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Stuart J Shankland

Publications and source records attributed to Stuart J Shankland.

At least 37 records · Page 2Linked to original sources
PubMed2004

Differential expression of d-type cyclins in podocytes in vitro and in vivo.

The proliferative response of podocytes to injury determines the histological phenotype. Moreover, an apparent lack of podocyte proliferation may underlie the development of glomerulosclerosis. Podocyte proliferation is closely linked with its state of differentiation. However, the mechanisms regulating these processes are not fully elucidated. Because D-type cyclins have been shown to be important in the regulation of proliferation and differentiation, we examined their expression in podocytes in vitro and in vivo. The glomerular expression of cyclins D1 and D3 was examined in vitro in cultured immortalized podocytes by immunostaining and Western blot analysis, and in embryonic mice and rats, the passive Heymann nephritis model of experimental membranous nephropathy in rats, and human immunodeficiency virus (HIV)-transgenic mice. Kidneys from cyclin D1 knockout mice were also examined. Cyclin D1 was abundant in cultured proliferating podocytes, but not in quiescent differentiated podocytes. In contrast, cyclin D3 was abundant in differentiated, but not proliferating podocytes. Cyclin D1 was expressed in embryonic mouse and rat glomeruli during the S- and comma-shaped stages, and was absent in podocytes at the capillary loop stage and in mature rodent glomeruli. Cyclin D1 protein increased after injury in passive Heymann nephritis rats and in HIV-transgenic mice. Cyclin D3 was constitutively and specifically expressed in podocytes in normal rodent glomeruli, and decreases during dedifferentiation and proliferation in HIV-transgenic mice. Kidneys from cyclin D1-/- mice were normal with the podocytes expressing specific differentiation markers. Cyclin D1 is not necessary for the terminal differentiation of podocytes, and expression coincides with cell-cycle entry. In contrast, cyclin D3 expression coincides with podocyte differentiation and quiescence.

Animals↗
PubMed2004

Cyclin-dependent kinase 5 is a regulator of podocyte differentiation, proliferation, and morphology.

Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in renal physiology, including the prevention of proteinuria. Cyclin-dependent kinase 5 (CDK5) has been shown to influence several cellular processes in other terminally differentiated cells, in particular neurons. In this study, we examined the role of CDK5 in podocyte differentiation, proliferation, and morphology. In conditionally immortalized mouse podocytes in culture, CDK5 increased in association with podocyte differentiation. During mouse glomerulogenesis in vivo, CDK5 expression was predominantly detected in podocytes from the capillary loop stage to maturation and persisted in the podocytes of adult glomeruli. In contrast, CDK5 was markedly decreased in the proliferating and dedifferentiated podocytes of mice with anti-glomerular basement membrane nephritis and in human immunodeficiency virus transgenic mice. p35, the activator of CDK5, was also detected in podocytes and the p35/CDK5 complex was active. Cell fractionation studies showed that active p35/CDK5 was mainly localized to the plasma membrane. Specific inhibition of CDK5 in differentiated cultured podocytes, either pharmacologically or with siRNA, induced shape changes, with cellular elongation and loss of process formation compared to the characteristic arborized phenotype. These data suggest a role for CDK5 as a regulator of podocyte differentiation, proliferation, and morphology.

Animals↗
PubMed2004

The rapamycin derivative RAD inhibits mesangial cell migration through the CDK-inhibitor p27KIP1.

