Evaluation of a New Tool for Exploring Podocyte Biology: Mouse Nphs1 5′ Flanking Region Drives LacZ Expression in Podocytes

Identification and Cloning of the Nphs1 Gene

A 0.5-kb mouse nephrin cDNA fragment (10) was used to identify two mouse 129-SvJ mouse genomic BAC clones (235 and 135 kb; Genome Systems Inc., St. Louis, MO). Upon restriction mapping, these clones were found to contain the entire Nphs1 coding region and at least 60 kb of genomic sequence 5′ to the initiation codon.

Construction of the Reporter Transgene

The β-galactosidase (β-gal) reporter plasmid used in this study was derived from pnlacF (12). pnlacF encodes Escherichia coli β-gal possessing a nuclear localization signal from simian virus fused to its amino terminus. A 14-kb HindIII cDNA fragment containing the 5′ nephrin coding region was cloned into puc18. To remove most of the nephrin coding region, a 5.5-kb SalI fragment was released using a SalI site 0.24-kb 3′ to the initiation codon and a SalI site in the multiple cloning region of puc18. The resulting construct (Nphs1-PE-puc18) containing an 8.7-kb insert with 8.3 kb of the promoter-enhancer region 5′ of the initiation codon and 0.2 kb of the nephrin coding region was DNA sequenced using the GPS™-1 Genome Priming System (New England Biolabs, Beverly, MA) for random primer insertion and an automated DNA sequencer. An NcoI site was created at the initiation codon ATG by PCR-based mutagenesis and was verified by DNA sequencing (TGATG to CCATG). The 8.3-kb full-length nephrin promoter-enhancer was released using NcoI and HindIII. After blunt-ending the HindIII site (DNA Polymerase I Large [Klenow], Promega, Madison, WI), the 8.3-kb fragment was cloned into the SmaI and NcoI sites of the pnlacF reporter vector (p8.3N-nlacF). A second, shorter 5.4-kb fragment of the nephrin promoter-enhancer was subcloned into pnlacF using XbaI and NcoI (p5.4N-nlacF).

Sequence Analysis

The identified murine Nphs1 5′ flanking sequence (8298 bp) was compared with the 5′ flanking sequence of the human NPHS1 gene (41.5 kb of cosmid clone R33502, Genbank accession number AC002133) using BLASTN 2.0.11 (default parameters) to identify conserved areas. With the use of the MatInspector V2.2 search engine and the TRANSFAC database (core sim = 1, matrix sim > 0.97; maximal stringency), potential transcription factor binding sites were identified in these conserved areas (13).

Generation of Transgenic Mice

The p8.3N- and p5.4N-nlacF construct (11.7 and 8.8 kb) were liberated from the plasmid vector backbone by digestion with KpnI and HindIII, separated by agarose gel electrophoresis, isolated from the gel, and purified by Nuclespin columns (Clontech, Palo Alto, CA). The purified DNA fragment was microinjected into F2 hybrid eggs from (C57BL/6J X SJL/J) F1 parents at a concentration of 2 to 3 ng/μl (14). Eggs were transferred to day 0.5 postcoitus (dpc) pseudopregnant ICR females. Founder transgenic mice were mated to C57BL/6J or SJL/J wild-type mice. (C57BL/6J X SJL/J)F1 mice were obtained from The Jackson Laboratory (Bar Harbor, ME); ICR mice were obtained from Harlan (Indianapolis, IN). The University of Michigan Committee on Use and Care of Animals approved all procedures that used mice. All work was conducted in accordance with the principles and procedures outlined in the NIH Guidelines for the Care and Use of Experimental Animals.

Identification of Transgenic Mice

Transgenic mice were identified using a PCR strategy on DNA recovered from tail biopsies. The tails of 3-wk-old mice were lysed in TNES solution (10 mM Tris [pH 7.5], 400 mM NaCl, 100 mM ethylenediaminetetraacetate, 0.6% sodium dodecyl sulfate) containing 0.55 mg/ml proteinase K overnight at 55°C. The DNA was isolated using the DNeasy Tissue Kit (Quiagen, Valencia, CA). The transgene was identified by PCR (Taq DNA Polymerase, Promega) using the primers LacZ.fwd: TTC ACT GGC CGT CGT TTT ACA ACG TCG TGA and LacZ.rev: ATG TGA GCG AGT AAC AAC CCG TCG GAT TCT (200 ng of genomic DNA, 30 cycles, Tm = 60°C). The PCR product was detected on a standard 1% TAE-agarose (4.84 g Tris-Base, 1.142 ml glacial acetic acid, 0.744 g Na₂ EDTA-2H₂O to 1 L H₂O) gel as a 364-bp fragment.

