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Novel approach for the detection of tubular cell migration into the interstitium during renal fibrosis in rats.

Nakasatomi M, Maeshima A, Mishima K, Ikeuchi H, Sakairi T, Kaneko Y, Hiromura K, Nojima Y - Fibrogenesis Tissue Repair (2015)

Bottom Line: The number of BrdU-positive cells migrating into the interstitium significantly increased and peaked at 8 days after UUO.Long-term BrdU labeling marked some of the proximal tubular cells and enabled us to detect tubular cell migration into the interstitium after UUO.This simple method might be useful to detect EMT in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Clinical Science, Gunma University Graduate School of Medicine, 3-39-15 Showa, Maebashi, 371-8511 Japan.

ABSTRACT

Background: The process of epithelial-mesenchymal transition (EMT), which is generally defined by phenotypic changes of injured tubules such as loss of epithelial markers or acquisition of mesenchymal markers, implies various activating steps, including proliferation, migration, and ability to produce extracellular matrix proteins. We established here a novel approach for the detection of tubular cell migration into the interstitium during renal fibrosis in vivo.

Results: Using an osmotic pump, bromodeoxyuridine (BrdU) was continuously given to 7-week-old Wistar rats for 4 weeks, and BrdU-positive cells were detected by immunostaining. BrdU-positive cells were present in aquaporin-1-positive proximal tubules, but not in the interstitium of BrdU-treated rat kidneys. After unilateral ureteral obstruction (UUO), some BrdU-positive tubular cells protruded from the basement membrane and migrated into the interstitium. Interstitial BrdU-positive cells were co-localized with alpha-smooth muscle actin, fibroblast specific protein-1, vimentin, and type I collagen, but not with CD68 or CD3. No BrdU-positive cells were observed in the interstitium of sham-operated kidneys. The number of BrdU-positive cells migrating into the interstitium significantly increased and peaked at 8 days after UUO.

Conclusions: Long-term BrdU labeling marked some of the proximal tubular cells and enabled us to detect tubular cell migration into the interstitium after UUO. This simple method might be useful to detect EMT in vivo.

No MeSH data available.


Related in: MedlinePlus

Localization of BrdU-positive cells in the normal rat kidneys after long-term BrdU labeling. a–c BrdU was given intraperitoneally to normal rats for 4 weeks. Double staining of BrdU with several tubular (a), glomerular (b), and interstitial (c) markers was performed. Markers: aquaporin-1 (AQP-1), lotus tetragonolobus agglutinin (LTA), Tamm-Horsfall glycoprotein (THP), aquaporin-2 (AQP-2), alpha-SMA, vimentin, CD31, WT1, and aminopeptidase P. DAPI (blue). Magnification, ×1000. d Quantitative analysis of percentage of BrdU-positive cells per total marker-positive cells. Values are means ± SE (n = 5)
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Fig2: Localization of BrdU-positive cells in the normal rat kidneys after long-term BrdU labeling. a–c BrdU was given intraperitoneally to normal rats for 4 weeks. Double staining of BrdU with several tubular (a), glomerular (b), and interstitial (c) markers was performed. Markers: aquaporin-1 (AQP-1), lotus tetragonolobus agglutinin (LTA), Tamm-Horsfall glycoprotein (THP), aquaporin-2 (AQP-2), alpha-SMA, vimentin, CD31, WT1, and aminopeptidase P. DAPI (blue). Magnification, ×1000. d Quantitative analysis of percentage of BrdU-positive cells per total marker-positive cells. Values are means ± SE (n = 5)

Mentions: Using several nephron markers, we also examined the localization of BrdU-positive cells in the kidney after long-term BrdU labeling. Most BrdU-positive cells were present in aquaporin (AQP)-1 or lotus tetragonolobus agglutinin (LTA)-positive proximal tubules, but not in other nephron segments (Fig. 2a). A very small number of BrdU-positive cells were detected in the glomerulus and were co-localized with aminopeptidase P-positive endothelial cells and vimentin-positive mesangial cells, but not with WT1-positive podocytes (Fig. 2b). Few BrdU-positive cells were observed in the interstitium of the normal rat kidney. No BrdU-positive cells were co-localized with vimentin-positive fibroblasts, CD31-positive capillary endothelial cells, or α-SMA-positive smooth muscle cells of vessels (Fig. 2c). Quantitative analysis demonstrated that nearly all BrdU-positive cells were proximal tubules (Fig. 2d).Fig. 2


Novel approach for the detection of tubular cell migration into the interstitium during renal fibrosis in rats.

