Identification and characterization of a glomerular-specific promoter from the human nephrin gene

M. Andrew Wong1, Shiying Cui1, and Susan E. Quaggin1,2

1 Department of Maternal and Fetal Health, The Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5; and 2 Division of Nephrology, St. Michael's Hospital, Toronto, Ontario, Canada M5B 1W8


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Podocytes are highly specialized cells that make up a major portion of the glomerular filtration barrier in the kidney. They are also believed to play a pivotal role in the progression of chronic renal disease due to diverse causes that include diabetes (3, 20, 24) and aging (1, 7). Despite the importance of podocytes for kidney function and disease, studies of this cell type have been hindered due to a lack of model systems. Recently, the gene responsible for congenital Finnish nephropathy was identified and named nephrin (13). Nephrin expression is restricted to slit diaphragms of podocytes (11, 30). Infants with congenital Finnish nephropathy develop massive proteinuria and subsequent kidney failure due to podocyte injury. We have identified a 1.25-kb DNA fragment from the human nephrin promoter and 5'-flanking region that is capable of directing podocyte-specific expression in transgenic mice; this represents the first glomerular-specific promoter to be identified. Use of this transgene will facilitate studies of the podocyte in vivo and allow the identification of transacting factors that are required for podocyte-specific expression.

podocyte; glomerulus; transgene; tissue-specific expression


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PROGRESSIVE RENAL DAMAGE OCCURS in a variety of disease states, even when the initiating injury is removed. Glomerulosclerosis and secondary tubulointerstitial fibrosis is the most common pathologic lesion identified in progressive renal failure (4). Together, these observations suggest that there is a common final pathway for many kidney diseases.

A number of studies have implicated the podocyte as a key cell in the progression of renal disease toward glomerulosclerosis (15, 16). Podocytes are mesodermally derived cells that are highly specialized and found only in the renal glomerulus. They exhibit unique characteristics such as foot processes and slit diaphragms, which are critical for glomerular filtration (21, 33). When podocytes are damaged, the foot processes fuse and eventually detach from the underlying glomerular basement membrane, leaving fewer cells to cover the capillary loops (16). Terminally differentiated podocytes are believed to be unable to divide in the adult kidney. Instead, they respond to glomerular injury through hypertrophy. Kriz has proposed that eventual loss of these hypertrophied podocytes leads to direct apposition of glomerular capillary endothelial cells to the overlying parietal epithelium, and obliteration of the filtration space (15, 16). Careful morphological studies have demonstrated a strong correlation between podocyte number and progression of glomerulosclerosis in diabetes (20). Although it has been reported that "dysregulated" glomerular visceral epithelial cells (podocytes) can proliferate in specific conditions such as collapsing glomerulopathy (2), these results remain controversial (14).

Despite the clinical importance of podocytes, their biology is still poorly understood. Previously, developing appropriate model systems to study podocytes in vitro has been difficult, as glomerular epithelial cells dedifferentiate in culture and lose their podocyte-specific markers. Recently, the gene responsible for congenital Finnish nephropathy was identified and named nephrin (13). Nephrin is a 135-kDa protein with homology to the immunoglobulin superfamily of cell adhesion molecules and is specifically located in the slit diaphragms of podocytes. Children who have mutations in the nephrin gene develop massive proteinuria and renal failure before age 2 yr (32).

Shih et al. (32) reported proteinuria and glomerular damage in mice that are homozygous null mutant for CD2AP (CD2-associated protein). These investigators demonstrated that CD2AP is expressed in podocytes and can associate with nephrin in vitro. The authors speculate that CD2AP links nephrin in the slit diaphragm to the intracellular cytoskeleton.

