Re-expression of the developmental gene Pax-2 during experimental acute tubular necrosis in mice 1.

BACKGROUND: The transcription factor Pax-2 is known to play a key regulatory role during embryonic development of the nervous and excretory systems in mammals and flies. During mouse kidney development, Pax-2 is expressed in the undifferentiated mesenchyme in response to ureter induction and continues to be expressed in the developing comma- and s-shaped bodies. These structures harbor the immediate precursors of the proximal tubular epithelial cells. Pax-2 expression is down-regulated as the differentiation of the functional units of the nephron proceeds. In the adult mammalian kidney, the Pax-2 protein is detectable exclusively in the epithelium of the collecting ducts. We sought to test the hypothesis that tissue regeneration is characterized by re-expression of developmentally important regulatory genes such as Pax-2. METHODS: The expression pattern of Pax-2 in kidneys after experimentally-induced acute tubular necrosis caused by intraperitoneally injected folic acid in mice was tested by indirect immunofluorescence, Western blotting, reverse transcriptase-polymerase chain reaction, and in situ hybridization analysis. RESULTS: A transient, temporally and locally restricted re-expression of Pax-2 in regenerating proximal tubular epithelial cells was observed following kidney damage. CONCLUSIONS: These data indicate that during the regeneration processes, developmental paradigms may be recapitulated in order to restore mature kidney function.

Pax-2 is required for mesenchyme-to-epithelium conversion during kidney development.

The conversion of mesenchyme to epithelium during the embryonic development of the mammalian kidney requires reciprocal inductive interactions between the ureter and the responding metanephric mesenchyme. The Pax-2 gene is activated in the mesenchyme in response to induction and is subsequently down-regulated in more differentiated cells derived from the mesenchyme. Pax-2 belongs to a family of genes, at least three of which encode morphogenetic regulatory transcription factors. In order to determine the role of Pax-2 during kidney development, we have generated a loss-of-function phenotype using antisense oligonucleotides in mouse kidney organ cultures. These oligonucleotides can specifically inhibit Pax-2 protein accumulation in kidney mesenchyme cells, where the intracellular concentrations are maximal. The kidney organ cultures were stained with uvomurulin and laminin antibodies as markers for epithelium formation. With significantly reduced Pax-2 protein levels, kidney mesenchyme cells fail to aggregate and do not undergo the sequential morphological changes characteristic of epithelial cell formation. The data demonstrate that Pax-2 function is required for the earliest phase of mesenchyme-to-epithelium conversion.

Differential distribution of oligodeoxynucleotides in developing organs with epithelial-mesenchymal interactions.

The use of antisense oligodeoxynucleotides (ODNs) to inhibit gene expression is a usefull method for determining protein function and has potential therapeutic applications. However, there is still great variability in the successfull application of antisense technology to individual systems. In order to assess the ability of different cell types to take up ODNs, developing embryonic tissues were cultured in vitro in the presence of fluoresceine labelled, phosphorothioate substituted ODNs. The distribution of ODNs in individual cell populations was assayed by fluorescent microscopy and the tissue sections were counterstained for epithelial basement membrane formation. High intracellular levels of ODNs were observed in all mesenchymal cells of the lung, salivary gland, kidney, ovary and testis. However, a significant decrease in ODN levels was observed with the formation of new epithelium in kidney and gonads, whereas mature epithelial cells in all tissues had no detecable levels of ODNs. The ability to inhibit gene expression in mesenchymal cells, but not in epithelial cells, was consistent with the distribution pattern of labeled ODNs. These results may indicate a general resistance of epithelial cells to take up ODNs in culture and bear directly on the ability of ODNs to affect gene expression in complex organs with epithelial components.

Deregulation of Pax-2 expression in transgenic mice generates severe kidney abnormalities.

The Pax genes comprise a family of transcription factors active in specific tissues during embryonic development and are associated with at least three developmental mutations in mouse and man. In the developing kidney, Pax-2 is expressed in the induced mesenchyme, in the ureter epithelium, and in early epithelial structures derived from the mesenchyme. Pax-2 expression is repressed upon terminal differentiation of the renal tubule epithelium, but persists in the undifferentiated epithelium of human Wilms' tumours. We have produced a dominant gain-of-function mutation in transgenic mice by deregulating the expression of the mouse Pax-2 gene. The data obtained with four independently derived transgenic embryos and with one transgenic line demonstrate that deregulated Pax-2 expression results in histologically abnormal and dysfunctional renal epithelium with properties similar to congenital nephrotic syndrome. Thus, repression of Pax-2 is required for normal kidney development and persistent expression of Pax-2 may restrict the differentiation potential of renal epithelial cells.