Nakasatomi M, Maeshima A, Mishima K, Ikeuchi H, Sakairi T, Kaneko Y, Hiromura K, Nojima Y - Fibrogenesis Tissue Repair (2015)

Localization of BrdU-positive cells in the normal rat kidneys after long-term BrdU labeling. a–c BrdU was given intraperitoneally to normal rats for 4 weeks. Double staining of BrdU with several tubular (a), glomerular (b), and interstitial (c) markers was performed. Markers: aquaporin-1 (AQP-1), lotus tetragonolobus agglutinin (LTA), Tamm-Horsfall glycoprotein (THP), aquaporin-2 (AQP-2), alpha-SMA, vimentin, CD31, WT1, and aminopeptidase P. DAPI (blue). Magnification, ×1000. d Quantitative analysis of percentage of BrdU-positive cells per total marker-positive cells. Values are means ± SE (n = 5)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4496823&req=5

Fig2: Localization of BrdU-positive cells in the normal rat kidneys after long-term BrdU labeling. a–c BrdU was given intraperitoneally to normal rats for 4 weeks. Double staining of BrdU with several tubular (a), glomerular (b), and interstitial (c) markers was performed. Markers: aquaporin-1 (AQP-1), lotus tetragonolobus agglutinin (LTA), Tamm-Horsfall glycoprotein (THP), aquaporin-2 (AQP-2), alpha-SMA, vimentin, CD31, WT1, and aminopeptidase P. DAPI (blue). Magnification, ×1000. d Quantitative analysis of percentage of BrdU-positive cells per total marker-positive cells. Values are means ± SE (n = 5)
Mentions: Using several nephron markers, we also examined the localization of BrdU-positive cells in the kidney after long-term BrdU labeling. Most BrdU-positive cells were present in aquaporin (AQP)-1 or lotus tetragonolobus agglutinin (LTA)-positive proximal tubules, but not in other nephron segments (Fig. 2a). A very small number of BrdU-positive cells were detected in the glomerulus and were co-localized with aminopeptidase P-positive endothelial cells and vimentin-positive mesangial cells, but not with WT1-positive podocytes (Fig. 2b). Few BrdU-positive cells were observed in the interstitium of the normal rat kidney. No BrdU-positive cells were co-localized with vimentin-positive fibroblasts, CD31-positive capillary endothelial cells, or α-SMA-positive smooth muscle cells of vessels (Fig. 2c). Quantitative analysis demonstrated that nearly all BrdU-positive cells were proximal tubules (Fig. 2d).Fig. 2

Bottom Line: The number of BrdU-positive cells migrating into the interstitium significantly increased and peaked at 8 days after UUO.Long-term BrdU labeling marked some of the proximal tubular cells and enabled us to detect tubular cell migration into the interstitium after UUO.This simple method might be useful to detect EMT in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Clinical Science, Gunma University Graduate School of Medicine, 3-39-15 Showa, Maebashi, 371-8511 Japan.

ABSTRACT

Background: The process of epithelial-mesenchymal transition (EMT), which is generally defined by phenotypic changes of injured tubules such as loss of epithelial markers or acquisition of mesenchymal markers, implies various activating steps, including proliferation, migration, and ability to produce extracellular matrix proteins. We established here a novel approach for the detection of tubular cell migration into the interstitium during renal fibrosis in vivo.

Results: Using an osmotic pump, bromodeoxyuridine (BrdU) was continuously given to 7-week-old Wistar rats for 4 weeks, and BrdU-positive cells were detected by immunostaining. BrdU-positive cells were present in aquaporin-1-positive proximal tubules, but not in the interstitium of BrdU-treated rat kidneys. After unilateral ureteral obstruction (UUO), some BrdU-positive tubular cells protruded from the basement membrane and migrated into the interstitium. Interstitial BrdU-positive cells were co-localized with alpha-smooth muscle actin, fibroblast specific protein-1, vimentin, and type I collagen, but not with CD68 or CD3. No BrdU-positive cells were observed in the interstitium of sham-operated kidneys. The number of BrdU-positive cells migrating into the interstitium significantly increased and peaked at 8 days after UUO.

Conclusions: Long-term BrdU labeling marked some of the proximal tubular cells and enabled us to detect tubular cell migration into the interstitium after UUO. This simple method might be useful to detect EMT in vivo.

No MeSH data available.


Related in: MedlinePlus