The purpose of the present study was to identify and characterize the podocyte-specific elements of the nephrin promoter. This promoter will be a valuable tool to study podocyte biology in vivo, with the hope of understanding its role in the development of glomerulosclerosis and ultimately enabling repopulation of the damaged glomerulus.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning the human nephrin promoter. The National Center for Biotechnology Information BLAST program was used to search the human chromosome 19 cosmid (Genbank accession no. AC002133)1 for location of the human nephrin cDNA. The predicted nephrin initiation codon was identified at position 27,309. We designed PCR oligonucleotide primers (shown below) to amplify 4 kb of the proximal 5' flanking sequence of the nephrin gene that includes the predicted initiation ATG and 57 bp of the first exon. DNA was amplified by PCR from human genomic DNA, as previously described (27). A 4-kb fragment was excised and subcloned into the PCR2.1 vector (Clontech). Subsequent sequence analysis revealed that only 1.25 kb of the proximal promoter and 5' flanking region was present in the plasmid, which suggests that DNA was lost during bacterial growth. The final nephrin transgene fragment begins at position 28,505 (GenBank accession no. AC002133)1 and ends at position 27,253. A 3.34-kb Sal I fragment from the pSDKlacZpA vector containing the lacZ-expression cassette was excised and cloned into the Xho I site of the nephrin/PCR2.1 vector to create the final nephrin-transgenic construct (26): sense oligonucleotide, 5' CTGGCTGAGACGCTGATGGCCTGA 3', and antisense oligonucleotide, 5' CCTTCAGTCAGCAGCCCCAGGAGCA 3'.

Generation of transgenic lines. The nephrin construct was linearized with NotI, and gel purified by using a Bio101 Geneclean kit. Transgenic DNA at a concentration of 2 ng/µl was injected into the pronucleus of one-cell embryos and transferred to psuedopregnant female CD1 mice as described (10). Genomic DNA was isolated from tails of transgenic mice and used for genotype analysis as described (26). DNA was digested with EcoR I, and Southern blot analysis was performed by using a probe for the beta -galactosidase gene.

In situ analysis of embryonic kidneys. Kidneys from 18.5-day postcoitum (dpc) embryos were dissected and fixed in 4% paraformaldehyde overnight at 4°C, cryopreserved in 30% sucrose overnight at 4°C, embedded in OCT compound (Tissue-Tek 4583), and frozen at -70°C. Twelve-micrometer cryosections were cut on a Leica Cryostat (model CM3050), and in situ hybridization was performed as described elsewhere (12, 31). A 2.3-kb fragment of the mouse nephrin gene from bp 1810 to 3728 (GenBank accession no. AF168466)2 in pBluescriptKS+ was used as a template for antisense- and sense digoxigenin-labeled RNA probes that were prepared according to manufacturer's (Boehringer Mannheim) instructions.

beta -Galactosidase staining of embryos and kidneys. Kidneys or whole embryos from 9.5 to 18.5 dpc were fixed in 4% paraformaldehyde and 1% glutaraldehyde and stained for beta -galactosidase activity as described (25). Kidneys and a variety of other tissues (lung, heart, gut, etc.) were also dissected from postnatal day 0 and postnatal day 14 mice, cut into small pieces, and fixed and stained with beta -galactosidase by whole mount as described above. Tissues were then embedded in paraffin, and 5-µm sections were cut.

Sequence analysis of the promoter region. The 1.25-kb fragment of nephrin DNA that was used to generate transgenic lines was analyzed for transcription-factor binding sites by using the TRANSFAC Web site and the internet browser3 (9).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
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REFERENCES

LacZ expression is restricted to podocytes in transgenic lines. Six founder lines were identified by Southern blot analysis (Fig. 1). Three of these lines had a single chromosomal integration of the transgene, while three had two or more integrations of the transgene. Kidneys from one newborn founder male and embryos and kidneys from the offspring of the other five founder lines were dissected and stained for galactosidase activity. Offspring and kidneys from three of the six founder lines demonstrated lacZ expression. Of note, only one of the expressing lines had two sites of integration of the transgene; the other two lines had a single integration of the transgene. Offspring from the other three founder lines and two nontransgenic-control littermates exhibited no lacZ expression at any stage of development. In the three positive lines, lacZ expression was found exclusively in podocytes of capillary-loop stage and mature glomeruli (Fig. 2). No staining was identified in podocyte precursors in S-shaped bodies or in any other tissues at any embryonic or postnatal day studied. In comparison, endogenous nephrin mRNA was detected in podocyte precursors in S-shaped bodies, in podocytes from capillary-loop stage glomeruli, and in mature podocytes (Fig. 3).


