Broadening the Spectrum of Diseases Related to Podocin Mutations
Gianluca Caridi*,
Roberta Bertelli*,
Marco Di Duca*,
Monica Dagnino*,
Francesco Emma,
Andrea Onetti Muda,
Francesco Scolari¶,
Nunzia Miglietti||,
Gianna Mazzucco#,
Luisa Murer@,
Alba Carrea*,
Laura Massella,
Gianfranco Rizzoni,
Francesco Perfumo and
Gian Marco Ghiggeri*,
*Laboratory on Pathophysiology of Uremia, and Department of Nephrology, Istituto G. Gaslini, Genova, Italy; Section of Nephrology, Bambin Gesù Children Hospital, Rome, Italy; Department of Experimental Medicine and Pathology, University "La Sapienza," Rome, Italy; ¶Unit of Nephrology, Spedali Civili di Brescia, Italy; ||Department of Pediatrics, University of Brescia, Italy; #Pathology Section, Department of Biomedical Sciences and Human Oncology, University of Torino, Italy; @Department of Pediatrics, University of Padova, Italy.
Correspondence to Dr. Gian Marco Ghiggeri, Laboratorio di Fisiopatologia dell’Uremia, Istituto G. Gaslini, 16148 Genova, Italy. Phone: 39-010-380742; Fax: 39-010-395214;
ABSTRACT. A total of 179 children with sporadic nephrotic syndromewere screened for podocin mutations: 120 with steroid resistance,and 59 with steroid dependence/frequent relapses. Fourteen steroid-resistantpatients presented homozygous mutations that were associatedwith early onset of proteinuria and variable renal lesions,including one case with mesangial C3 deposition. Single mutationsof podocin were found in four steroid-resistant and in foursteroid-dependent; five patients had the same mutation (P20L).Among these, two had steroid/cyclosporin resistance, two hadsteroid dependence, and one responded to cyclosporin. The commonvariant R229Q of podocin, recently associated with late-onsetfocal segmental glomerulosclerosis, had an overall allelic frequencyof 4.2% versus 2.5% in controls. To further define the implicationof R229Q, a familial case was characterized with two nephroticsiblings presenting the association of the R229Q with A297Vmutation that were inherited from healthy mother and father,respectively. Immunohistochemistry with anti-podocin antibodiesrevealed markedly decreased expression of the protein in theirkidneys. All carriers of heterozygous coding podocin mutationor R229Q were screened for nephrin mutation that was found inheterozygosity associated with R229Q in one patient. Finally,podocin loss of heterozygosity was excluded in one heterozygouschild by characterizing cDNA from dissected glomeruli. Thesedata outline the clinical features of sporadic nephrotic syndromedue to podocin mutations (homozygous and heterozygous) in arepresentative population with broad phenotype, including patientswith good response to drugs. The pathogenetic implication ofsingle podocin defects per se in proteinuria must be furtherinvestigated in view of the possibility that detection of asecond mutation could have been missed. A suggested alternativeis the involvement of other gene(s) or factor(s). E-mail: labnefro@ospedale-gaslini.ge.it
The discovery of molecular defects of podocyte components (1–4)causing familial forms of nephrotic syndrome (NS) suggestedthe role of podocytes as the site of permselectivity in thekidney (5,6). Since then, knockout models of podocyte componentsand further molecular genetic screenings definitely consolidatedthis concept (7). Nephrin (NPHS1) was the first podocyte proteinto be found mutated in association with a rare form of congenitalNS with autosomal recessive inheritance (CNF) (1,2). Patientswith CNF present massive proteinuria starting in utero and progressto end-stage renal failure within a few years (8,9). Renal histopathologicchanges include immature glomeruli with fusion of foot processesand pseudocystic dilation of the proximal tubules (10). Podocin(NPHS2) was the second recognized protein to cause proteinuriain familial cases with recessive inheritance and in sporadicpatients (3,11,12). The clinical picture of nephrotic syndromecaused by podocin mutations ranges from an early onset, thusresembling CNF, to a late onset in the second decade of life,resembling idiopathic focal segmental glomerulosclerosis (FSGS).Finally, -actinin 4 was the most recently recognized structuralcomponent of the podocyte causing proteinuria in rare casesof dominant NS in humans (4). Recent data on nephrin/podocin/-actinin4 interaction underline the putative importance of protein-proteininteractivity within this network and suggest a structural modelin which nephrin represents the repulsion site within the slitdiaphragm and is anchored by podocin to the cytoskeleton where-actinin 4 is localized (13,14). The coexistence of mutationsaffecting both nephrin and podocin in three patients with early-onsetNS (15) reinforces this concept and provides further evidencefor a digenic inheritance model in which mutation hints occurin one gene, with the second mutant acting as a modifier.
