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Stuart J Shankland

Publications and source records attributed to Stuart J Shankland.

42 records · Page 3Linked to original sources
PubMed2002

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.

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PubMed2002

Insulin is a potent survival factor in mesangial cells: role of the PI3-kinase/Akt pathway.

BACKGROUND: Elucidating the mechanisms of apoptosis is important for understanding the molecular mechanisms underlying glomerular disease. The phosphatidylinositol 3 kinase (PI3-kinase)/Akt pathway is essential for survival signaling in non-renal cells. However, little is known about the anti-apoptotic effect of insulin and the role of the PI3-kinase/Akt pathway in mesangial cells (MC) apoptosis. METHODS: Apoptosis was induced in wild type, p27Kip1 (p27) -/- and p21Cip1/Waf1 (p21) -/- mouse MC by survival factor withdrawal, actinomycin D, ultraviolet (UV)-B irradiation and cycloheximide in the presence or absence of insulin (1 micromol/L) or insulin-like growth factor-I (IGF-I; 100 ng/mL). The activation and levels of Akt, extracellular signal regulated kinase (ERK) and specific cell cycle proteins were determined by Western blot analysis. RESULTS: Insulin and IGF-I inhibited wild-type MC apoptosis induced by survival factor withdrawal, actinomycin D, ultraviolet-B irradiation and cycloheximide and in p27 -/- MC when apoptosis was induced by survival factor withdrawal. Akt was activated by insulin and IGF-I during apoptosis. Blocking PI3-kinase with LY294002 reduced Akt activation and abrogated the anti-apoptotic effect of insulin. ERK was activated during apoptosis and blocking ERK activation with U0126 or PD98059 partially rescued MC from apoptosis. Moreover, insulin also suppressed ERK activation during apoptosis. Our results also showed that the CDK-inhibitor p21 was increased by insulin and that p21 up-regulation was PI3-kinase/Akt pathway dependent. Furthermore, p21 -/- MC apoptosis induced by survival factor withdrawal was not rescued by insulin in contrast to the wild-type and p27 -/- MC. These data suggest that p21 may have a critical role in the anti-apoptotic effect of insulin. CONCLUSIONS: Insulin is a potent survival factor for MC in response to a number of different apoptotic triggers, and this effect is mediated through the PI3-kinase/Akt pathway. Moreover, ERK and p21 may be involved in anti-apoptotic effect of insulin in MC.

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PubMed2002

Effects of cyclosporine in osteopontin null mice.

BACKGROUND: Osteopontin (OPN) is a macrophage adhesive and cell survival factor that is up-regulated in tubules in tubulointerstitial disease. We have previously reported that rats with cyclosporine (CsA) nephropathy have increased tubular osteopontin that correlates with the infiltration of macrophages and interstitial fibrosis. This study tested the hypothesis that the absence of OPN would ameliorate CsA nephropathy. METHODS: OPN knockout (-/-) and wild type (+/+) mice were fed a low salt diet (Na+ 0.01%) for one week and then received daily CsA injections (30 mg/kg, SC) until sacrifice at two weeks. Afferent arteriolopathy, tubulointerstitial injury, macrophage infiltration, collagen III deposition, transforming growth factor-beta (TGF-beta) expression, and tubular and interstitial cell proliferation and apoptosis were evaluated. RESULTS: Wild type mice developed early features of CsA nephropathy, with arteriolar hyalinosis and cortical and tubulointerstitial fibrosis. Despite comparable CsA levels, OPN-/- mice had less arteriolopathy (15 vs. 24%, P < 0.05), a 20% reduction in cortical macrophage infiltration (P < 0.05), and 20% reduction in interstitial collagen deposition (P < 0.05). OPN-/- mice also showed less cortical interstitial cell proliferation but no differences in tubular cell proliferation or apoptosis. OPN+/+ mice also developed some neurotoxicity, consisting of ataxia, and this was associated with increased mortality at two weeks. CONCLUSION: OPN partially mediates arteriolopathy, early macrophage recruitment and fibrosis in murine CsA nephropathy. OPN also may be involved in CsA associated neurotoxicity.

