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The molecular and functional phenotype of glomerular podocytes reveals key features of contractile smooth muscle cells.

Saleem MA, Zavadil J, Bailly M, McGee K, Witherden IR, Pavenstadt H, Hsu H, Sanday J, Satchell SC, Lennon R, Ni L, Bottinger EP, Mundel P, Mathieson PW - Am. J. Physiol. Renal Physiol. (2008)

Bottom Line: We further examined the smooth muscle phenotype and showed that podocytes consistently express the differentiated smooth muscle markers smoothelin and calponin and the specific transcription factor myocardin, both in vitro and in vivo.We demonstrated using two novel techniques that podocytes contract vigorously in vitro when differentiated and in real time were able to demonstrate that angiotensin II treatment decreases monolayer resistance, morphologically correlating with enhanced contractility.We conclude that the mature podocyte in vitro possesses functional apparatus of contractile smooth muscle cells, with potential implications for its in vivo ability to regulate glomerular dynamic and permeability characteristics.

View Article: PubMed Central - PubMed

Affiliation: Academic and Children's Renal Unit, University of Bristol, Lifeline Bldg., Southmead Hospital, Bristol, BS10 5NB, United Kingdom. m.saleem@bristol.ac.uk

ABSTRACT
The glomerular podocyte is a highly specialized cell, with the ability to ultrafilter blood and support glomerular capillary pressures. However, little is known about either the genetic programs leading to this functionality or the final phenotype. We approached this question utilizing a human conditionally immortalized cell line, which differentiates from a proliferating epithelial phenotype to a differentiated form. We profiled gene expression during several time points during differentiation and grouped the regulated genes into major functional categories. A novel category of genes that was upregulated during differentiation was of smooth muscle-related molecules. We further examined the smooth muscle phenotype and showed that podocytes consistently express the differentiated smooth muscle markers smoothelin and calponin and the specific transcription factor myocardin, both in vitro and in vivo. The contractile contribution of the podocyte to the glomerular capillary is controversial. We demonstrated using two novel techniques that podocytes contract vigorously in vitro when differentiated and in real time were able to demonstrate that angiotensin II treatment decreases monolayer resistance, morphologically correlating with enhanced contractility. We conclude that the mature podocyte in vitro possesses functional apparatus of contractile smooth muscle cells, with potential implications for its in vivo ability to regulate glomerular dynamic and permeability characteristics.

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Glomerular immunofluorescence. A: smoothelin (green). Left: linear, capillary loop pattern. Center: nephrin staining (red). Right: double immunostaining with nephrin demonstrating colocalization (yellow, arrow shows an example in a capillary loop). B: calponin (green). Left: linear, capillary loop pattern. Center: nephrin staining (red). Right: double immunostaining with nephrin demonstrating colocalization (yellow, arrow shows an example in a capillary loop). C: α-SMA (green). Left: mesangial cell pattern of distribution, with relatively weak podocyte staining. Center: double immunolabeling with nephrin to localize podocytes (red). Right: the same at higher magnification, demonstrating absence of colocalization. D: myosin heavy chain (green). Left: mesangial cell pattern of distribution, with relatively weak podocyte staining. Center: double immunolabeling with nephrin to localize podocytes (red). Right: the same at higher magnification, demonstrating absence of colocalization. E: myocardin (green). Left: nuclear cell staining. Center: WT-1 staining (a nuclear podocyte marker). Right: double staining with WT-1 colocalizing with myocardin (yellow). Magnification ×40 to ×100. Far right-hand column shows the level of expression of each protein in vascular smooth muscle in the same sections, illustrating greater level of expression compared with glomeruli. Where a glomerulus is present in the same panel, this is indicated by the solid arrow. Stippled arrows indicate blood vessels. For SMA (C), the inset is the same view as the main panel and shows the same artery, at lower fluorescence intensity.
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f3: Glomerular immunofluorescence. A: smoothelin (green). Left: linear, capillary loop pattern. Center: nephrin staining (red). Right: double immunostaining with nephrin demonstrating colocalization (yellow, arrow shows an example in a capillary loop). B: calponin (green). Left: linear, capillary loop pattern. Center: nephrin staining (red). Right: double immunostaining with nephrin demonstrating colocalization (yellow, arrow shows an example in a capillary loop). C: α-SMA (green). Left: mesangial cell pattern of distribution, with relatively weak podocyte staining. Center: double immunolabeling with nephrin to localize podocytes (red). Right: the same at higher magnification, demonstrating absence of colocalization. D: myosin heavy chain (green). Left: mesangial cell pattern of distribution, with relatively weak podocyte staining. Center: double immunolabeling with nephrin to localize podocytes (red). Right: the same at higher magnification, demonstrating absence of colocalization. E: myocardin (green). Left: nuclear cell staining. Center: WT-1 staining (a nuclear podocyte marker). Right: double staining with WT-1 colocalizing with myocardin (yellow). Magnification ×40 to ×100. Far right-hand column shows the level of expression of each protein in vascular smooth muscle in the same sections, illustrating greater level of expression compared with glomeruli. Where a glomerulus is present in the same panel, this is indicated by the solid arrow. Stippled arrows indicate blood vessels. For SMA (C), the inset is the same view as the main panel and shows the same artery, at lower fluorescence intensity.