β-Gal Assays

Assays of β-gal activity in tissue homogenates were performed using a chemiluminescence assay as described by Shaper et al. (15). Fresh tissues (100 mg) were dissected from transgenic and nontransgenic mice and were homogenized in 1 ml of lysis buffer containing 100 mM potassium phosphate (pH 7.8), 0.2% Nonidet P-40, 1 mM dithiothreitol, and 1 tablet/50 ml of Complete EDTA-free Protease Inhibitor Cocktail (#1836170, Roche Molecular Biochemicals, Indianapolis, IN). Homogenization was performed for 20 s on ice using a Micro Ultrasonic Cell Disrupter (Kontes, Vineland, NJ). After centrifugation at 12,500 × g for 10 min, the supernatants were heated at 48°C for 50 min to inactivate endogenous mammalian β-gal activity (16). After a second centrifugation at 12,500 × g for 5 min, the protein concentration in the supernatants was determined using the Bradford Protein Assay Reagent (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard. Aliquots of heat-inactivated lysate containing 10 mg of total protein was incubated for 60 min at 25°C with 300 μl of reaction buffer containing Galacto-Star (Tropix, Bedford, MA), 100 mM sodium phosphate (pH 7.5), 1 mM MgCl₂, and 5% Sapphire-II (Tropix). Light output was integrated in mV over 20 s using a 1251 luminometer (BioOrbit, London, UK). Assays of β-gal activity in situ were performed by perfusing freshly euthanized mice with ice-cold 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.8) intracardially for 10 min (4.4 ml/min) and with 18% sucrose in PBS (pH 7.8) for 10 min (4.4 ml/min). Successful fixation was indicated by rapid blanching of the liver and kidneys and stiffening of the skeletal muscles.

The kidneys and brain were resected and incubated overnight in 30% sucrose in PBS (pH 7.8) on ice as cryoprotection. The tissues were embedded (Tissue-Tek, Miles Inc., Iowa City, IA) and 8- to 10-μm cryosections were cut. The sections were postfixed in 4% paraformaldehyde in PBS (pH 7.8) for 5 min and washed in PBS (pH 7.8). The samples were then incubated overnight at 30°C in a humidified atmosphere in staining solution (1 mg/ml X-gal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl₂ in PBS [pH 7.8]), washed with tap water, stained with Nuclear Fast Red Staining Solution (Vector Laboratories Inc., # H-3403, Burlingame, CA) for 1 min, washed with tap water for 10 min, and dehydrated through grades of ethanol and xylene. Newborn kidneys were resected and fixed for 25 min in 1.5% paraformaldehyde and 0.2% glutaraldehyde in PBS (pH 7.8) on ice with continuous agitation, washed thrice in PBS, and embedded, cut, and stained as described above. Whole mounts (up 13 dpc) were generated with timed pregnancies by mating heterozygous transgenic males with wild-type females. The yolk sack was recovered to extract DNA to identify transgenic embryos by PCR as described above. Whole mounts were fixed for 15 to 35 min (depending on gestational age) in 1.5% paraformaldehyde and 0.2% glutaraldehyde in PBS (pH 7.8) on ice with continuous agitation, washed thrice in PBS, and stained overnight at 30°C in X-gal staining solution (plus 0.02% Nonidet P-40 and 0.01% deoxycholate) as described above. Eleven dpc embryos were dehydrated in 100% methanol and cleared in benzyl benzoate: benzyl alcohol 2:1 (vol/vol).

