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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 8, 2004 as doi:10.1096/fj.03-0939fje. |
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* INSERM U508, Pasteur Institute, Lille, France;
Protein Biochemistry and Proteomics Laboratory, University of Paris 13, Bobigny, France;
INSERM U36, Collège de France, Paris, France;
INSERM U489, Hopital Tenon, Paris, France; and
|| INSERM U460, Hopital Bichat, Paris, France
2Correspondence: INSERM U508, Pasteur Institute, 1 rue du professeur Calmette, 59019 Lille cedex, France. E-mail: florence.pinet{at}pasteur-lille.fr
SPECIFIC AIMS
Renovascular hypertension is a renin-dependent form of hypertension caused by a stenosis of the renal artery. The renal phenotype is characterized by a recruitment of renin-producing cells.
The purpose of this work was to determine the factors and proteins involved in the process of smooth muscle cell differentiation by recruitment of renin-producing cells along the renal afferent arterioles during renovascular hypertension.
To understand the molecular mechanisms involved in this process, we used an innovative approach—proteomic analysis to demonstrate differential renal protein expression using an experimental model of renovascular hypertension.
PRINCIPAL FINDINGS
Our animal model of renovascular hypertension was the Goldblatt, two-kidney, one clip (2K1C) rat model in which one renal artery is constricted to chronically reduce renal perfusion, and the other kidney remains untouched. The 2K1C model is characterized by contrasting behavior of the two kidneys. In clipped kidneys, renin expression, storage and release is up-regulated, whereas in contralateral kidneys exposed to hypertension, renin expression is down-regulated.
We used a syngenic strain, the Lewis rat, to avoid genetic heterogeneity from one animal to another. Extraction of renal proteins was performed 2 wk after clipping to limit the adaptive responses to blood pressure elevation. Several hemodynamics parameters were measured (blood pressure, heart rate, organ weight, and renin plasma elevation) to ensure the quality of the 2K1C rats used for these experiments. Conventional histology of both kidneys was also performed to ensure the absence of renal necrosis.
We did not perform differential proteomic analysis of proteins from the whole clipped or contralateral kidneys. Our innovation was to compare the afferent arterioles isolated from clipped and contralateral kidneys. This enabled us to analyze the dynamic process affecting the afferent arterioles during development of renovascular hypertension involving recruitment of renin-producing cells. Protein profiles of afferent arterioles from clipped and contralateral kidneys were compared by proteomics.
This proteomic analysis was performed using several techniques: 1) 2-dimensional electrophoresis with separation of the proteins, 2) by their isoelectric point, and 3) by their molecular mass, followed by silver staining. Two-dimensional gels were then digitized for their bioinformatic analysis performed using software adequate for 2-dimensional gels (Melanie). The polypeptides identified as differentially expressed were analyzed by mass spectrometry to identify the proteins.
For the first time, proteomic mapping of renal afferent arterioles has been performed. Approximately 1,000 protein spots were visualized for each sample (clipped and contralateral kidneys) with a reproducible pattern among all 2-dimensional gels performed.
The comparative analysis evidenced differential expression of fourteen proteins. Ten could be identified by mass spectrometry. Five of these proteins are involved in metabolism and three are of muscular origin. We focused on the latter proteins because stenosis of the renal artery is accompanied by a phenotypic modification of smooth muscle cells.
The most striking protein differentially expressed was troponin T. We performed immunocytochemistry to confirm this data, first in kidneys from untreated rats. Troponin T was observed in the smooth muscle cells along the afferent arterioles in the kidneys.
We further examined the hypothesis generated by the results of differential proteomic analysis, that a decrease in troponin T in afferent arterioles of clipped kidneys could be a marker of smooth muscle cell differentiation into myoepithelioïd cells by confocal imaging. The data obtained are consistent with the proteomic data.
CONCLUSIONS AND SIGNIFICANCE
In renovascular hypertension, the decreased staining of troponin T was correlated with the acquisition of a secretory phenotype by the smooth muscle cells, which in turn synthesized renin. Renin and troponin T have been shown to be inversely regulated during the development of hypertension and the two proteins were never expressed within the same cells in the kidney.
We propose that troponin T, whose presence in renal afferent arterioles is shown for the first time, is a marker of smooth muscle cell differentiation in this particular arterial location (Fig. 1
).
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0939fje; 
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