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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 19, 2002 as doi:10.1096/fj.02-0125fje. |
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Department of Anatomy and Cell Biology, University of Heidelberg, D-69120 Heidelberg, Germany;
* Max Planck Institute for Molecular Genetics, D-14195 Berlin, Germany; and
Medical Research Center (ZMF), Klinikum Mannheim, University of Heidelberg, D-68167 Mannheim, Germany
4Correspondence: Institut für Anatomie und Zellbiologie I, INF 307, D-69120 Heidelberg, Germany. E-mail: karlhans.endlich{at}urz.uni-heidelberg.de
SPECIFIC AIMS
To understand the molecular alterations of podocytes in glomerular hypertension, the aim of the present study was to identify genes that are differentially regulated by mechanical stress in podocytes.
PRINCIPAL FINDINGS
1. Identification of differentially regulated genes in stretched podocytes using cDNA arrays
Differentiated mouse podocytes of a conditionally immortalized cell line were cultured on flexible silicone membranes stretched in a biaxial (5% linear strain) and cyclic (0.5 Hz) fashion for 3 days. Total RNA isolated from stretched podocytes and unstretched controls was hybridized on nylon membranes, on which cDNA of 6144 clones of a mouse library were spotted in duplicate (Fig. 1
A). Expression levels (averaged normalized intensities) on stretch vs. control membranes clustered around a straight line with r2 = 0.93 (Fig. 1B
). Sixteen genes were differentially expressed in response to mechanical stress in podocytes. Up-regulated genes were osteopontin (OPN), tumor susceptibility gene TSG101, pyruvate kinase M2, heat shock cognate protein of 73 kDa (hsc73), 28S rRNA, ribosomal proteins L7 and L12, RNA binding protein DEF-3, calmodulin I. Down-regulated genes were ferritin heavy and light chain, NADH dehydrogenase subunits 4 and 4L, glutathione S-transferase (GST1–1), tissue transglutaminase (TGM2), EST AI465668. OPN showed the strongest up-regulation (2.9-fold, Fig. 1C
).
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2. Up-regulation of OPN in stretched podocytes and glomeruli of DOCA salt-treated rats
OPN up-regulation in stretched podocytes was confirmed by RT-PCR and Northern blot. To extend this finding to the in vivo situation, expression of OPN was examined by RT-PCR in glomeruli isolated from desoxycorticosterone acetate (DOCA) salt-treated rats (2.5 wk treatment) and untreated controls. DOCA salt treatment of rats is a well-characterized model to induce focal segmental glomerular sclerosis (FSGS) through elevation of glomerular capillary pressure. Systemic blood pressure in DOCA salt-treated rats is already elevated after 2 wk of treatment, when glomerular damage is practically absent. Compared with unchanged glyceraldehyde-3-phosphate dehydrogenase expression, OPN expression was increased 2.9 ± 0.4-fold (P<0.05; n=4) in glomeruli of DOCA salt-treated rats, suggesting that OPN expression was increased primarily through the elevated glomerular pressure at this early point. Treatment of cultured podocytes with DOCA (1 µM, 3 days) did not change OPN expression.
3. OPN alters the actin cytoskeleton in podocytes
Because of the possible effect of OPN on podocytes as a secreted matrix protein, OPN was studied in greater detail. Podocytes cultured for 3 days on OPN-coated coverslips showed a marked decrease in the number of stress fibers and size of focal adhesions (vinculin staining) compared with cells on collagen IV. At the same time, OPN coating increased the number of cells with lamellipodia and enhanced localization of actin nucleation proteins (cortactin and ARP2/3 complex) to the cell margins. Thus, OPN induced a migratory phenotype in podocytes with an increase of dynamic F-actin structures.
4. OPN enhances the adaptation of podocytes to mechanical stress
We have shown recently that mechanical stress induces a unique reorganization of the actin cytoskeleton in podocytes: thick and dense bundles of transversal stress fibers are replaced by a few radially oriented stress fibers and an actin-rich center (ARC) is formed in every cell. (cf. Fig. 2
e, f). Whether OPN, which augments dynamic F-actin structures, facilitates this stretch-induced actin reorganization in podocytes was tested in stretch experiments at a higher strain of 7%. Since OPN could act on podocytes as a matrix component or a soluble factor, three protocols were used: 1) collagen IV coating; 2) collagen IV coating + OPN coating and OPN medium supplementation (15 nM); 3) collagen IV coating + OPN coating. Within 1 day, mechanical stress induced an ARC in 85% of podocytes in the presence of OPN, but only in 45% of podocytes when OPN was absent. After 1 day of stretching, we did not note a striking effect of OPN on the number of podocytes that were attached to the substratum. After application of mechanical stress for 2 days, actin reorganization of podocytes on collagen IV was again incomplete (Fig. 2d
), whereas ARCs and radial stress fibers had formed in almost all cells in the presence of OPN (Fig. 2e, f
). The number of podocytes was about threefold lower on membranes coated only with collagen IV compared with those to which OPN had been added (Fig. 2B, C
). Thus, OPN accelerated the stretch-induced actin reorganization in podocytes and enhanced the ability of podocytes to resist stretch, with OPN coating alone being sufficient to elicit this effect.