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Fig. 1.   Transgenic construct and Southern blot analysis of transgenic mice. A: schematic diagram of the human nephrin lacZ-transgene construct. The gray-shaded box represents the 1.25-kb fragment of genomic DNA from the promoter region of the human nephrin gene. The arrow represents the transcription initiation site (TIS); nucleotide positions are indicated relative to the TIS. The lacZ-expression cassette contains an SDK oligonucleotide and is followed by the polyadenylation signal from SV40 (pA). B: Southern blot analysis of genomic DNA from 2 founder lines. Genomic DNA was isolated from mouse tails and digested with EcoR I, which cuts the transgene once internally. The blot was hybridized with a lacZ probe. Lanes 2 and 7 demonstrate founder lines with 1 and 2 sites of chromosomal integration, respectively. Both of these founder lines expressed the lacZ reporter gene specifically in podocytes (po). Left: DNA molecular wt standards for 7 and 3 kb.



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Fig. 2.   LacZ expression in transgenic mouse kidneys at embryonic day 18.5. Embryonic mouse kidneys from offspring of the founder line shown in Fig. 1 (lane 2) were dissected at day 18.5 and stained with beta -galactosidase by whole mount. The kidneys were embedded in paraffin and sectioned. A: the 1.25-kb nephrin fragment directs expression of the lacZ reporter gene to glomeruli and specifically to po (B, C). pa, Parietal epithelial cells; me, mesangial cells.



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Fig. 3.   mRNA expression of the endogenous murine nephrin gene. A digoxigenin-labeled antisense 2.3-kb probe from the 3' end of the murine nephrin cDNA was used for in situ analysis of embryonic day 18.5 mouse kidneys. Murine nephrin is weakly expressed in po precursors of an S-shaped body (developing glomerulus, A), but is highly expressed in po in capillary-loop stage (B) and mature glomeruli (C). In contrast, no staining was seen in samples hybridized with the sense digoxigenin-labeled probe (not shown).

Transcription factor binding sites in the podocyte-specific promoter. The DNA fragment capable of directing podocyte-specific expression in the glomerulus was searched for potential transcription factor binding sites (Fig. 4). Within this 1.25-kb fragment there is no TATA box, but GATA binding sites are seen. Of note, there are several E-box consensus sequences that are recognition sites for basic-helix-loop-helix proteins and a potential Pax-2 binding site. The transcription initiation site has been determined by primer extension and is reported to occur 156 bp upstream of the initiating codon (17).


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Fig. 4.   DNA sequence of the 1.25-kb po-specific promoter. The sequence of the proximal promoter region of the human nephrin gene is shown. The predicted initiation ATG is shown in bold with an asterisk. The reported transcription start site is shown with double asterisks. Within this region we were unable to identify a TATA box, but several GATA and E-box consensus sites are shown. In addition, a putative Pax-2 binding site was identified and is shown. The GA repeat sequence is underlined. Mutations in this region of the promoter were reported in 2 patients with congenital Finnish nephropathy (17).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Podocytes are an integral component of the glomerular filter and play a pivotal role in the progression of many renal diseases. In contrast to the mesangial cell, which is also found in the glomerulus but is not a structural component of the renal filter, the biological role of the podocyte in renal disease has not been studied extensively. In part, this has been due to the lack of cell-culture models. Although it has been possible to isolate glomerular epithelial cells from primary cultures, these cells lose their podocyte-specific characteristics after a few passages (23). Mundel et al. (22) have isolated SV40-transformed murine-podocyte cell lines that retain certain characteristics and markers of podocytes but cannot replace in vivo studies. As a first step in the development of molecular tools to study the podocyte in vivo, we report the first glomerular-specific promoter to be identified. In this paper, we demonstrate that a 1.25-kb fragment of genomic human DNA, which includes the predicted initiation codon and immediate 5'-flanking region of nephrin, directs podocyte-specific expression in vivo.