The initial studies on podocin mutations in sporadic NS (11,12)were conducted on selected populations with particular clinicalpictures characterized by early onset and malignant outcome,mimicking familial cases. It is clear that determination ofthe actual impact of podocin mutations on sporadic NS requiresthat we extend screening in patients with variable phenotypeand increase the number of observations. In this article, wereport the results of a screening for podocin mutations in asufficiently vast cohort of children with sporadic NS and differentphenotype, including a variable response to drugs (from steroidsensitivity to strict resistance). In parallel, we also screenedour patients for nephrin and for the exon 8 hot-spot -actinin4 mutations.
Patients
We enrolled 179 nephrotic children for the screening of podocinmutations. All were presenting or had presented proteinuriafrom moderate to severe before age 18 yr and had received (orwere receiving) modular therapies according to their sensitivityto different drugs. As a rule, the therapeutic approach startedwith steroids following consolidated schemes (16) (2 mg/kg for30 to 60 d); in case of unresponsiveness (partial or global),steroids were associated or substituted with cyclophosphamide(2 mg/kg for 60 d) and/or with cyclosporin (5 mg/kg startingdose, followed by tapering to reach the minimum dose requiredfor maintaining cyclosporin serum levels between 50 and 100ng/ml). In case of persistent steroid-cyclosporin resistance,methyl-prednisolone was given in pulses (10 mg/kg, 6 cycles).According to the scheme above, patients were subdivided in corticoresistant(n = 120) and/or corticodependent/frequent relapsers (n = 59).The relevant clinical and pathologic features (i.e., gender,age at onset of proteinuria, evolution toward renal failure,renal transplant) are reported in Table 1. Renal histology wasavailable in 128 cases. Overall, 91 children had a diagnosisof FSGS based on the histologic evidence of at least one segmentalarea of glomerulosclerosis; 22 presented mesangial IgM deposition,and 15 minimal change nephropathy. In two cases with podocinmolecular defects, the pathologic features were not specificfor FSGS and were therefore further investigated with electronmicroscopy. Most FSGS cases presented strict resistance to corticosteroidsfollowing the scheme reported above, but other FSGS cases werealso observed who presented steroid sensitivity or belongedto the variant with corticodependence or with frequent relapsesof proteinuria (more than three episodes of proteinuria in 1yr). Podocin/nephrin and -actinin mutations were also evaluatedin 100 normal controls (45 male controls, 55 female controls;age, 3 to 40 yr) enrolled among the blood donors in our hospital.
Table 1. General features and clinical details of 179 children with sporadic nephrotic syndrome subdivided in two subgroups according to the response to steroidsa
Mutational Analyses
With the informed consent, we obtained peripheral blood samplesfor genetic analyses from the enrolled patients and from selectedparents and siblings. Genomic DNA was extracted according tostandard procedures. Molecular analyses of podocin and nephrinwere performed by direct sequencing as already described (1,3).Primer sequences for podocin were selected on the basis of whatis already reported in the literature (3,12). For exons 2 and6, primer design followed Karle et al. (12) to avoid the presenceof a recognized SNP. Exons were amplified by PCR using flankingintronic primers and subjected to automatic sequence analysisby dye-terminator reaction (Automated sequencer ABI 3100; Applera,Milan Italy). For -actinin 4, the hot-spot A682G and C695T mutationsat exon 8 were detected by restriction site analyses as describedby Kaplan et al. (4).
Glomerular cDNA for Molecular Analyses
After biopsy, the renal fragment was immediately placed at 4°Cin an RNase inhibitor solution (Vanadyl ribonuclease complex,20 mM; Life Technologies BRL) and then transferred to a micro-dissectingdish and cooled at 4°C, in which glomeruli were separatedfrom tubules. After micro-dissection, isolated glomeruli werewashed and transferred to a PCR tube that contained a humanplacental RNase inhibitor 40 U. Microdissected glomeruli werepermeabilized immediately before RT in a mixture containing0.2% Triton X-100, 40 U of RNase inhibitor, and 5 mM dithiothreitol(Sigma-Aldrich, St. Louis, Missouri). RT was performed utilizinga cDNA kit, according to the manifacturer’s instructions(Roche Diagnostics GmbH, Mannheim, Germany) for 90 min at 37°C.cDNA amplification was performed as described by Boute et al.(3).