Mentions: We then examined the expression of smoothelin and calponin (CNN1) at the protein level using immunofluorescence and Western blotting (Figs. 2–5), showing expression of calponin, and both “visceral” (59 kDa, also known as smoothelin-A) and “vascular” (110 kDa, also known as smoothelin-B) isoforms of smoothelin (25) in differentiated podocytes. Interestingly, the vascular isoform was upregulated in differentiated cells (Fig. 5B). The immunofluorescence (IF) appearance of smoothelin was filamentous in podocytes, consistent with the observation that in some cell types it has a filamentous distribution (46) and in others it is stress fiber associated (7). The antibody we used has been used to detect human smoothelin transfected into COS7 cells (46), yielding the same IF distribution. Calponin had the typical stress fiber distribution in cultured podocytes (Fig. 2B). In human glomerular sections, double stained with the podocyte marker nephrin, both proteins were found specifically in podocytes (Figs. 3, A and B). Expression of smoothelin in podocytes was noticeably weaker than in arteriolar SMCs in the same sections. We further examined the smooth muscle phenotype of undifferentiated and differentiated podocytes by examining other known myofibroblastic markers: smooth muscle myosin heavy chain (MHC) and α-smooth muscle actin (SMA) (Figs. 2 and 5). There was expression of SMA by IF and Western blotting in vitro and no detectable expression by IF of MHC at 33°C, with expression at 37°C. Weak nuclear staining of SMA was also seen in vitro, compared with control, of uncertain significance. MHC and SMA were detected strongly in a mesangial cell distribution in vivo, with comparatively weak podocyte expression (Fig. 3).


The molecular and functional phenotype of glomerular podocytes reveals key features of contractile smooth muscle cells.

Saleem MA, Zavadil J, Bailly M, McGee K, Witherden IR, Pavenstadt H, Hsu H, Sanday J, Satchell SC, Lennon R, Ni L, Bottinger EP, Mundel P, Mathieson PW - Am. J. Physiol. Renal Physiol. (2008)

Glomerular immunofluorescence. A: smoothelin (green). Left: linear, capillary loop pattern. Center: nephrin staining (red). Right: double immunostaining with nephrin demonstrating colocalization (yellow, arrow shows an example in a capillary loop). B: calponin (green). Left: linear, capillary loop pattern. Center: nephrin staining (red). Right: double immunostaining with nephrin demonstrating colocalization (yellow, arrow shows an example in a capillary loop). C: α-SMA (green). Left: mesangial cell pattern of distribution, with relatively weak podocyte staining. Center: double immunolabeling with nephrin to localize podocytes (red). Right: the same at higher magnification, demonstrating absence of colocalization. D: myosin heavy chain (green). Left: mesangial cell pattern of distribution, with relatively weak podocyte staining. Center: double immunolabeling with nephrin to localize podocytes (red). Right: the same at higher magnification, demonstrating absence of colocalization. E: myocardin (green). Left: nuclear cell staining. Center: WT-1 staining (a nuclear podocyte marker). Right: double staining with WT-1 colocalizing with myocardin (yellow). Magnification ×40 to ×100. Far right-hand column shows the level of expression of each protein in vascular smooth muscle in the same sections, illustrating greater level of expression compared with glomeruli. Where a glomerulus is present in the same panel, this is indicated by the solid arrow. Stippled arrows indicate blood vessels. For SMA (C), the inset is the same view as the main panel and shows the same artery, at lower fluorescence intensity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2576149&req=5