The link between mesangial cell (MC) proliferation and migration during glomerular repair in the experimental mesangial proliferative glomerulonephritis suggests that cell cycle regulation and cell migration require similar pathways, such as cell cycle proteins. The immunosuppressant RAD inhibits mesangial cell (MC) proliferation via G1/S arrest. Moreover, RAD dramatically impairs glomerular healing in the anti-Thy1 model. We tested the hypothesis that RAD alters MC migration in vitro and that this effect was mediated by the CDK-inhibitors p21(CIP1) and p27(KIP1). Using a modified Boyden chamber in vitro migration assay, our results showed that RAD dose dependently (1-50 nM) inhibited fibronectin-induced chemotaxis in wild-type (wt) MC. RAD treatment prevented the decrease in p27(KIP1) induced by mitogenic growth factors, but had no effect on p21(CIP1) by Western blot analysis. The antimigratory effect of RAD in wt MC was substantially dependent on p27(KIP1), but not p21(CIP1), since the inhibitory effects of 1-10 nM RAD on MC migration were similar in p21(CIP1) deficient and wild-type MC. The effect of RAD on MC migration was also examined in the anti-Thy1 model by BrdU-labeling of proliferating MC on day 3 that typically repopulate the glomerulus from the hilus. A control biopsy on day 3 was taken to define the starting point prior to the initiation of RAD (3 mg/kg or placebo). MC migration was determined on day 7 by measuring the distances of BrdU-labeled MC (OX-7+/BrdU+cells) from the glomerular hilus using computerized morphometry. RAD significantly reduced the migratory response of BrdU-labeled MC compared to controls. We conclude that the immunosuppressant RAD effectively inhibits MC migration in vivo and in vitro thereby limiting the normal glomerular repair process after severe injury. Moreover, RAD-induced inhibition of MC migration in vitro is partially mediated by the CDK-inhibitor p27(KIP1), but not p21(CIP1).

Animals↗
PubMed2004

Activation of a local tissue angiotensin system in podocytes by mechanical strain.

BACKGROUND: Glomerular capillary hypertension, a common denominator in various forms of progressive glomerular disease, results in mechanical distention of the capillary tuft, and subsequent injury of the overlying podocyte layer. The mechanisms by which elevated intraglomerular pressure is translated into a maladaptive podocyte response remain poorly understood. Angiotensin II plays a central role in the pathogenesis of chronic renal injury, largely through its actions on the subtype 1 receptor. Accordingly, we have tested the hypothesis that mechanical strain up-regulates local angiotensin II in podocytes, thereby resulting in a progressive reduction in podocyte number. METHODS: Conditionally immortalized mouse podocytes were subjected to cyclical stretch of 10% amplitude. Nonstretched podocytes served as controls. Angiotensin II levels were measured in whole cell lysate by competitive enzyme-linked immunosorbent assay (ELISA). Expression of angiotensin II receptors (AT1R, AT2R) was measured by quantitative polymerase chain reaction (PCR) and Western blot analysis. Apoptosis was measured by Hoechst staining. Immunostaining for AT1R was performed in tissue sections from rats with 5/6 remnant kidney disease, a model of glomerular hypertension. RESULTS: Mechanical strain increased angiotensin II production in podocytes at 24, 48, and 72 hours (P < 0.05 vs. nonstretched controls). Stretching podocytes resulted in a fivefold increase in AT1R mRNA expression at 24 hours and a twofold increase in protein levels vs. controls (P < 0.05), and also an increase in transforming growth hormone-beta (TGF-beta) mRNA expression. AT1R staining was increased in a podocyte distribution in the 5/6 remnant kidney, consistent with our in vitro findings. Mechanical strain resulted in a 2.5-fold increase in apoptosis (P < 0.001 vs. nonstretched controls) in an angiotensin II-dependent fashion. CONCLUSION: Mechanical strain leads to up-regulation of the AT1R and increased angiotensin II production in conditionally immortalized podocytes. The resulting activation of a local tissue angiotensin system leads to an increase in podocyte apoptosis, mainly in an AT1R-mediated fashion.

Angiotensin II↗
PubMed2004

Viable podocytes detach in experimental diabetic nephropathy: potential mechanism underlying glomerulosclerosis.