Anti-Podocin Antibodies
Polyclonal anti-podocin antibodies were raised in rabbits immunizedwith the peptide GPEPSGSGRAGTP covering the amino acid sequencefrom 42 to 55 of podocin. This region is human-specific, withonly 30% homology versus rat and mouse. Specificity of antibodieswas controlled by two-dimensional electrophoresis (2-D) andimmunoblot (Figure 1) of human podocyte (kindly provided byProf. Giovanni Camussi) extracts in -hexyl-glucopyranoside.Two-dimensional electrophoresis, preparation, and rehydrationof immobilized gradients (IPG) and polyacrylamide gels havebeen described in detail elsewhere (17). Briefly, the IPG stripswere rehydrated overnight at 4°C in 9 M urea, 2% wt/volCHAPS, 0.6% wt/vol carrier ampholytes (IPG; Amersham PharmaciaBiotech), and a trace of bromophenol blue. Proteins, 30 µg,were solubilized with a solution containing 9 M urea, 4% wt/volCHAPS, and 40 mM Tris.
Figure 1. Two-dimensional electrophoresis and immunoWestern with polyclonal anti-podocin antibodies of cellular extracts from human podocytes. These antibodies recognized microheterogeneous spots with a molecular weight of 42 kD and pH around 7 that correspond to podocin.
Isoelectric focusing was performed at 18°C. SDS-PAGE inthe second dimension was performed following the original techniquedescribed by Bjellqvist et al. (18). The applied voltage forelectrophoresis was increased from 300 to 3500 V during thefirst 5 h, followed by 5000 V for a total of 100 kV/h. Beforethe 2-D run, IPG strips were equilibrated within the strip trayfor 30 min with a solution of 0.05 M Tris-HCl buffer, pH 6.8,6 M urea, 30% vol/vol glycerol, 2% wt/vol SDS, and a trace ofbromophenol blue. The second dimension was performed on 180x 160 x 1.5–mm slabs of polyacrylamide gradient gels (%T,8 to 16) using piperazine diacrylamide (PDA) as a cross-linkingagent. The gels were run at 45 mA/gel constant current and maintainedat a temperature of 12°C.
For Western blot, proteins were transblotted to Hybond nitrocellulosemembranous (Amersham Pharmacia Biotech) with a Novablot semidrysystem using a continuous buffer system with 38 mM Tris, 39mM glycine, 0.035% SDS, and 20% methanol. The transfer was achievedat 1.55 mA/cm2 for 3.5 h.
Immunofluorescence
Podocin expression in the kidney was studied by indirect immunofluorescencein a few renal biopsies of patients carrying the R229Q. Cryosectionswere fixed in cold acetone, rinsed, and sequentially incubatedwith the primary rabbit polyclonal antibody against podocin,followed by FITC-labeled goat anti-rabbit secondary antibody(Zymed; Histoline, Milan, Italy). Specificity of labeling wasdemonstrated by the lack of staining after substituting phosphate-bufferedsaline (PBS) and proper control immunoglobulins (Zymed) forthe primary antibody. Control kidneys represented by normalportion of nephrectomy for cancer were mounted on the same slideand processed in parallel.
Microalbuminuria Assay
Microalbuminuria was determined with Albumin Kit purchased fromRoche (Roche Diagnostic, Milan, Italy).
Statistical Analyses
Age at onset of proteinuria in subgroups was compared usingthe one-way ANOVA. Data on haplotype frequency in controls andnephrotic patients and in different pathologic subgroups werecompared with the 2 test. Data are given as mean ± SEM.