Indirect Immunofluorescence

Indirect immunofluorescence was performed on 8-μm cryosections of kidneys from freshly euthanized transgenic or nontransgenic mice. Adult mice were intracardially perfused with ice-cold 4% paraformaldehyde in PBS (pH 7.4, 5 min) and washed with 18% sucrose in PBS, and their kidneys were frozen in liquid nitrogen immediately after resection. Sections were fixed with ice-cold acetone for 2 min, washed and blocked with 10% donkey serum, and incubated with the following antibodies: goat anti-β-gal polyclonal antibody (1:100 dilution, Biogenesis #4600-1409), Fluorescein (FITC)-conjugated AffiniPure Donkey Anti-Goat IgG (H+L) (1:100 dilution, Jackson Labs, #705-095-147), rabbit anti-WT-1 polyclonal antibody (1:100 dilution in 10% goat-serum, C-19, #sc-192, Santa Cruz) in 10% goat serum, and Cy3-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (1:200 dilution, Jackson Labs, #111-165-144).

Isolation, Molecular Cloning, and Sequence Analysis

An 8.3-kb fragment that included the 5′ untranslated region and the 5′ flanking region of the murine Nphs1 gene was identified, cloned, and entirely sequenced as described in the Materials and Methods section. With the use of computer algorithms and visual inspection, this mouse sequence was aligned with the published human NPHS1 genomic sequence (Figure 1). Like the human gene, no classical TATA box was found in the mouse gene (17). Eighteen conserved regions of 50 to 556 bp were identified. These conserved regions occurred in an array, the ordering of which was also conserved between the two species. Regions of conserved sequence extended to -7.3 kb in the mouse (+1 defined as the A at the translation initiation codon) and extended to -10.5 kb in the human gene. Within these conserved regions, consensus transcription factor binding sites were identified using the MatInspector search engine and the TRANSFAC database (Figure 1).

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Figure 1.

Analysis of the identified 8.3-kb 5′ flanking region of the murine Nphs1 gene. Conserved regions between the murine and human genome are indicated as rectangles. The order of the conserved regions is preserved in both species, regions 5′ of the XbaI site (used to clone the p5.4-nlacF construct) are shown as white rectangles. The percentage sequence identity between murine and human conserved regions is indicated under each region. Potential transcription factor binding sites in the conserved regions of the murine genome and their nucleotide positions relative to the translation initiation site (ATG) are indicated. The +1 site of the human gene is marked with an arrow (17).

Generation of Transgenic Mice

The ability of the identified Nphs1 5′ flanking region to drive appropriate tissue-specific expression of a reporter gene was tested in mice. Two transgenes were created containing either 8.3 kb or 5.4 kb of the 5′ flanking region and the entire 5′ untranslated region of the Nphs1 gene placed upstream to a bacterial lacZ gene encoding β-gal (Figure 2). This lacZ gene possessed a nuclear localization signal. Transgenic mice were generated by pronuclear injection, and founders were identified by PCR analysis of genomic DNA isolated from tail biopsies. Nine transgenic founders of 50 littermates (18%) were obtained after injection of the p8.3N-nlacF construct, and 24 founders of 84 littermates (29%) were obtained after injection of the p5.4-nlacF construct.

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Figure 2.

Map of the two reporter constructs used for the generation of transgenic mice containing 8.3 (p8.3-nlacF) and 5.4 kb (p5.4-nlacF) of the 5′ flanking region and the 5′ untranslated region of the murine Nphs1 gene. The promoter constructs drive expression of an Escherichia coli lacZ reporter gene with a nuclear localization signal (nlacZ). Unique restriction sites and their nucleotide positions relative to the translation initiation site (ATG) are indicated. HindIII and KpnI were used to release the constructs for microinjection.

Tissue Screen for β-Gal Activity

Transgenic F₀ founders initially were examined at 6 to 8 wk postgestation using a sensitive chemiluminescence assay to detect β-gal activity in a survey of multiple tissues (Figure 3). Homogenates were examined from the tissues of three groups of animals. These groups included three independent adult F₀ transgenic founders obtained from each of the two transgenes (two female, one male for each transgene) and a group of three adult wild-type littermates. Endogenous β-gal activity was quenched by heating homogenates to 48°C before analysis. Remaining β-gal activity significantly above background was detected in kidneys and brains of founder mice transgenic for both transgenes but was not detected in other tissues. Indeed, when examined by X-gal staining of cryosections, 9 of 9 of p8.3N-nlacF founders examined and 19 of 20 of p5.4N-nlacF founders expressed β-gal in their kidneys (95 to 100% penetrance). Particularly elevated β-gal activity was measured in the kidneys of founders transgenic for p5.4N-nlacF compared with nontransgenic littermates. With the exception of small intestine, low activities were observed in all other tissues; these activities were not different than those of wild-type controls. Subsequent evaluation of sections of intestinal structures stained with X-gal did not demonstrate nuclear localization of β-gal activity in the small intestine or elsewhere in the gut (data not shown).