BACKGROUND: A decrease in podocyte number contributes to the development of glomerulosclerosis in diabetic nephropathy. Although podocytes have been detected in the urine in certain glomerular diseases, their viability is poorly understood. METHODS: Diabetes was induced in rats with streptozotocin. Urine was collected from control rats (given citrate), and rats with diabetic nephropathy, and cells obtained by centrifugation were resuspended in tissue culture media, and seeded onto collagen-coated tissue culture plates. Cells were grown under standard cell culture conditions ex vivo. Cell number was measured, the cell type in the urine was identified by immunostaining with specific antibodies, and morphology was assessed by light and electron microscopy. RESULTS: Within 24 h, cells obtained from the urine of diabetic rats attached to tissue culture plates ex vivo. Cells were not detected in the urine from control rats. All cells from diabetic rats stained positive for the podocyte-specific proteins synaptopodin, nephrin, podocin and Glepp-1 and negative for mesangial (OX-7), tubular (Tamm-Horsfall protein) and endothelial (RECA) cell antigens. The cell number increased daily, which is consistent with cell growth ex vivo. CONCLUSIONS: Rats with diabetic nephropathy shed podocytes into the urine that attach and grow ex vivo. These results are consistent with the detachment of viable podocytes in diabetes and add new perspectives into our understanding of development of glomerulosclerosis in diabetes mellitus.

Animals↗
PubMed2003

Cell cycle control in glomerular disease.

The sequential activation of the cyclin-dependent kinases by their partner cyclins underlies the progression of the cell cycle from quiescence through growth to cell division. More recently a role for these proteins and their inhibitors has been appreciated in several diverse renal and non-renal cell processes, including proliferation, development, differentiation, hypertrophy and apoptosis. The glomerulus represents a unique micro-environment in which to study the cellular outcome following injury, as each of the three resident cell types undergoes a specific and distinct response to a given stimulus. The mesangial cell is capable of marked proliferation, often accompanied by the deposition of extracellular matrix. In contrast, the podocyte has previously been considered a relatively inert cell, and the reparative proliferation of glomerular endothelial cells following injury has recently been described. There is currently increasing awareness of the need to prevent, control and ameliorate the progression of renal diseases. Knowledge of the cell cycle and an understanding of how this may be beneficially manipulated may be crucial to improving the outlook for patients with both diabetic and non-diabetic glomerular disease.

Animals↗
PubMed2003

Angiotensin II-induced hypertrophy of proximal tubular cells requires p27Kip1.

BACKGROUND: Angiotensin II (Ang II), as a single factor, induces hypertrophy of cultured proximal tubular cells of various species. Cells undergoing hypertrophy are arrested in the G1 phase of the cell cycle. Ang II also stimulated the expression of p27Kip1, an inhibitor of cyclin-dependent kinases (CDK). Although previous studies inhibiting p27Kip1 expression with antisense oligonucleotides suggested that this CDK inhibitor is important for Ang II-induced hypertrophy of proximal tubular cells, nonspecific effects of antisense technology, and the inability to transfect 100% of cells raised concerns about the true role of p27Kip1 in tubular hypertrophy. METHODS: Proximal tubular cells were isolated and cultured from wild-type (p27Kip1+/+) and knockout (p27Kip1-/-) mice. p27Kip1 genomic and protein expression was evaluated. Proximal tubular cell origin was confirmed by expression of various markers [3M-1 antigen, gamma-glutamyltransferase, angiotensin-converting enzyme (ACE)]. Cell proliferation (cell number, 3[H]thymidine incorporation) and hypertrophy (de novo protein synthesis as measured by 3[H]leucine incorporation, hypertrophy index, cell size) were evaluated. CDK2 and CDK4 activities were determined by an in vitro kinase assay. In addition, cell cycle analysis was performed by flow cytometry. p27Kip1 expression was reconstituted in two different clones of p27Kip1-/- proximal tubular cells using an inducible vector system based on ecdysone response elements. RESULTS: In accordance with previous studies, 10-7 mol/L Ang II induces hypertrophy and cell cycle arrest of p27Kip1+/+ proximal tubular cells. In contrast, Ang II facilitated cell cycle progression of two p27Kip1-/- proximal tubular cell lines without inducing hypertrophy. Ang II activates CDK4/cyclin D kinase activity in p27Kip1+/+ and -/- tubular cells, but stimulates CDK2/cyclin E activity only in wild-type cells. However, in the presence of Ang II, reconstituting p27Kip1 expression in p27Kip1-/- tubular cells using an inducible expression system, restored G1 phase arrest and the hypertrophic phenotype. Ang II did not induce apoptosis of either p27Kip1+/+ or -/- tubular cells. CONCLUSION: Our findings are the first clear evidence that p27Kip1 is required for Ang II-induced hypertrophy of proximal tubular cells. However, although p27Kip1 expression is an absolute requirement for this hypertrophy, reconstitution experiments revealed that other factors induced by Ang II contribute to this hypertrophy.