The expression pattern of our human lacZ transgene recapitulates the expression pattern of the endogenous murine nephrin gene with a few exceptions. In the mouse, nephrin mRNA is weakly expressed in podocyte precursors at the S-shaped body stage (Fig. 3A) and is strongly expressed in podocytes in capillary-loop stage and mature glomeruli (11; Fig. 3, B and C). In addition, Holzman et al. (11) describe nephrin expression in the spleen, and we also see weak expression in developing pancreas (data not shown). In contrast, we did not observe any expression of the nephrin transgene outside the renal podocyte. Furthermore, we did not detect lacZ expression until the capillary-loop stage podocyte; no lacZ expression was detected in S-shaped bodies. This may represent sensitivity of the lacZ-expression assay, absence of a regulatory element in the transgene, and/or interspecies expression differences as the human promoter was used for the transgenic studies. Because three of the six transgenic founder lines did not demonstrate any lacZ expression, it follows that expression of the 1.25-kb promoter of human nephrin is influenced by the chromosomal integration site. Although we observed these stable position effects, we did not observe any heterocellular expression of the transgene (19).

Given the limited size (1.25 kb) of the podocyte-specific promoter, it is of interest to identify putative cis-binding elements. Of note, a putative Pax-2 binding element and multiple canonical E-box consensus sequences exist. Pax-2 is a member of the paired box family of transcription factors; it is expressed in renal vesicles and is specifically downregulated in podocyte precursors at the S-shaped-body stage (6). Studies have shown that Pax-2 can act as both a transcriptional activator and a repressor (5, 8). Thus one might speculate that Pax-2 actively represses transcription of the nephrin gene in epithelial cells of the renal vesicle prior to podocyte differentiation. In addition, we and others have identified a basic-helix-loop-helix transcription factor, Pod1/capsulin/epicardin (18, 27, 29), which is highly expressed in podocyte precursors and mature podocytes. Similar to other bHLH proteins, Pod1 can bind to E-box consensus sequences in vitro (18). Although podocytes fail to differentiate terminally beyond the capillary-loop stage in Pod1 mutant mice, nephrin is still expressed in the mutant podocytes. These results demonstrate that Pod1 is not required to activate transcription of the nephrin gene (28; data not shown).

Lenkkeri et al. (17) reported promoter deletion mutations in two patients with Finnish nephropathy. These occurred in the GA repeat sequence between bp -292 to -327 of the proximal promoter. One of these patients presented with an atypical course and did not require renal transplantation until age 5 yr (17). It will be of interest to determine whether mutations in this region affect expression of our transgene.

We have identified and begun to characterize the first glomerular-specific and podocyte-specific promoter. Identification and characterization of cis-acting elements in this promoter fragment will be useful to identify transcription factors required for podocyte-specific expression. Use of this transgene will allow genetic manipulation of the podocyte in vivo, characterization of its biological role in renal function and disease, and ultimately, testing of the hypothesis that the podocyte plays a pivotal role in the progression of renal injury toward glomerulosclerosis.


    ACKNOWLEDGEMENTS

We thank Johanne Pellerin and Lois Schwartz for expert technical assistance.


    FOOTNOTES

S. E. Quaggin is the recipient of a Clinician Scientist Award from the Medical Research Council of Canada, the Carl Gottschalk Scholar Award from the American Society for Nephrology, and is a Canadian Foundation for Innovation Researcher. This work was supported by a Medical Research Council of Canada grant to S. E. Quaggin.

Address for reprint requests and other correspondence: S. E. Quaggin (E-mail: quaggin{at}mshri.on.ca).

1 The nucleotide sequence for the human chromosome 19 cosmid can be accessed through the National Center for Biotechnology Information (NCBI) nucleotide database www.ncbi.nlm.nih.gov under NCBI accession no. AC002133.

2 The nucleotide sequence for the murine nephrin cDNA can be accessed through the NCBI nucleotide database under NCBI accession no. AF168466.

3 The TRANSFAC transcription factor site database can be accessed on: http://transfac.gbf.de/TRANSFAC (9).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 15 May 2000; accepted in final form 18 August 2000.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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