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PubMed2002

The hypertrophic effect of transforming growth factor-beta is reduced in the absence of cyclin-dependent kinase-inhibitors p21 and p27.

Transforming growth factor-beta (TGF-beta) has both antiproliferative and hypertrophic effects on mesangial cells (MC). However, it is not known if these processes are independent or if they share common signaling pathways. Proliferation and hypertrophy are regulated by specific cell-cycle regulatory proteins, where the cyclin-dependent kinase (CDK) inhibitors inhibit target cyclin-CDK complexes. This study examined whether the growth regulatory effects of TGF-beta were determined by the CDK inhibitors p21 and p27. Accordingly, cultured MC from wild type (+/+) and single and double null (-/-) p21 and p27 mice were grown in 5% serum in the presence or absence of TGF-beta1 (2 ng/ml). Proliferation ([(3)H]-thymidine incorporation, cell number, cell cycle) and hypertrophy ([(3)H]-leucine incorporation, total protein content, forward light scatter) were measured after 24 h, 48 h, and 96 h. TGF-beta inhibited proliferation in +/+ and p21/p27 double -/- MC to a similar extent. TGF-beta induced hypertrophy in +/+ MC (18.0% increase at 48 h), and to lesser extent in p21 -/- (12.8%) and p27 -/- MC (11.5%) measured by forward light scatter analysis. In p21/p27 double -/-, the hypertrophic effects of TGF-beta were significantly reduced (3.9% at 48 h). Similar results were obtained by measuring hypertrophy by total protein and [(3)H]-leucine incorporation. In conclusion, the CDK inhibitors p21 and p27 are not required for the antiproliferative effects of TGF-beta. However, the hypertrophic growth effects of TGF-beta are reduced in the absence of both p21 and p27. These data suggest that the regulation of the antiproliferative and hypertrophic effects of TGF-beta may be distinct processes.

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PubMed2001

Cyclooxygenase-2 overexpression inhibits platelet-derived growth factor-induced mesangial cell proliferation through induction of the tumor suppressor gene p53 and the cyclin-dependent kinase inhibitors p21waf-1/cip-1 and p27kip-1.

Cyclooxygenase-2 (COX-2) is an inducible enzyme and serves as a source of paracrine prostaglandin E2 (PGE2) formation in many tissues. In glomerular immune injury COX-2 formation is up-regulated in association with increased mesangial cell growth. To examine whether COX-2 exerts growth modulating effects on glomerular cells, we established two separate COX-2-overexpressing mesangial cell lines (COX-2+) and assessed their proliferative response to the potent mesangial cell growth-promoting factor, platelet-derived growth factor (PDGF). PDGF increased proliferation in mock-transfected cells. In contrast, PDGF did not induce proliferation in COX-2+ cells. Our results also showed that the tumor suppressor protein p53 and the cyclin-dependent kinase inhibitors p21(cip-1) and p27(kip-1) were up-regulated in COX-2+ cells de novo as well as under PDGF-stimulated conditions. To study whether COX-2 products are required for these effects, COX-2+ cells were treated with indomethacin (1 microg/ml) or NS-398 (3 microm). Unexpectedly, both COX inhibitors had no significant effect on cell proliferation, not on the protein levels of p53, p21(cip-1), or p27(kip-1). To evaluate the role of p21(cip-1) and p27(kip-1), COX-2 was overexpressed in mesangial cells derived from p21(cip-1) (p21-/- COX-2+) and p27(kip-1) (p27-/- COX-2+) null mice. In contrast to the wild type COX-2+ cells, p21-/- COX-2+ and p27-/- COX-2+ cells proliferated in response to PDGF. These data suggest that COX-2 inhibits mesangial cell proliferation by a novel mechanism that is independent of prostaglandin synthesis, but involves p53, p21(cip-1), and p27(kip-1).

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