Angiotensin II↗
PubMed2003

Podocytes that detach in experimental membranous nephropathy are viable.

BACKGROUND: Podocyte loss contributes to the development of glomerulosclerosis. Although podocytes have been detected in the urine in certain glomerular diseases, the viability of detached cells is not known. METHODS: Urine was collected from rats with experimental membranous nephropathy [passive Heymann nephritis (PHN) model], centrifuged, and following resuspension in tissue culture media, cells were seeded onto collagen-coated tissue culture plates. Cells were grown under typical cell culture conditions. Cell number was measured, the cell type was identified by immunostaining with specific antibodies, and cell morphology was assessed by light and electron microscopy. RESULTS: Cells obtained in the urine from PHN rats were positive for synaptopodin, nephrin, podocin, WT-1, and GLEPP1 (podocyte-specific antigens). When grown ex vivo under cell culture conditions, cells obtained in the urine from PHN rats adhered to tissue culture plates, and expressed podocyte-specific proteins at the mRNA [reverse transcription-polymerase chain reaction (RT-PCR)] and protein (immunostaining) level. Cells did not stain with antibodies to mesangial (OX-7), tubular (Tamm-Horsfall protein) and endothelial (RECA) cells. Electron microscopy showed the presence of foot processes, and podocytes from PHN rats stained positive for C5b-9. Although podocyte number increased transiently during the first 5 days ex vivo, apoptosis increased significantly thereafter, reducing overall cell number. CONCLUSION: Rats with experimental membranous nephropathy shed podocytes into the urine that attach to tissue culture plates ex-vivo, and proliferate. These results suggest that detached podocytes are viable. These results add new perspectives into our understanding of podocyte loss in the development of glomerulosclerosis.

Animals↗
PubMed2003

Mitotic cell cycle proteins increase in podocytes despite lack of proliferation.

BACKGROUND: Podocyte proliferation is an uncommon response to glomerular injury and its lack may underlie the development of glomerulosclerosis. However, whether podocytes have the capacity to enter and finish mitosis and cytokinesis is not known. METHODS: The expression of mitotic cell cycle proteins (phosphorylated Histone 3, Cdc2, cyclin B1 and B2) was examined by immunohistochemistry in kidneys of embryonal mice, transgenic HIV-mice, and rats with experimental membranous nephropathy (passive Heymann nephritis, PHN). Mitotic proteins also were measured by Western blot in glomerular protein from PHN-rats and the activity of mitotic cyclins was quantified by histone kinase assay. RESULTS: Mitotic proteins were increased in embryonal mouse glomeruli during the S- and comma-shaped stages and were absent at the capillary loop stage and in mature rodent glomeruli. There was an increase in podocyte expression of Cdc2, cyclin B1 and B2 and phosphorylated histone 3 in PHN rats, and in HIV transgenic mice. CONCLUSIONS: Podocytes have the ability to increase cell cycle proteins required for mitosis. Without obvious differences in the expression of the major mitotic proteins in PHN- and HIV-nephropathy, a regulatory disturbance in cytokinesis might be responsible for the development of polynucleated cells and a lack of podocyte proliferation in experimental glomerular disease.

AIDS-Associated Nephropathy↗
PubMed2003

The role of cell cycle proteins in Glomerular disease.

Although initially identified and characterized as regulators of the cell cycle and hence proliferation, an extended role for cell cycle proteins has been appreciated more recently in a number of physiologic and pathologic processes, including development, differentiation, hypertrophy, and apoptosis. Their precise contribution to the cellular response to injury appears to be dependent on both the cell type and the nature of the initiating injury. The glomerulus offers a remarkable situation in which to study the cell cycle proteins, as each of the 3 major resident cell types (the mesangial cell, podocyte, and glomerular endothelial cell) has a specific pattern of cell cycle protein expression when quiescent and responds uniquely after injury. Defining their roles may lead to potential therapeutic strategies in glomerular disease.