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CONCLUSIONS AND SIGNIFICANCE
Glomerular hypertension is a major factor that accelerates the progression to end-stage renal failure. Increased systemic blood pressure, disbalance between pre- and postglomerular vascular resistances, and impaired renal hemodynamic autoregulation have been shown to lead to increased levels or fluctuations in glomerular pressure in DOCA salt-treated, diabetic, and fawn-hooded hypertensive rats. Progressive nephron degeneration in theses models follows the pattern of "classic" FSGS at the glomerular vascular pole. The concept has emerged that progressive loss of podocytes initiates the development of FSGS through formation of tuft adhesions to Bowman’s capsule. Podocytes, which are thought to counteract pressure-mediated capillary expansion as a kind of pericyte, are increasingly challenged in glomerular hypertension. We have recently shown that podocytes are mechano-sensitive and that mechanical stress induces a reorganization of the actin cytoskeleton in podocytes. However, changes at the molecular level in podocytes in response to mechanical stress are unknown.
In the present study, we uncover molecular mechanisms that are likely to be operative in glomerular hypertension. Using cDNA array analysis, several genes were discovered to be up- or down-regulated in podocytes in response to mechanical stress. The known functions of these differentially regulated genes suggest that mechanical stress affects the following aspects in podocytes (Fig. 3
): cell–matrix interaction, signal transduction, mitochondrial and metabolic function, oxidative stress, protein synthesis, and degradation.
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OPN, whose expression is known to be increased likewise in stretched osteoblasts and proximal tubular cells, was the most strongly up-regulated gene in podocytes in response to mechanical stress. By sensitive RT-PCR, we detected an increase in glomerular OPN mRNA already after 2.5 wk of DOCA salt treatment, arguing for a primary role of increased glomerular pressure in up-regulating glomerular OPN in vivo. In support of our findings, enhanced glomerular OPN expression in antiglomerular basement membrane and DOCA salt-treated rats after 6 wk of treatment has been visualized in podocytes and parietal glomerular epithelial cells.
In response to mechanical stress, podocytes exhibit a unique reorganization of the actin cytoskeleton, consisting of ARCs and radially arranged stress fibers. This stretch-induced actin reorganization is likely to be necessary for podocytes to withstand mechanical stress. In OPN-treated podocytes, this reorganization was markedly accelerated when cells were subjected to mechanical stress, since OPN reduced stress fibers, diminished the size of focal adhesions, and increased dynamic actin structures containing actin nucleation proteins (ARP2/3 complex, cortactin). Accelerated formation of ARCs and radial stress fibers in the presence of OPN markedly enhanced the ability of podocytes to withstand mechanical stress. SPARC (secreted protein acidic and rich in cysteine), whose expression was shown to be up-regulated in stretched podocytes in a preliminary report, is known to decrease stress fibers and could enhance the resistance of podocytes to mechanical stress through a similar mechanism as presented for OPN (Fig. 3)
.
In summary, we identified several genes that are differentially regulated by mechanical stress in podocytes. Differential expression of these genes points to specific alterations in podocyte function, guiding further investigations into the molecular mechanisms of glomerular hypertension. Specifically, our results demonstrate that OPN, being up-regulated in stretched podocytes and in glomeruli of DOCA salt-treated rats, induces a shift to dynamic actin structures, thereby enhancing adaptation of podocytes to mechanical stress.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0125fje; to cite this article, use FASEB J. (September 19, 2002) 10.1096/fj.02-0125fje 
2 Present address: Department of Anatomy, School of Dentistry at Tokyo, The Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan.
3 Present address: Scienion AG, Volmerstraße 7b, D-12489 Berlin, Germany.
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) Up-regulation/increase; (
) down-regulation/decrease; (OPN) osteopontin; (TGM2) tissue transglutaminase; (SPARC) secreted protein acidic and rich in cysteine.