RAPID COMMUNICATION |
Correspondence to: Adel Giaid, Montreal General Hospital, Suite L3-314, Montreal, Quebec H3G 1A4, Canada. E-mail: adel.giaid@mcgill.ca
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Summary |
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Human urotensin-II (U-II) is a cyclic 11-amino-acid residue peptide with a wide range of vasoactive properties dependent on the anatomic site and the species studied. The purpose of this study was to determine the localization of human U-II in normal human kidneys and in renal carcinoma. Normal human kidneys (n=11) and eight cases of clear-cell carcinoma were immunostained with a polyclonal antibody to human U-II. In normal human kidneys, U-II was mostly present in the epithelial cells of tubules and ducts, with greater intensity in the distal convoluted tubules. Moderate U-II immunoreactivity was seen in the endothelial cells of renal capillaries, but only focal immunoreactivity was found in the endothelial cells of the glomeruli. No staining was found in the veins. All tumors expressed moderate U-II immunoreactivity in the cancer cells and vasculature. Here we demonstrate abundant expression of U-II in normal human kidneys and renal carcinoma. These findings suggest that the vasoactive and growth-mediator peptide U-II may contribute to the pathophysiology of the human renal system.
(J Histochem Cytochem 50:885889, 2002)
Key Words: epithelial cells, endothelial cells, clear cells, tubules
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Introduction |
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UROTENSIN-II (U-II) was first isolated from an extract of the urophysis of the goby, which led to the discovery of its structure (
A recent study has demonstrated expression of U-II and its receptor mRNAs in many types of tumors, such as T98G glioblastoma cells, IMR32 neuroblastoma cells, NB69 neuroblastoma cells, BeWo choriocarcinoma cells, SW-13 adrenocortical carcinoma cells, DLD-1 colorectal adenocarcinoma cells, and HeLa cervical cancer cells (
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Materials and Methods |
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Tissues
Normal human kidney specimens (seven male, four female) collected at the Montreal General Hospital were fixed in 10% formalin, embedded in paraffin, and processed for immunohistochemistry (IHC). Renal carcinoma (clear-cell carcinoma; n=8, six male and two female) were all collected at surgery. Eight of the normal cases studied were collected at surgery and three at autopsy.
Antibody Generation
Female New Zealand White rabbits were dosed (100 µg SC followed by two booster injections at 4 and 8 weeks) with KLH-conjugated human urotensin-II (ETPD [CFWKYC] V). Antisera were subjected to protein A purification (ImmunoPure; Pierce, Rockford, IL), ammonium sulfate precipitation, and dialysis (25C in PBS overnight). The human urotensin-II polyclonal antibody (PAb) was further purified using an agarose-immobilized diaminodipropylamine column to which 2 mg human UR-II had been coupled [1 hr, 25C; EDC/DADPA immobilization, as described previously (
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Immunohistochemistry
Paraffin sections of 5-µm thickness were immunostained by the avidinbiotinperoxidase complex (ABC) method. Sections were deparaffinized and rehydrated in alcohol. After washing in PBS, the sections were placed in a Triton X-100 solution (0.2% in PBS) for 30 min at room temperature (RT). After washing in PBS, the sections were placed in a hydrogen peroxide solution (5% in PBS) for 30 min at RT. They were then washed in PBS and incubated for 30 min at RT in 10% normal goat serum (Vector Labs; Burlingame, CA). The sections were incubated at 4C overnight (18 hr) in U-II polyclonal antibody (1:75 in PBS) in 0.1% BSA. After washing again in PBS, they were then incubated for 45 min at RT in biotin-conjugated goat anti-rabbit IgG (Vector). After washing in PBS, the slides were incubated at RT for 45 min with the avidinbiotin complex (Vector). Sites of U-II immunoreactivity were developed in 3,3-diaminobenzidine (DAB) solution and hydrogen peroxide. After washing in PBS, sections were counterstained with hematoxylin. Negative controls were immunostained with Ag/Ab mixture or with the non-immune serum in place of the primary antiserum.
