shown that conditionally immortalized glomerular podocytes from a mouse model of HIVassociated nephropathy (HIVAN) are substantially softer than normal podocytes (by 25%), as assessed by atomic force microscopy (AFM) and microaspiration (Tandon et al, 2006). At a stage of the disease that showed no detectable pathological changes, the glomeruli from these mice were 30% softer than normal glomeruli (1.5 kPa compared with 2.5 kPa, as measured by AFM) (Wyss et al, 2011).…”