Biochemical and Cellular Determinants of Renal Glomerular Elasticity

Abstract

The elastic properties of renal glomeruli and their capillaries permit them to maintain structural integrity in the presence of variable hemodynamic forces. Measured by micro-indentation, glomeruli have an elastic modulus (E, Young’s modulus) of 2.1 kPa, and estimates from glomerular perfusion studies suggest that the E of glomeruli is between 2 and 4 kPa. F-actin depolymerization by latrunculin, inhibition of acto-myosin contractility by blebbistatin, reduction in ATP synthesis, and reduction of the affinity of adhesion proteins by EDTA reduced the glomerular E to 1.26, 1.7, 1.5, and 1.43 kPa, respectively. Actin filament stabilization with jasplakinolide and increasing integrin affinity with Mg2+ increased E to 2.65 and 2.87 kPa, respectively. Alterations in glomerular E are reflected in commensurate changes in F/G actin ratios. Disruption of vimentin intermediate filaments by withaferin A reduced E to 0.92 kPa. The E of decellularized glomeruli was 0.74 kPa, indicating that cellular components of glomeruli have dominant effects on their elasticity. The E of glomerular basement membranes measured by magnetic bead displacement was 2.4 kPa. Podocytes and mesangial cells grown on substrates with E values between 3 and 5 kPa had actin fibers and focal adhesions resembling those of podocytes in vivo. Renal ischemia and ischemia-reperfusion reduced the E of glomeruli to 1.58 kPa. These results show that the E of glomeruli is between 2 and 4 kPa. E of the GBM, 2.4 kPa, is consistent with this value, and is supported by the behavior of podocytes and mesangial cells grown on variable stiffness matrices. The podocyte cytoskeleton contributes the major component to the overall E of glomeruli, and a normal E requires ATP synthesis. The reduction in glomerular E following ischemia and in other diseases indicates that reduced glomerular E is a common feature of many forms of glomerular injury and indicative of an abnormal podocyte cytoskeleton.

Similar Biophysical Abnormalities in Glomeruli and Podocytes from Two Distinct Models

“…Podocytes have an important role in determining the elastic and structural characteristics of glomerular capillaries and glomeruli, although the contributions of podocytes and the GBM have not been resolved definitively. 4,5,9,39 Although mechanisms of injury differ, podocytes are also the primary glomerular cell injured in many diseases and disease models, including the Tg26 mouse and protamine administration. 13,17 In response to injury, podocytes rearrange their cytoskeletons with effacement of foot processes and loss of filtration barrier function, and this response to injury may correspond to increased deformability of podocytes and glomeruli.…”

“…Gene expression was probed with the oligos listed in Supplemental Material and normalized with glyceraldehyde-3-phosphate dehydrogenase control. 5,27 Measurement of Cell Adhesion Adhesion was measured by washing equally seeded cells after plating on collagen-or poly-L-lysine-coated 96-well plates for 30 minutes and related to paxillin phosphorylation. Adherent ).…”

“…The E of glomeruli reflects the E of capillary walls (WT 2.3 kPa) (Figure 2A, Supplemental Material). 5,26,29 With disease progression, Tg26 glomeruli become increasingly deformable. Although E decreased, extracellular matrix collagen staining increased with more advanced disease ( Figure 1).…”

“…The cytoskeletal structure of podocytes supports glomerular capillary structure in response to normal and pathologic hemodynamic forces, although the relative contributions of podocytes and the glomerular basement membrane (GBM) are not fully resolved. 4 Additionally, mechanical environment can affect the differentiated state of numerous cell types, including podocytes, [5][6][7] which are determinants of the mechanical properties of glomerular capillaries and glomeruli. 8,9 To understand the biophysical characteristics of glomeruli and podocytes in disease, we studied the mechanical properties of glomeruli and podocytes in two mechanistically distinct, established models of extreme and mild glomerular disease: Tg26 mice (podocytes expressing HIV, severe) and protamine sulfateinduced minimal change disease (acute, reversible, mild).…”

A High-Content Screening Technology for Quantitatively Studying Podocyte Dynamics

“…The function of this elegant filtration system is maintained by the interplay among these core constituents as well as the immaculate arrangement of the structural proteins within the membrane. The integrity 12 and elasticity 13 are other fundamental concepts since the capillary pressures far exceed those in other organs. The mechanical support required for glomerular capillaries are mainly provided by podocytes 14 since GBM and its associated cells are not rigid, but rather flexible 15 .…”