f3: Glomerular immunofluorescence. A: smoothelin (green). Left: linear, capillary loop pattern. Center: nephrin staining (red). Right: double immunostaining with nephrin demonstrating colocalization (yellow, arrow shows an example in a capillary loop). B: calponin (green). Left: linear, capillary loop pattern. Center: nephrin staining (red). Right: double immunostaining with nephrin demonstrating colocalization (yellow, arrow shows an example in a capillary loop). C: α-SMA (green). Left: mesangial cell pattern of distribution, with relatively weak podocyte staining. Center: double immunolabeling with nephrin to localize podocytes (red). Right: the same at higher magnification, demonstrating absence of colocalization. D: myosin heavy chain (green). Left: mesangial cell pattern of distribution, with relatively weak podocyte staining. Center: double immunolabeling with nephrin to localize podocytes (red). Right: the same at higher magnification, demonstrating absence of colocalization. E: myocardin (green). Left: nuclear cell staining. Center: WT-1 staining (a nuclear podocyte marker). Right: double staining with WT-1 colocalizing with myocardin (yellow). Magnification ×40 to ×100. Far right-hand column shows the level of expression of each protein in vascular smooth muscle in the same sections, illustrating greater level of expression compared with glomeruli. Where a glomerulus is present in the same panel, this is indicated by the solid arrow. Stippled arrows indicate blood vessels. For SMA (C), the inset is the same view as the main panel and shows the same artery, at lower fluorescence intensity.
Mentions: We then examined the expression of smoothelin and calponin (CNN1) at the protein level using immunofluorescence and Western blotting (Figs. 2–5), showing expression of calponin, and both “visceral” (59 kDa, also known as smoothelin-A) and “vascular” (110 kDa, also known as smoothelin-B) isoforms of smoothelin (25) in differentiated podocytes. Interestingly, the vascular isoform was upregulated in differentiated cells (Fig. 5B). The immunofluorescence (IF) appearance of smoothelin was filamentous in podocytes, consistent with the observation that in some cell types it has a filamentous distribution (46) and in others it is stress fiber associated (7). The antibody we used has been used to detect human smoothelin transfected into COS7 cells (46), yielding the same IF distribution. Calponin had the typical stress fiber distribution in cultured podocytes (Fig. 2B). In human glomerular sections, double stained with the podocyte marker nephrin, both proteins were found specifically in podocytes (Figs. 3, A and B). Expression of smoothelin in podocytes was noticeably weaker than in arteriolar SMCs in the same sections. We further examined the smooth muscle phenotype of undifferentiated and differentiated podocytes by examining other known myofibroblastic markers: smooth muscle myosin heavy chain (MHC) and α-smooth muscle actin (SMA) (Figs. 2 and 5). There was expression of SMA by IF and Western blotting in vitro and no detectable expression by IF of MHC at 33°C, with expression at 37°C. Weak nuclear staining of SMA was also seen in vitro, compared with control, of uncertain significance. MHC and SMA were detected strongly in a mesangial cell distribution in vivo, with comparatively weak podocyte expression (Fig. 3).

Bottom Line: We further examined the smooth muscle phenotype and showed that podocytes consistently express the differentiated smooth muscle markers smoothelin and calponin and the specific transcription factor myocardin, both in vitro and in vivo.We demonstrated using two novel techniques that podocytes contract vigorously in vitro when differentiated and in real time were able to demonstrate that angiotensin II treatment decreases monolayer resistance, morphologically correlating with enhanced contractility.We conclude that the mature podocyte in vitro possesses functional apparatus of contractile smooth muscle cells, with potential implications for its in vivo ability to regulate glomerular dynamic and permeability characteristics.

View Article: PubMed Central - PubMed

Affiliation: Academic and Children's Renal Unit, University of Bristol, Lifeline Bldg., Southmead Hospital, Bristol, BS10 5NB, United Kingdom. m.saleem@bristol.ac.uk

ABSTRACT
The glomerular podocyte is a highly specialized cell, with the ability to ultrafilter blood and support glomerular capillary pressures. However, little is known about either the genetic programs leading to this functionality or the final phenotype. We approached this question utilizing a human conditionally immortalized cell line, which differentiates from a proliferating epithelial phenotype to a differentiated form. We profiled gene expression during several time points during differentiation and grouped the regulated genes into major functional categories. A novel category of genes that was upregulated during differentiation was of smooth muscle-related molecules. We further examined the smooth muscle phenotype and showed that podocytes consistently express the differentiated smooth muscle markers smoothelin and calponin and the specific transcription factor myocardin, both in vitro and in vivo. The contractile contribution of the podocyte to the glomerular capillary is controversial. We demonstrated using two novel techniques that podocytes contract vigorously in vitro when differentiated and in real time were able to demonstrate that angiotensin II treatment decreases monolayer resistance, morphologically correlating with enhanced contractility. We conclude that the mature podocyte in vitro possesses functional apparatus of contractile smooth muscle cells, with potential implications for its in vivo ability to regulate glomerular dynamic and permeability characteristics.

Show MeSH
Related in: MedlinePlus