Animals↗
PubMed2003

Podocyte proliferation and differentiation in glomerular disease: role of cell-cycle regulatory proteins.

Injury to the podocyte underlies many forms of glomerular disease. In contrast to mesangial and endothelial cells, podocytes do not typically proliferate. Moreover, the lack of proliferation is thought to underlie the development of glomerulosclerosis. Studies have recently shown that the lack of podocyte proliferation is due to an increase in cyclin-dependent kinase inhibitors, which arrest the cell cycle. Current work is aimed at further delineating the mechanisms regulating podocyte proliferation.

Cell Cycle↗
PubMed2003

Hematopoietic stem cells contribute to the regeneration of renal tubules after renal ischemia-reperfusion injury in mice.

Ischemia-reperfusion injury (I/R injury) is a common cause of acute renal failure. Recovery from I/R injury requires renal tubular regeneration. Hematopoietic stem cells (HSC) have been shown to be capable of differentiating into hepatocytes, cardiac myocytes, gastrointestinal epithelial cells, and vascular endothelial cells during tissue repair. The current study tested the hypothesis that murine HSC can contribute to the regeneration of renal tubular epithelial cells after I/R injury. HSC isolated from male Rosa26 mice that express beta-galactosidase constitutively were transplanted into female nontransgenic mice after unilateral renal I/R injury. Four weeks after HSC transplantation, beta-galactosidase-positive cells were detected in renal tubules of the recipients by X-Gal staining. PCR analysis of the male-specific Sry gene and Y chromosome fluorescence in situ hybridization confirmed the presence of male-derived cells in the kidneys of female recipients. Antibody co-staining showed that beta-galactosidase was primarily expressed in renal proximal tubules. This is the first report to show that HSC can differentiate into renal tubular cells after I/R injury. Because of their availability, HSC may be useful for cell replacement therapy of acute renal failure.

Animals↗
PubMed2003

DNA damage is a novel response to sublytic complement C5b-9-induced injury in podocytes.

In response to Ab-complement-mediated injury, podocytes can undergo lysis, apoptosis, or, when exposed to sublytic (<5% lysis) amounts of C5b-9, become activated. Following the insertion of sublytic quantities of C5b-9, there is an increase in signaling pathways and growth factor synthesis and release of proteases, oxidants, and other molecules. Despite an increase in DNA synthesis, however, sublytic C5b-9 is associated with a delay in G(2)/M phase progression in podocytes. Here we induced sublytic C5b-9 injury in vitro by exposing cultured rat podocytes or differentiated postmitotic mouse podocytes to Ab and a complement source; we also studied the passive Heymann nephritis model of experimental membranous nephropathy in rats. A major finding was that sublytic C5b-9-induced injury caused an increase in DNA damage in podocytes both in vitro and in vivo. This was associated with an increase in protein levels for p53, the CDK inhibitor p21, growth-arrest DNA damage-45 (GADD45), and the checkpoint kinases-1 and -2. Sublytic C5b-9 increased extracellular signal-regulated kinase-1 and -2 (ERK-1 and -2), and inhibiting ERK-1 and -2 reduced the increase in p21 and GADD45 and augmented the DNA damage response to sublytic C5b-9-induced injury. These results show that sublytic C5b-9 induces DNA damage in vitro and in vivo and may explain why podocyte proliferation is limited following immune-mediated injury.

Animals↗
PubMed2003

Cell cycle control in glomerular disease.

Glomerular diseases are a leading caused of kidney failure. The three resident glomerular cell types respond differently to injury, which includes proliferation, hypertrophy, apoptosis and de-differentiation. Each leads to glomerular scarring, and a decline in renal function. Studies have shown that these events are critically controlled by cell cycle regulatory proteins, providing potential targets for the development of future therapeutics.

Animals↗