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Figure 3.

Tissue screen for β-galactosidase (β-gal) activity. Tissues from three transgenic founders of each construct were homogenized, and β-gal activity was measured in the heat-inactivated lysates using a chemiluminescence assay (first bar, p8.3-nlacF construct; second bar, p5.4-nlacF construct; third bar, wild type). Data are shown as means of mV of three independent experiments with the maximal and minimal value indicated by a vertical line. β-gal activity above background could be detected in the kidney and brain in both constructs. Kidney lysates from p5.4-nlacF founders contained higher β-gal activity than lysates from p8.3-nlacF founder animals. Activity in the brain was comparable in founders of both constructs. Elevated activity in the gut could be attributed to nonspecific endogenous β-gal activity (not shown).

Expression of Nphs1-Driven Transgenes in Adult Kidney and Brain

Because β-gal activity was confined to adult kidney and brain in the initial screen, these tissues were examined microscopically for β-gal activity after X-gal staining of serial 8-μm cryosections. Nuclear lacZ staining was detected in all glomeruli in a characteristic peripheral distribution consistent with that of podocyte nuclei (Figure 4, A and B). This characteristic staining pattern was observed in all adult kidneys examined derived from multiple independent founders and carrying either transgene (6 of 6 of p8.3N-nlacF founders examined and 15 of 15 of p5.4N-nlacF founders). No β-gal activity was identified elsewhere within kidney or within associated adrenal gland or in kidneys of wild-type littermates. To confirm that Nphs1 transgenes were indeed expressed in podocyte nuclei, sections of adult transgenic kidneys were double labeled with anti—β-gal and anti—WT-1 antibodies (Figure 4, C through E). Chosen as a marker because it is expressed solely in podocyte nuclei in adult kidney, WT-1 expression co-localized with that of bacterial β-gal (18). Therefore, both Nphs1 transgenes are expressed like endogenous nephrin, in a podocyte-specific distribution in the adult kidney.

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Figure 4.

β-gal expression in the kidney and brain of adult transgenic mice. (A) β-gal in situ staining on an 8-μm cryosection of the kidney. β-gal is expressed exclusively in glomerular nuclei in the renal cortex in founder animals of both constructs (arrows). No β-gal activity can be detected elsewhere in the kidney. (B) Higher magnification of a single glomerulus of the kidney. β-gal expressing nuclei are distributed in a peripheral pattern in the glomerulus consistent with podocyte nuclei. (C through E) Cryosections of adult transgenic founder animals were double-stained with FITC-labeled antibacterial β-gal (C) and Cy3-labeled anti—WT-1, a marker specific for podocyte nuclei in adult kidneys (E). Superimposition of the two labels demonstrates that β-gal is expressed exclusively in podocyte nuclei expressing WT-1 (D). (F) Nuclear β-gal expression in the area postrema detected by in situ X-gal staining on serial cryosections of the brain of an p5.4-nlacF founder animal. The insert shows a higher magnification of the area postrema. The plexus choroideus is negative. (G) Nuclear β-gal expression could also be detected in nuclei of the medulla lateral to the midline and caudal to the fourth ventricle (insert shows higher magnification).

The brain of an adult p5.4-nlacF F₀ founder was sectioned serially and was stained with X-gal. On microscopic examination, nuclear lacZ staining was detected in two regions of the medulla oblongata. Here, staining was observed in the nuclei within the area postrema at the caudal extremity of the fourth ventricle (Figure 4F). Staining was also observed in nuclei in an area of the medulla lateral to the midline and caudal to the fourth ventricle (Figure 4G). Isolated nuclei in the nucleus medialis of the cerebellum (2 to 20 nuclei/slide) also stained positive for X-gal (not shown). A similar pattern of nuclear localized β-gal activity was observed in two randomly sectioned brain specimens obtained from mice expressing each transgene. No X-gal staining was identified elsewhere in the brain or in the choroid plexus of these mice.

Expression of Transgenes during Embryonic Development

Whole-mount embryos were examined for β-gal activity after X-gal staining and tissue clearing. Embryos from four independent transgenic founders (embryos obtained from two independent founders for each transgene) were analyzed at 8.5 d postcoitus (dpc) and 11.5 dpc. Wild-type littermates served as negative controls. All transgenic embryos examined had similar tissue-specific expression patterns that were independent of the type of transgene expressed. At 8.5 dpc, β-gal activity was present in the neural tube in the region of the hindbrain in transgenic embryos (Figure 5, B and C). β-gal activity extended caudally from the brain and could be detected in the cranial first third to one half of the neural tube. Among littermates, the extent and intensity of β-gal activity in the neural tube increased in a manner directly proportional to the degree of maturity. At 11.5 dpc, β-gal activity was again most prominent within the met- and myelencephalic part of the rhombencephalon, the neural tube structures of the hindbrain. At this time point, β-gal activity was now observed over the entire extent of the developing spinal cord (Figure 5, D and F), suggesting that neural tube Nphs1 transgene expression extends caudally with developmental age. LacZ staining was also identified in the thin roof of the fourth ventricle (Figure 5F). Notably, β-gal activity could also be detected in the notochord of embryos at 11 dpc. β-gal activity was not detected elsewhere in embryos at 8.5 or 11.5 dpc.

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Figure 5.

Embryonic expression of the transgenes in whole mounts at 8.5 and 11.5 days postcoitus (dpc). Transgenic embryos were obtained from timed pregnancies, fixed, and submitted to whole-mount X-gal staining overnight. Transgenic embryos were identified by PCR. A and the insert in C show 8.5 dpc wild-type littermate controls. At 8.5 dpc, β-gal activity is present in the region of the hindbrain and extends caudally along the first third of the neural tube (arrowheads in B). A dorsal view of the same 8.5 dpc embryo is shown in C (arrowheads identify β-gal expression). Embryos at 11.5 dpc were cleared before imaging (D through F). Wild-type littermates at 11.5 dpc are shown as controls in E and in the insert in D. At 11.5 dpc, β-gal is expressed in the met- and myelencephalic (arrowhead in F) region of the hindbrain. Arrows in F indicate faint β-gal expression in the roof of the fourth ventricle; stars indicate expression in the notochord. β-gal expression along the entire length of the neural tube can be seen on a dorsal view in D (arrows). In D, the star indicates the notochord; arrowheads point to the myelencephalon. Bar = 1 mm.

Expression of the Transgene in the Neonatal Kidney

Nephrogenesis occurs in a telescoped manner in newborn kidney that allows the evaluation of all developmental stages in a single section. To determine when the Nphs1 transgenes initiate expression of β-gal, newborn kidneys from several mice carrying either Nphs1 transgene were sectioned and examined after X-gal staining. As shown in Figure 6, nuclear localized β-gal activity was observed in early capillary loop stage glomeruli and in glomeruli in later developmental stages. β-gal activity was not observed in more primitive nephric structures, such as comma- and S-shaped figures, or elsewhere in these kidneys. These results demonstrate that both the 5.4-kb and 8.3-kb Nphs1 transgenes drive lacZ expression in a pattern identical to that described for endogenous nephrin during metanephric development.

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Figure 6.

β-gal expression in the developing kidney. Eight-μm cryosections of kidneys of transgenic newborns were stained with X-gal and were counterstained with nuclear fast red. β-gal expression arises during early capillary loop stage of glomerular development in podocyte nuclei (open arrowheads) and persists in podocytes of mature glomeruli (arrowheads) in animals transgenic for both constructs. β-gal expression was not detected elsewhere in earlier forms of developing nephrons, such as pretubular condensation (short arrow), comma-shaped (arrow) and S-shaped bodies (arrow with open head), or elsewhere in the kidney.