| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 93 === Acute tubular necrosis (ATN) is the most common pathologic entity responsible for the clinical state of acute renal failure (ARF). Quiescent cells in the renal tubule would normally divide and differentiate during ATN via as yet unclear mechanism to restore the functional integrity of the nephron. The purpose of this study is to identify biomarkers and explore the pathophysiology of the disease. In a mouse model of toxic nephropathy induced by the administration of uranyl nitrate, we observed the development of ATN 3-7 days after induction, followed by regeneration of renal tubular epithelial cells approximately 2 weeks after induction, which was characterized by increased dedifferentiated and mitotic cells in the damaged tubules. The whole study included three parts.
In the Part A, as DNA microarray technology now offers the advantages of monitoring global gene expression patterns and providing a powerful approach to molecular dissection of cells and tissues by comparing expression levels of up to thousands of gene simultaneously. We used the uranyl nitrate induced nephrotoxic ATN animal models for global gene analysis. With analyzing the large-scale changes in gene expression in the uranyl-nitrated induced nephrotoxic ATN model using cDNA microarray technology and identified altered over-expressions of 35 gene spots and identified several novel genes that may be important in renal repair after nephrotoxic ATN.
In Part B, we used the real-time PCR analysis of mRNA expression of a large family of intracellular calcium and phospholipases-binding proteins demonstrated selective expression of Calcyclin (S100A6) and Annexin A2 (Anxa2) in the renal cortex was greatly elevated on day 3, and gradually declined on day 7 and was only slightly elevated on day 14. Similarly, the expression of both proteins, as demonstrated by immunohistochemistry (IHC) and Western blot analysis, was increased and reached the peak level on day 7 and then gradually declined by day14. As demonstrated by IHC, vimentin, a marker of dedifferentiated cells, was highly expressed during the recovery phase. By combined in situ hybridization and IHC, co-localization of S100A6 & proliferating cell nuclear antigen (PCNA), and Anxa2 & PCNA was identified, respectively, indicating that most PCNA-positive tubular cells expressed both S100A6 and Anxa2. The universality of this phenomenon was confirmed in two other mouse ATN models, the ischemic-reperfusion injury and folic acid-induced ARF. The pattern of mRNA expression of the two genes was very rapid and similar in pattern to that of ATN induced by uranyl nitrate. With these findings demonstrate an important role for S100A6 and Anxa2 in sensing injury and recovery of tubular cells by regeneration in acute renal failure.
In the Part C, we used quantitative RT-PCR, in situ hybridization and immunohistochemistry to demonstrate that nephronectin was modulated in a temporal pattern concomitant with the initiation and recovery of acute renal failure. In the nephrotoxic uranyl nitrate model elevated levels of nephronectin were significantly elevated at 3 days post-induction and returned to normal level by 14 days. Thus these findings suggest nephronectin could be involved in the proliferative process that results in repopulation of tubular cells and recovery from acute renal failure.
During the course of our studies on global gene expression profiling in a mouse model of uranyl nitrate-induced acute renal failure, we observed dramatic up-regulation in the expression of many genes. Accordingly, we infer that these interactive effector molecules might play a role in the pathophysiology of acute renal failure and their expression might be useful biomarkers of the temporal events and processes of the tubular epithelial cell in ATN.
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