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Similar Biophysical Abnormalities in Glomeruli and Podocytes from Two Distinct Models.

ABSTRACT: Background FSGS is a pattern of podocyte injury that leads to loss of glomerular function. Podocytes support other podocytes and glomerular capillary structure, oppose hemodynamic forces, form the slit diaphragm, and have mechanical properties that permit these functions. However, the biophysical characteristics of glomeruli and podocytes in disease remain unclear.Methods Using microindentation, atomic force microscopy, immunofluorescence microscopy, quantitative RT-PCR, and a three-dimensional collagen gel contraction assay, we studied the biophysical and structural properties of glomeruli and podocytes in chronic (Tg26 mice [HIV protein expression]) and acute (protamine administration [cytoskeletal rearrangement]) models of podocyte injury.Results Compared with wild-type glomeruli, Tg26 glomeruli became progressively more deformable with disease progression, despite increased collagen content. Tg26 podocytes had disordered cytoskeletons, markedly abnormal focal adhesions, and weaker adhesion; they failed to respond to mechanical signals and exerted minimal traction force in three-dimensional collagen gels. Protamine treatment had similar but milder effects on glomeruli and podocytes.Conclusions Reduced structural integrity of Tg26 podocytes causes increased deformability of glomerular capillaries and limits the ability of capillaries to counter hemodynamic force, possibly leading to further podocyte injury. Loss of normal podocyte mechanical integrity could injure neighboring podocytes due to the absence of normal biophysical signals required for podocyte maintenance. The severe defects in podocyte mechanical behavior in the Tg26 model may explain why Tg26 glomeruli soften progressively, despite increased collagen deposition, and may be the basis for the rapid course of glomerular diseases associated with severe podocyte injury. In milder injury (protamine), similar processes occur but over a longer time.

SUBMITTER: Embry AE

PROVIDER: S-EPMC5967771 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Similar Biophysical Abnormalities in Glomeruli and Podocytes from Two Distinct Models.

Embry Addie E AE Liu Zhenan Z Henderson Joel M JM Byfield F Jefferson FJ Liu Liping L Yoon Joonho J Wu Zhenzhen Z Cruz Katrina K Moradi Sara S Gillombardo C Barton CB Hussain Rihanna Z RZ Doelger Richard R Stuve Olaf O Chang Audrey N AN Janmey Paul A PA Bruggeman Leslie A LA Miller R Tyler RT

Journal of the American Society of Nephrology : JASN 20180323 5

<b>Background</b> FSGS is a pattern of podocyte injury that leads to loss of glomerular function. Podocytes support other podocytes and glomerular capillary structure, oppose hemodynamic forces, form the slit diaphragm, and have mechanical properties that permit these functions. However, the biophysical characteristics of glomeruli and podocytes in disease remain unclear.<b>Methods</b> Using microindentation, atomic force microscopy, immunofluorescence microscopy, quantitative RT-PCR, and a thre ...[more]

PMID: 29572404

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Project description:This paper compares the biophysical and mechanistic properties of a typical type I dehydroquinase (DHQase), from the biosynthetic shikimate pathway of Escherichia coli, and a typical type II DHQase, from the quinate pathway of Aspergillus nidulans. C.d. shows that the two proteins have different secondary-structure compositions; the type I enzyme contains approx. 50% alpha-helix while the type II enzyme contains approx. 75% alpha-helix. The stability of the two types of DHQase was compared by denaturant-induced unfolding, as monitored by c.d., and by differential scanning calorimetry. The type II enzyme unfolds at concentrations of denaturant 4-fold greater than the type I and through a series of discrete transitions, while the type I enzyme unfolds in a single transition. These differences in conformational stability were also evident from the calorimetric experiments which show that type I DHQase unfolds as a single co-operative dimer at 57 degrees C whereas the type II enzyme unfolds above 82 degrees C and through a series of transitions suggesting higher orders of structure than that seen for the type I enzyme. Sedimentation and Mr analysis of both proteins by analytical ultracentrifugation is consistent with the unfolding data. The type I DHQase exists predominantly as a dimer with Mr = 46,000 +/- 2000 (a weighted average affected by the presence of monomer) and has a sedimentation coefficient s0(20,w) = 4.12 (+/- 0.08) S whereas the type II enzyme is a dodecamer, weight-average Mr = 190,000 +/- 10,000 and has a sedimentation coefficient, s0(20,w) = 9.96 (+/- 0.21) S. Although both enzymes have reactive histidine residues in the active site and can be inactivated by diethyl pyrocarbonate, the possibility that these structurally dissimilar enzymes catalyse the same dehydration reaction by the same catalytic mechanism is deemed unlikely by three criteria: (1) they have very different pH/log kcat. profiles and pH optima; (2) imine intermediates, which are known to play a central role in the mechanism of type I enzymes, could not be detected (by borohydride reduction) in the type II enzyme; (3) unlike Schiff's base-forming type I enzymes, there are no conserved lysine residues in type II amino acid sequences.

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Similar Biophysical Abnormalities in Glomeruli and Podocytes from Two Distinct Models.

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Similar Biophysical Abnormalities in Glomeruli and Podocytes from Two Distinct Models.

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