The Department of Maternal and Fetal Health, The Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, and The Division of Nephrology, St. Michael’s Hospital, University of Toronto, Canada.
Correspondence to Susan E. Quaggin, Samuel Lunenfeld Research Institute, Room 871Q, Mt. Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada. Phone: 416-586-4800; Fax: 416-586-8588; E-mail: Quaggin{at}mshri.on.ca
One of the many challenges facing researchers in the postgenomicera is the assignment of biologic functions to all the genesequences now publicly available. In many centers, the mouseis the experimental model of choice for studying the geneticsof human development and disease. The mouse is a powerful toolto study human biology because of the remarkable similaritiesof the genetic, cellular, and organ functions between thesetwo species. Furthermore, the ability to manipulate the murinegenome through targeted or random mutagenesis is unparalleledin other mammalian systems. Inactivation of genes in the germlinehas provided great insight into their function; however, theresulting phenotypes are often complex and may preclude analysisof the gene’s function in specific organs or tissues ofinterest. In part, investigators are able to overcome this problemby inactivating and/or overexpressing genes of interest in acell-specific manner. This has required the identification andcharacterization of tissue-specific promoters, genetic elementsthat direct the expression of genes in specific cell types.Using this approach, great strides have been made in the areasof cardiac, lung, and gut development and disease (1–7).However, progress in the renal field has been slower due tothe lack of useful tissue-specific promoters. In this issueof JASN, Moeller et al. (8) report the identification and characterizationof two podocyte-specific promoters that promise to provide acatalyst to the study of gene function in the kidney.
The authors identified regulatory regions from two genes, nphs1and NPHS2, which are responsible for congenital Finnish nephropathy(9) and autosomal recessive steroid-resistant nephrotic syndrome(10), respectively, and are able to direct the expression ofbeta-galactosidase specifically to glomerular visceral epithelialcells (podocytes). In transgenic mouse models, Holzman and hiscolleagues elegantly demonstrate that 1.25-kb of the proximal5' flanking region of the murine nphs1 gene and 2.5-kb of thehuman podocin (NPHS2) promoter contain all of the regulatorysequences required for podocyte-specific expression. Wong etal. (11) have previously shown that 1.25-kb of the human NPHS1gene also provides podocyte-specific expression, whereas largerfragments that measure 8.3-kb, 5.4-kb, or 4.125-kb direct expressionto neural subsets in the developing hindbrain in addition topodocytes (12,13). In their article, Moeller et al. (8) showthat only 30% of the nphs1 founder lines express the transgene,suggesting that activity of the 1.25-kb nphs1 promoter is dependenton the site of chromosomal integration. In contrast, 100% ofthe NPHS2 transgenic founder lines express beta-galactosidasein podocytes. The property of integration-independence is obviouslyimportant when considering which promoter to choose in generatingfuture transgenic lines.
To date, a handful of cell-specific promoters have been identifiedin the kidney; these include a 1542-bp fragment of the 5' flankingregion of the KAP gene (kidney androgen-regulated promoter)(14) and 346-bp of the gamma-Glutamyl Transpeptidase Type IIpromoter (15), which direct expression to the proximal tubule,3.0-kb of the Tamm Horsfall Protein (THP) promoter, which directsexpression to the thick ascending limb of the loop of Henle(TAL) and early distal convoluted tubules (16), 1.34-kb of theKsp-cadherin promoter, which directs expression to the TAL andcollecting ducts of the adult nephron and weakly in other celltypes and to the ureteric bud, Wolffian duct, Mullerian duct,and developing tubules in the mesonephros and metanephros (17)while a 324-bp fragment limits expression to tubular epitheliaof the developing kidney and GU tract (18), the HoxB7 promoter,which marks the ureteric bud and its derivatives (19), and 1.25-kbof the human NPHS1 and 8.3-kb, 5.4-kb, 4.125-kb, and 1.25-kbof the murine nphs1 promoters, which direct expression to podocytes(11–13). The present article adds NPHS2, another podocyte-specificpromoter, to the list.
How will the identification of these tissue-specific regulatorysequences help nephrologists and researchers interested in kidneybiology? The most obvious answer is the ability to express genesof interest in specific cell types within the kidney and lookat the resulting phenotypes. For example, increased expressionof numerous growth factors has been reported to occur in podocytesduring glomerular injury (20,21). Using the promoters describedby Moeller et al. (8) to direct the expression of these growthfactors to podocytes, it will now be possible to test whetherincreased expression of these factors in podocytes underliesthe pathogenesis of glomerular scarring or is simply a markerof disease (i.e., do the mice that overexpress a growth factorin podocytes develop glomerular scarring?) (Figure 1). Althoughthis is a straightforward experiment, caution must be used ininterpretation of any overexpression study. Most importantly,the relevance of overexpressing a gene under the regulationof a heterologous promoter to supraphysiologic levels and/orin the presence of two normal copies of the endogenous genemust be established. In addition, one has to be aware of thedevelopmental stage at which the promoter becomes active, asthis may affect the phenotype.
Figure 1. The podocyte-specific promoters reported in the manuscript by Moeller et al. (8) were used to drive the expression of beta-galactosidase in glomerular visceral epithelial cells. Upon lacZ staining, the transgenic podocytes turn blue. Investigators will be able to use these same promoters to direct expression of candidate "disease genes" to the podocyte to determine whether their overexpression leads to kidney disease.
