J Vet Sci.  2016 Sep;17(3):269-277. 10.4142/jvs.2016.17.3.269.

Hydration status affects osteopontin expression in the rat kidney

• Lee SY
• Lee SJ
• Piao HL
• Yang SY
• Weiner ID
• Kim J
• Han KH

• KMID: 2413125
• DOI: http://doi.org/10.4142/jvs.2016.17.3.269

Abstract

Osteopontin (OPN) is a secretory protein that plays an important role in urinary stone formation. Hydration status is associated with the development of urolithiasis. This study was conducted to examine the effects of dehydration and hydration on OPN expression in the rat kidney. Animals were divided into three groups, control, dehydrated, and hydrated. Kidney tissues were processed for light and electron microscope immunocytochemistry, in situ hybridization, and immunoblot analysis. Dehydration induced a significant increase in OPN protein expression, whereas increased fluid intake induced a decrease in protein expression. Under control conditions, OPN protein and mRNA expression were only detected in the descending thin limb (DTL). Dehydration induced increased expression in the DTL and the development of detectable expression in the thick ascending limb (TAL). In contrast, OPN expression levels declined to less than the controls in the DTL after hydration, while no expression of either protein or mRNA was detectable in the TAL. Immunoelectron microscopy demonstrated that hydration status altered tubular ultrastructure and intracellular OPN expression in the Golgi apparatus and secretory cytoplasmic vesicles. These data confirm that changes in oral fluid intake can regulate renal tubular epithelial cell OPN expression.

Keyword

hydration; osteopontin; renal stone; tubular epithelial cells

MeSH Terms

Animals
Desiccation
Gene Expression Regulation/*physiology
Immunohistochemistry
Kidney/cytology/*metabolism/physiopathology/ultrastructure
Kidney Calculi/*genetics/physiopathology/ultrastructure
Male
Microscopy, Electron, Transmission
Osteopontin/*genetics/metabolism
Rats
Rats, Sprague-Dawley
Osteopontin

Figure

• 

  Fig. 1 (A) Immunoblots of proteins (10 µg per lane) from renal medulla of control (C), dehydrated (D) and hydrated (H) rats. A distinct band of 70 kD corresponding to the molecular weight of OPN is present. Blots demonstrate increased OPN expression in dehydrated and decreased expression in hydrated kidneys based on comparison of relative optical densities to those of controls (B). *p < 0.05 by t-test.

• 

  Fig. 2 Light micrographs illustrating OPN immunostaining and in situ hybridization in the kidney from control (A–C), dehydrated (D–F) and hydrated (G–I) rats. The OPN immunostaining and hybridization signal increased remarkably in dehydrated animals, while it decreased in hydrated animals relative to control animals. OPN expression was primarily observed in the tubular profiles in the inner stripe of the outer medulla (ISOM). Note that the OPN immunostaining and hybridization signal were not only observed in the descending thin limb of Henle's loop (DTL), but also in the thick ascending limb (TAL) in dehydrated animals (E and F). Arrows indicate an abrupt transition from the OPN-negative proximal tubule (PT) to the OPN-positive descending thin limb of Henle's loop. Quantitative analysis of OPN protein and mRNA expression (J). OSOM, outer stripe of the outer medulla; IM, inner medulla. *p < 0.05 by two-tailed t-test versus control. Scale bars = 1 mm (G), 20 µm (H and I).

• 

  Fig. 3 Light micrographs illustrating OPN immunostaining in the cortex (A). Occasionally, OPN expression was observed in Bowman's capsules (B, arrows) and distal tubules (inset, arrow). Rectangles mark areas magnified. G, glomerulus; PT, proximal tubule.

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  Fig. 4 Transmission electron micrographs illustrating OPN immunostaining in the descending thin limb from control (A), dehydrated (B), and hydrated (C) rats. OPN immunoreactivity increased in the Golgi apparatus and secretory vesicles in dehydrated animals. In contrast, after hydration, the labeling intensity declined to less than the controls. Note that the intercellular spaces between the thin epithelial cells were remarkably widened (asterisk) in hydrated rats. Scale bar = 2 µm.

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  Fig. 5 Transmission electron micrographs illustrating OPN immunostaining in the thick ascending limb (asterisk) from dehydrated (A) and hydrated (B) rats. OPN immunoreactivity is observed in the subapical secretory vesicles in dehydrated animals. Note a rough surface apical membrane in hydrated animals. Scale bar = 5 µm.

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  Fig. 6 Scanning electron micrographs showed crystal-like structures (arrow) in the tubule lumen in dehydrated rats. The inset shows a magnified view of the field marked with a rectangle. Scale bar = 20 µm.

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