Mechanical stress reduces podocyte proliferation in vitro.
BACKGROUND
Mechanical stretch, a consequence of capillary glomerular hypertension, is thought to be the common final pathway for glomerulosclerosis in systemic hypertension, diabetes, reduced nephron number and focal segmental glomerulosclerosis. However, the effects of stretch on podocyte growth and the mechanisms that underlie this have not been elucidated.


METHODS
Mouse podocyte growth (3H-thymidine, MTT-assay, FACS) was measured following the application of mechanical stretch created by vacuum. The expression of specific cell cycle regulatory proteins was examined by RNAse protection assay and Western blot analysis. Control cells were grown under similar conditions, but were not exposed to stretch.


RESULTS
Mechanical stretch decreased DNA-synthesis (3H-thymidine incorporation) and cell number (MTT-assay) in podocytes at 24, 48 and 72 hours (P < 0.001 vs. control non-stretched cells), which was not due to apoptosis (Hoechst staining) nor cell detachment. Stretch decreased the mRNA and protein levels of cyclins D1, A and B1 within 24 hours. Stretching cells decreased the activity of Cdk2 (measured by histone H1 kinase assay) at 48 and 72 hours and Cdc2 at 72 hours. In contrast, stretch increased the protein levels of the cyclin dependent kinase inhibitors (CKI) p21Cip/Kip/Waf (p21) and p27Kip1 (p27) within the first 24 hours, and increased the mRNA levels of p57Kip2 (p57) at 72 hours. To examine the role of p21 in inhibiting proliferation induced by stretch, we studied p21-/- podocytes in culture. Stretch did not reduce proliferation in p21-/- podocytes (P> 0.05 vs. non-stretched podocytes; P < 0.001 vs. stretched p21+/+ podocytes).


CONCLUSIONS
In contrast to mesangial cells, mechanical stretch decreases the growth of podocytes. This effect is mediated through the regulation of specific cell cycle regulatory proteins. These events may explain the apparent lack of podocyte proliferation in diseases correlated with capillary glomerular hypertension.
Petermann
Arndt T
AT
Division of Nephrology, Department of Medicine, University of Washington School of Medicine, Seattle, Washington 98195, USA.
Hiromura
Keiju
K
Blonski
Mary
M
Pippin
Jeffrey
J
Monkawa
Toshi
T
Durvasula
Raghu
R
Couser
William G
WG
Shankland
Stuart J
SJ
D000818:Animals; D015153:Blotting, Western; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins / Q000032:analysis / Q000235:genetics; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D019925:Cyclin A / Q000032:analysis / Q000235:genetics; D019926:Cyclin B / Q000032:analysis / Q000235:genetics; D056744:Cyclin B1; D019938:Cyclin D1 / Q000032:analysis / Q000235:genetics; D056743:Cyclin D3; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D050761:Cyclin-Dependent Kinase Inhibitor p57; D018844:Cyclin-Dependent Kinases / Q000032:analysis / Q000378:metabolism; D016213:Cyclins / Q000032:analysis / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression / Q000502:physiology; D066298:In Vitro Techniques; D007678:Kidney Glomerulus / Q000737:chemistry / Q000166:cytology* / Q000201:enzymology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009687:Nuclear Proteins / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D013314:Stress, Mechanical; D025521:Tumor Suppressor Proteins / Q000032:analysis / Q000235:genetics
C534700:Ccnb1 protein, mouse; C534697:Ccnd3 protein, mouse; C493957:Cdkn1a protein, mouse; C493961:Cdkn1b protein, mouse; C493965:Cdkn1c protein, mouse; D018797:Cell Cycle Proteins; D019925:Cyclin A; D019926:Cyclin B; D056744:Cyclin B1; D056743:Cyclin D3; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050761:Cyclin-Dependent Kinase Inhibitor p57; D016213:Cyclins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases