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*
Division of Nephrology, Department of Internal Medicine, University of
Michigan Medical School, Ann Arbor, Michigan.
Department of Veterans Affairs, Ann Arbor, Michigan.
Correspondence to Dr. Lawrence B. Holzman, University of Michigan Medical School, 1560 Medical Science Research Building II, Ann Arbor, MI 48109-0676. Phone: 734-764-3157; Fax: 734-763-0982; E-mail: lholzman{at}umich.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In response to glomerular injury, podocytes undergo a dramatic change in morphology, termed foot process effacement, resulting in retraction and spreading of foot processes and alteration in their intercellular junctions (4). Foot process effacement is a fluid and reversible process that correlates closely with the development of proteinuria both in human disease and in experimental models (2, 5). The cellular and molecular mechanisms that govern these changes in podocyte structure are incompletely defined, although recent progress has ignited significant interest in this area (6,7,8). Clearly, foot process effacement requires the interplay of multiple cellular events, including rearrangement of the structure of the cytoskeleton, spreading or dynamic cell adhesion of the foot process over the basement membrane, and disassembly or reassembly of the intercellular junction that comprises the slit diaphragm. Given the correlation between alterations in podocyte morphology and the occurrence of proteinuria, better understanding the molecular mechanisms that govern these changes should provide insight into the mechanisms of glomerular disease.
The study of podocyte biology has been hampered by limitations in available experimental models that both recapitulate the complex in vivo phenotypes of this cell and can be readily and specifically manipulated at the molecular level. Recent work has provided conditionally immortalized mouse cell lines that express molecular markers consistent with the in situ podocyte phenotype (2, 9). However, these cell lines do not reproduce the complex three-dimensional cyto-architecture of the podocyte in situ and lack typical foot processes that possess the specialized intracellular junction structures of this cell. These shortcomings limit the utility of these cell lines for studying the functional role of specific molecules in governing podocyte morphology and the maintenance of glomerular filter integrity.
Transgenic manipulation of the podocyte is one approach that might circumvent these limitations. The purpose of this study was to identify a promoter-enhancer that would direct the expression of transgenes in a podocyte-specific manner for later use in experiments aimed at manipulating the podocyte in situ. The murine nephrin (Nphs1) promoter was considered a good candidate for this purpose, because previous studies suggested that nephrin was expressed exclusively in podocytes (6, 10, 11).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction of the Reporter Transgene
The ß-galactosidase (ß-gal) reporter plasmid used in this study
was derived from pnlacF (12).
pnlacF encodes Escherichia coli ß-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 GPSTM-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.
ß-Gal Assays
Assays of ß-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 x g for 10 min, the supernatants were heated at 48°C
for 50 min to inactivate endogenous mammalian ß-gal activity
(16). After a second
centrifugation at 12,500 x 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 ß-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-ß-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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
ß-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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Tissue Screen for ß-Gal Activity
Transgenic F0 founders initially were examined at 6 to
8 wk postgestation using a sensitive chemiluminescence assay to detect
ß-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 ß-gal activity
was quenched by heating homogenates to 48°C before analysis. Remaining
ß-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 ß-gal in their kidneys (95 to 100% penetrance). Particularly
elevated ß-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 ß-gal activity in the small
intestine or elsewhere in the gut (data not shown).
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Expression of Nphs1-Driven Transgenes in Adult Kidney and Brain
Because ß-gal activity was confined to adult kidney and brain in the
initial screen, these tissues were examined microscopically for ß-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
ß-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—ß-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 ß-gal
(18). Therefore, both
Nphs1 transgenes are expressed like endogenous nephrin, in a
podocyte-specific distribution in the adult kidney.
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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 ß-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 ß-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, ß-gal activity was present in the neural
tube in the region of the hindbrain in transgenic embryos
(Figure 5, B and C). ß-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 ß-gal activity in the neural tube increased in a manner
directly proportional to the degree of maturity. At 11.5 dpc, ß-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, ß-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,
ß-gal activity could also be detected in the notochord of embryos at 11
dpc. ß-gal activity was not detected elsewhere in embryos at 8.5 or 11.5
dpc.
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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 ß-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
ß-gal activity was observed in early capillary loop stage glomeruli and
in glomeruli in later developmental stages. ß-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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nphs1-driven transgene expression was identified in the kidney and brain. In the kidney, the lacZ reporter transgene carrying a nuclear localization signal was expressed exclusively in podocyte nuclei. Expression of ß-gal arose during the early capillary loop stage of glomerular development mimicking the reported expression pattern of nephrin in the kidney (10).
ß-gal activity was observed in developing transgenic mouse brain at 8 dpc in the region of the hindbrain and extended down the entire length of the neural tube during maturation. Strongest ß-gal expression could be seen at 11.5 dpc in the met- and myelencephalic part of the rhombencephalon and in the roof of the fourth ventricle. During the execution of this study, it was reported by Putaala et al. (19) that the endogenous Nphs1 gene is expressed in an identical distribution in wild-type mouse embryos at 11 dpc. Similarly, expression of ß-gal expression in transgenic mice was observed in the lumbar area of the neural tube mimicking the distribution of the expression of the endogenous Nphs1 gene (19). Previously, the expression of nephrin in adult brain had not been reported. The identification of ß-gal activity in discrete nuclei of the adult medulla oblongata and cerebellum correlates appropriately with developmental expression in structures of the hindbrain.
The expression of nephrin in the central nervous system is an interesting finding and provides possible clues regarding the function of the protein. Because other members of the Ig superfamily of transmembrane proteins have been implicated as functionally important in neuronal growth and differentiation, it is plausible that nephrin, too, plays a similar role in development of the cerebellum, brainstem, and spinal cord (20, 21). Indeed, it has recently been communicated that a subset of patients with the congenital nephrotic syndrome of the Finnish type, who carry mutations of the nephrin gene, exhibit discrete extrapyramidal signs (19).
It is likely that all cis-regulatory elements necessary for cell-specific expression in the mouse are present in the identified 8.3-kb portion of the 5' flanking region of the Nphs1 gene, because this region directed expression of the reporter transgene in a manner similar to the endogenous Nphs1 gene. The high degree of sequence similarity between the human and murine 5' flanking region is not surprising. However, there clearly are portions of this region that are distinctly more highly conserved. It is possible that there exist elements within these more highly conserved regions that are necessary to direct Nphs1 gene expression. Lenkkeri et al. (17) reported the association between glomerular disease and deletions in a GA repeat sequence in the human Nphs1 promoter. Of note, this GA repeat sequence was not identified in the murine Nphs1 promoter, suggesting that the human GA repeat is not a functionally significant promoter element. Rather, this repeat may represent a polymorphism that cosegregates with the glomerular disease phenotype present in these patients.
The p5.4-nlacF reporter construct produced significantly higher expression of ß-gal activity in the kidney compared with that of the full-length p8.3-nlacF construct. Expression levels in the brain, conversely, were similar in both constructs. This observation might be explained by the occurrence of multiple integrations of the transgene or by the effect of the site of transgene integration. However, because transgene expression in the brain does not vary between constructs, one might postulate that deletion of a binding element for a kidney-specific transcriptional repressor has resulted in increased ß-gal expression in the shorter construct. Additional work is required to investigate these hypotheses.
It is important to note that the expression of both transgenes used in this study was characterized by a remarkably high penetrance. Moreover, the site of integration did not significantly influence expression of either transgene. No ectopic tissue expression was observed in this study. Such stable expression characteristics should prove to be valuable features when this promoter is used as a tool for the generation of transgenic mouse models.
The study of podocyte biology has been restricted by limitations imposed by available experimental models. Understanding the mechanisms that govern the unique podocyte cyto-architecture and the architecture of the specialized slit-diaphragm is central to the problem of glomerular pathophysiology. For this reason, experimental models of podocyte biology should preserve the physiologic three-dimensional in situ phenotype of these cells. To be useful, the ideal model should also be amenable to specific manipulations at the molecular level. The nephrin promoter constructs identified in this study provide unique reagents that will allow application of a transgenic approach for manipulating the podocyte in situ. In the kidney, the identified promoter directs expression solely in mature or maturing podocytes. Transgene expression arises late in nephrogenesis coinciding with the initiation of foot process formation. Therefore, future transgenes driven by this promoter should have little effect on early nephron developmental processes. In some circumstances, the use of Nphs1-driven transgenes to study the kidney may be limited by extrarenal expression in the region of the hindbrain and spinal cord. This may be particularly true during the gestational period, when aberrant expression of transgenes may result in embryonic lethal or severe developmental abnormalities that would preclude the study of mature kidneys. Ideally, identification of a region of the Nphs1 promoter that drives podocyte-specific transgene expression would circumvent this problem. Short of this, the use of an inducible system in which the identified Nphs1 promoter determines transgene expression should circumvent this problem.
In summary, the present study has identified the murine Nphs1 promoter as a useful reagent that can drive transgene expression in a podocyte-specific pattern in the kidney. The use of this tool to express dominant negative proteins or to create conditional gene deletion models—each in podocyte-specific patterns—should provide important new approaches for studying the molecular basis of podocyte pathophysiology.
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Acknowledgments |
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The present study was supported in part by a Merit Review Award from the Department of Veterans Affairs (to L.B.H.), a fellowship from the Deutsche Forschungsgemeinschaft (to M.J.M.), and by a George M. O'Brien Kidney Research Center (DK39255) and the University of Michigan Multipurpose Arthritis Center (NIH/NIAMS AR20557).
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O. Beltcheva, S. Kontusaari, S. Fetissov, H. Putaala, P. Kilpelainen, T. Hokfelt, and K. Tryggvason Alternatively Used Promoters and Distinct Elements Direct Tissue-Specific Expression of Nephrin J. Am. Soc. Nephrol., February 1, 2003; 14(2): 352 - 358. [Abstract] [Full Text] [PDF]
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 H. Pavenstadt, W. Kriz, and M. Kretzler Cell Biology of the Glomerular Podocyte Physiol Rev, January 1, 2003; 83(1): 253 - 307. [Abstract] [Full Text] [PDF]
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M. J. Moeller, S. K. Sanden, A. Soofi, R. C. Wiggins, and L. B. Holzman Two Gene Fragments that Direct Podocyte-Specific Expression in Transgenic Mice J. Am. Soc. Nephrol., June 1, 2002; 13(6): 1561 - 1567. [Abstract] [Full Text] [PDF]
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S. E. Quaggin A "Molecular Toolbox" for the Nephrologist J. Am. Soc. Nephrol., June 1, 2002; 13(6): 1682 - 1685. [Full Text] [PDF]
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V. Eremina, M. A. Wong, S. Cui, L. Schwartz, and S. E. Quaggin Glomerular-Specific Gene Excision In Vivo J. Am. Soc. Nephrol., March 1, 2002; 13(3): 788 - 793. [Abstract] [Full Text] [PDF]
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