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Results |
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In normal human kidney there was abundant expression of human U-II, mainly in the tubule epithelial cells. The cytoplasm of epithelial cells of distal convoluted tubules and collecting tubules exhibited the strongest U-II expression (Fig 2A and Fig 2C). The cells of the proximal convoluted tubules exhibited weaker U-II immunoreactivity compared to those of the distal convoluted tubules (Fig 2A). The collecting ducts were weakly immunostained for U-II (Fig 2C). The Henle loops showed weak immunoreactivity for U-II. In the glomeruli, only a small number of cells expressed strong U-II immunoreactivity (Fig 2B). There was abundant U-II immunoreactivity in the endothelial cells of renal capillaries. Endothelial cells and smooth muscle cells of renal arteries showed moderate U-II immunoreactivity (Fig 2E). No immunostaining was found in the endothelial cells of veins.
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In the cases with renal clear-cell carcinoma, we observed moderate levels of U-II immunoreactivity in the cancer cells (Fig 2F). The intensity of U-II immunoreactivity in the tubule epithelial cells of normal and cancer tissues appeared comparable. The vasculature of the tumors expressed moderate U-II immunoreactivity. We also observed U-II immunoreactivity in the inflammatory cells within and surrounding the tumor. In contrast, very little immunostaining was found in the inflammatory cells of the tissue free of tumor.
Negative control experiments showed no immunostaining, confirming the specificity of the antisera and techniques used in this study (Fig 2D, Fig 2G, and Fig 2H).
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Discussion |
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This is the first report demonstrating the presence of U-II by IHC in the kidneys and in clear-cell renal adenocarcinoma. Our results showed abundant expression of U-II in normal human kidney. The epithelial cells of distal convoluted tubules exhibited the most intense U-II expression, followed by collecting tubules, collecting ducts, and proximal convoluted tubules. U-II was also evident in all vascular endothelial cells except those of veins. In the renal cell carcinomas, there was abundant U-II immunoreactivity in both cancer cells and normal epithelial cells. These results demonstrate the presence of U-II protein in normal human kidneys and in renal carcinoma, suggesting that the presence of the peptide may contribute to the pathophysiology of human kidneys.
The kidneys are very vascular organs and play an important role in regulating arterial blood pressure. They do so by controlling water reabsorption and the glomerular filtration rate (GFR). The reninangiotensin complex is well known for its major role in water reabsorption and the control of cardiovascular peripheral resistance. Regulation of GFR is obtained by three main mechanisms: myogenic response, tubuloglomerular feedback, and reflex control of GFR. The tubuloglomerular feedback consists of a chemical message sent by the distal tubules to nearby afferent arterioles when the flow increases in the tubules. The chemicals sent produce a vasoconstriction in the arteriole, reducing GFR. It is uncertain which chemicals act in this feedback, but a paracrine vasoactive hormone is a likely candidate. Because U-II is a vasoactive peptide and is abundantly expressed in the tubules and the vasculature, it is reasonable to suggest that U-II plays a role in this mechanism by a paracrine or an endocrine action.
There is also a reflex control, which occurs when blood pressure falls, causing vasoconstriction and decreasing GFR. This decrease in GFR augments blood volume and systemic blood pressure. This process is regulated by different hormones such as angiotensin-II, a vasoconstrictor, and prostaglandins, which act as vasodilators. U-II is known for its very strong vasoactive effects and may be one of the hormones responsible for this reflex. This is supported by the recent findings of increased U-II levels in patients with renal failure (
U-II also modulates transepithelial [Na+]/[Cl-] ion transport in fish (skin epithelia, operculum, intestine, bladder), and it is therefore proposed that U-II might possess an osmoregulatory function in mammals (
Recent reports have shown a mitogenic role for U-II through induction of smooth muscle cell proliferation (
In summary, we report abundant expression of the human U-II peptide in the kidney tubules and vasculature and in renal carcinoma. These morphological findings suggest a possible role for this vasoactive and mitogenic peptide in the pathophysiology of the human renal system. Further studies are needed to address the role played by this peptide in this system and the mechanism of its regulation in health and disease.
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Acknowledgments |
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Supported by the Canadian Institute of Health Research and the Fond de la Recherche en Sante du Quebec.
We wish to thank Ghada Hassan, Fazila Chiouali, and Zhaohui Ao for their help during the course of the study.
Received for publication December 7, 2001; accepted February 20, 2002.
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