Using these same promoters and conditional gene targeting strategies,it will also be possible to control the temporal, spatial, and/orlevel of gene expression precisely. Investigators will be ableto "knock out" genes in specific renal cell types rather thanfrom the germline, allowing the study of genes that might playimportant roles during earlier stages of development or in multipleorgans. Several systems exist for generating conditional knockouts,including the Cre-loxP and flp-frt recombinase systems (22,23).To date, the Cre-loxP system has been most widely used in mammaliancells and tissues. Murine lines that demonstrate Cre-mediatedexcision from tubular epithelia and podocytes have already beenreported (13,24). The general strategy for Cre-mediated excisionis shown in Figure 2. Cre recombinase is a bacteriophage enzymethat causes site-specific recombination between loxP sites (34-bpDNA repeat sequences). Using 4.125-kb of the murine nphs1 and1.34-kb of the ksp-cadherin promoter, investigators have alreadygenerated transgenic mice that express Cre-recombinase specificallyin the podocyte or tubular epithelia, respectively. When thesemice are bred with a transgenic strain that carries ‘loxP’sites around a gene-of-interest, it will lead to the loss ofthe target gene ONLY from the cells that also express Cre-recombinase.In this manner, it will be possible to rescue early embryoniclethality that might occur when a gene is inactivated in thegermline and to look at the function of removing 1 or 2 copiesof a gene in specific cell types within the kidney. Furthermore,this strategy can also be used to activate the expression ofreporter genes to perform lineage-tracing studies in vivo. Excisionof a ‘STOP codon’ allows the expression of a cellmarker such as enhanced green fluorescent protein (EGFP), whichtags the cell at a specific time point and allows one to followits fate during development and aging or in disease. The identificationof truly podocyte-specific promoters as described in this issueof JASN should allow additional Cre-recombinase lines to begenerated. Of note, the 4.125-kb nphs1 promoter used to generatethe Cre-recombinase lines by Eremina et al. (13) also directsexpression in a small subset of neural cells that derive fromthe first and second rhombi, which might interfere with theanalyses of target genes that are also expressed there.
Figure 2. General scheme for Cre-mediated DNA excision in vivo. A transgenic mouse line that expresses Cre-recombinase under the regulation of a podocyte-specific promoter is crossed to a reporter strain (the Z/EG mouse [27]). In cells where the Cre-recombinase is active (i.e., ONLY in podocytes), site-specific recombination occurs between the loxP sites () and leads to excision of the Beta-geo cassette and 3 poly-A signals, which functions as a STOP signal for transcription. After excision, the enhanced green fluorescent protein gene is transcribed and the offspring demonstrate green glowing podocytes. In other tissues where the promoter is inactive, no excision occurs and no fluorescence is visible. P, podocyte-specific; , loxP sites; EGFP, enhanced green fluorescent protein; Beta-geo, beta galactosidase and neomycin cassette.
Perhaps of most value to adult nephrologists will be the generationof inducible knockout and overexpression systems in specificcell types within the kidney. Although several varieties exist,the tetracycline or estrogen-sensitive systems have both beensuccessfully employed (25,26). In the first, it is possibleto repress or activate the expression of a target gene whenthe transactivator tTA or ‘reverse’ rtTA is boundto the Tet operator (tetO). Placing tTA or rtTA under kidneyspecific control and crossing these transgenic lines with micecarrying Cre-recombinase or another gene of interest under thecontrol of the TetO sequence, allows the precise control oftarget gene expression upon administration of tetracycline.Similarly, the administration of an estrogen agonist to transgenicmice that carry a fusion protein consisting of Cre-recombinasefused to an estrogen receptor results in activation of the Crerecombinase (26). Thus, the investigator will be able to choosethe time of administration of these drugs to study gene functionin the developing and/or adult kidney.
Although these promoters represent powerful new tools for studiesin whole animals, they will also be valuable for in vitro applications.The identification of common regulatory elements between thehuman NPHS1, NPHS2, and mouse nphs1 genes should aid in theidentification of novel and known transcription factors thatare required for podocyte-specific expression as transcriptionfactors bind to "consensus" sequences encoded by the DNA. Knowledgeof these factors will lead to elucidation of the molecular pathwaysthat promote differentiation and maintenance of a "healthy podocytephenotype" and will help identify putative targets for therapyand treatment of patients with kidney disease. Interestingly,none of the knockouts of transcription factors that are knownto be expressed in the podocyte have shown changes in nephrinmRNA expression. Although other obvious candidates such as WT1exist, it is also possible that there will be novel factorsrequired for its regulation.
In summary, characterization of the murine nephrin and podocinpromoters adds two useful podocyte-specific promoters to thearmamentarium of molecular tools for the nephrologist. Researcherswill now be able to design elegant experiments and target geneexpression in the kidney in both temporal and cell-specificmanners.
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) and leads to excision of the Beta-geo cassette and 3 poly-A signals, which functions as a STOP signal for transcription. After excision, the enhanced green fluorescent protein gene is transcribed and the offspring demonstrate green glowing podocytes. In other tissues where the promoter is inactive, no excision occurs and no fluorescence is visible. P, podocyte-specific;