Prostaglandin E(2) is crucial in the response of podocytes to fluid flow shear stress

Abstract

Podocytes play a key role in maintaining and modulating the filtration barrier of the glomerulus. Because of their location, podocytes are exposed to mechanical strain in the form of fluid flow shear stress (FFSS). Several human diseases are characterized by glomerular hyperfiltration, such as diabetes mellitus and hypertension. The response of podocytes to FFSS at physiological or pathological levels is not known. We exposed cultured podocytes to FFSS, and studied changes in actin cytoskeleton, prostaglandin E(2) (PGE(2)) production and expression of cyclooxygenase-1 and-2 (COX-1, COX-2). FFSS caused a reduction in transversal F-actin stress filaments and the appearance of cortical actin network in the early recovery period. Cells exhibited a pattern similar to control state by 24 h following FFSS without significant loss of podocytes or apoptosis. FFSS caused increased levels of PGE(2) as early as 30 min after onset of shear stress, levels that increased over time. PGE(2) production by podocytes at post-2 h and post-24 h was also significantly increased compared to control cells (p < 0.039 and 0.012, respectively). Intracellular PGE(2) synthesis and expression of COX-2 was increased at post-2 h following FFSS. The expression of COX-1 mRNA was unchanged. We conclude that podocytes are sensitive and responsive to FFSS, exhibiting morphological and physiological changes. We believe that PGE(2) plays an important role in mechanoperception in podocytes.

Keywords: Actin; Cyclooxygenase; Mechanical strain; Prostaglandin E2; Shear stress.

Fig. 1

The figure on the left shows the arrangement of apparatus used to apply fluid flow shear stress (FFSS) to podocytes. It shows the ‘fluid device chamber’ in which six glass slides containing podocytes are placed for FFSS studies, and the 3-way stop cock attachment to collect media during application of FFSS. The figure on the right provides the experiment design for collection of culture media at 3 different time points

Fig. 2

The figure shows immunostaining for F-actin in control podocytes, and in podocytes exposed to FFSS at end of FFSS (120 min), during early recovery period following FFSS at 2 h (post-2 h FFSS) and 24 h (post-24 h FFSS) in the left column (400X). The right column shows podocytes pre-treated with indomethacin (2.5 μM for 1 h) prior to application of FFSS during early recovery period at 2 h (post-2 h FFSS) and 24 h (post-24 h FFSS) in the presence of indomethacin (200X). Indomethacin prevented FFSS-induced changes in F-actin

Fig. 3

The figure shows control podocytes, and podocytes exposed to FFSS (upper panel) stained with crystal violet for cellular morphology during early recovery period following FFSS at 2 h (post-2 h FFSS) and 24 h (post-24 h FFSS). Podocytes show an indistinct cellular margin and blunted cytoplasmic processes compared to control cells at post-2 h following application of FFSS, changes that are resolving by post-24 h FFSS. The lower panel shows podocytes pre-treated with indomethacin (2.5 μM for 1 h) prior to application of FFSS during early recovery period at 2 h (post-2 h FFSS) and 24 h (post-24 h FFSS). Indomethacin prevented FFSS-induced changes in cell morphology in podocytes

Fig. 4

Prostaglandin E₂ in media collected at time 0 min, 30 min and 120 min during application of fluid flow shear stress (FFSS) at 2 dynes/cm² are shown as bar figure with ±1 SE and the absolute difference in prostaglandin E₂ from 5 separate experiments are shown as a line figure (* denotes p < 0.05)

Fig. 5

Prostaglandin E₂ in culture media prior to FFSS, and following recovery at 2 and 24 h post application of FFSS from control and experimental podocytes are shown as bar figure with ±1 SE, and the absolute difference in prostaglandin E₂ from 5 separate control and FFSS experiments are shown as line figures (* denotes p < 0.05)

Fig. 6

The measured intracellular PGE₂/DNA and the change in PGE₂/DNA from control podocytes following FFSS from 5 separate experiments are shown as a bar figure with ±1 SE and as a line figure respectively (* denotes p < 0.05)

Fig. 7

The figure shows the gene expression for COX-1 and COX-2 in podocytes following application of FFSS. a The gel figure shows an increase in COX-2 gene expression at post-2 h following application of FFSS that begins to resolve at post-24 h FFSS. COX-1 expression is unchanged with FFSS. b The bar graph denotes the change in gene expression for COX-1 (n = 3) and COX-2 (n = 6) on RT-PCR at post-2 h and post-24 h FFSS (* denotes p < 0.05)

Fig. 8

The figure shows changes in F-actin on phalloidin staining and cell morphology on crystal violet staining in podocytes following continuous treatment with PGE₂ from 0–1.25–2.5–5–10–20 µM (from left to right) without FFSS for 2 h (upper two panels) and 24 h (lower two panels). The early discernable changes start to appear at 5 μM and are marked at 10 μM and 20 μM at both 2 h and 24 h