Confirmation of High Incidence of Homozygous Podocin Mutations of Podocin in Sporadic NS
After our original observation (11) of a high incidence of homozygouspodocin mutations in a small cohort of children with FSGS andrenal failure (44 patients), we extended the screening to further135 children (overall 179 patients studied) with NS and a morebenign phenotype that varied from steroid-dependence or frequentrelapses after steroid withdrawal to persistent steroid resistance(Table 1). Our results confirmed that homozygous podocin mutationsoccur frequently in patients with sporadic NS, and we extendedthe original observation to five new cases, the clinical datafor whom are reported in Table 2. The spectrum of histopathologyfeatures in patients with homozygous podocin mutations was variable.A renal biopsy was available in 12 patients; the most frequentpicture was consistent with FSGS (9 of 12); two children presentedMCN and mesangial proliferation with IgM deposits, respectively.In one case, carrying a composite R138Q-V180M mutation, renallesions were not reminiscent of a well-defined pathologic entity.Glomeruli (n = 18) appeared normal; at immunofluorescence, hepresented diffuse mesangial deposits of IgG(+++), C3(++), andC1q(+++). This picture was considered nonspecific, althoughindicative for a diffuse immunocomplex glomerulonephritis. Electronmicroscopy confirmed the presence of electron-dense depositsin mesangium with extension to subendothelial and subepithelialspaces (not shown).
Table 2. Type of mutations and clinical/pathological features in 22 carriers of podocin mutation and in 1 nephrin mutationa
Single Podocin Mutations
In eight patients, a single mutation of podocin was found; clinicalfeatures are reported in Table 2. As shown in Figure 2, threenew mutations were found (R291Q, 555delT, A242V). With the exceptionof P20L, none of the mutations found in heterozygosity haveever been observed in homozygous patients. The P20L was foundin five children. A relevant clinical characteristic of thiscohort of patients was that most had sensitivity to drugs (5of 8) and are currently presenting a normal renal function aftera long follow-up (Table 2). From a genetic point of view, renallesions due to podocin mutations should be inherited followinga recessive model that should produce an evident phenotype onlyin homozygosity. We cannot therefore exclude the possibilityof missing a second mutation in noncoding or regulatory regionsof podocin. Alternative possibilities are that a second mutationinvolves another podocyte gene that interacts with podocin orfocal loss of heterozygosity of podocin in glomeruli. This lastpossibility was excluded by sequencing podocin cDNA in microdissectedglomeruli in one child presenting A242V heterozygous mutationand found persistence of the expression of the normal allele(data not shown). All heterozygous carriers of podocin mutationand R229Q were screened for nephrin. A new nephrin mutation(A907T) was found in one case associated with the R229Q variantof podocin; this patient developed proteinuria with steroidresistance at the age of 17 mo but afterwards responded to cyclosporinand actually has a normal urinalysis at the age of 197 mo. However,the mother of this patient presented the same association andis healthy at the age of 55 yr. Two patients presented a nephrinmutation determining a nucleotide change in the promoter (-489delGA).In the literature, the presence of the above nucleotide changein the promoter is associated with nephrotic syndrome occurringbefore 5 yr and is actually considered a pathogenic mutation;however, we performed a screening of 50 controls and found thisnucleotide change in heterozygosity in 4. Moreover, the same-489delGA was found in association with a complex trait forpodocin in a simple family (see below, and Figure 3). They hadinherited this variant from their healthy father who was homozygousfor it. This observation supports the idea that this is indeeda polymorphism.
Figure 2. Electropherogram of three podocin (NPHS2) and one nephrin (NPHS1) new mutations described in this study. They occurred in heterozygosity in four sporadic children who presented variable clinical outcome. Mutated nucleotide and altered amino acids are reported.
Figure 3. Simple family consisting in two siblings with R229Q associated with A297V podocin mutation. They also presented the -489delGA of nephrin in heterozygosity. They inherited the A297V and the -489delGA from the healthy father and the R229Q from the healthy mother.
Podocin R229Q Variant
Podocin R229Q is a polymorphism that appears to enhance susceptibilityto FSGS in association with a second mutant NPHS2 allele (19).We found heterozygous R229Q in 5 of 100 normal controls (allelicfrequency 2.5%) and in 12 nephrotic patients, 5 of whom hadsteroid sensitivity. It was moreover associated with a mutantnephrin in one case (see above) and in one child homozygousR229Q was associated with homozygous R138X to give an overallfrequency 4.2% in our cohort of nephrotic patients. In a simplefamily (that was studied on an anecdotal basis), two siblingspresented an association of R229Q with the A297V mutation onthe other allele (inherited from the mother and the father,respectively) (Figure 3); moreover, as a part of a complex trait,these siblings also presented the -489delGA variant of the nephrinpromoter that they inherited from a homozygous healthy father.The glomerular expression of podocin was evaluated by polyclonalanti-podocin antibodies in one of the two siblings for whoma renal bioptic fragment was available. Immunofluorescence demonstratedthe absence of podocin in glomeruli (Figure 4)
Figure 4. Immnunofluorescence with anti-human podocin antibodies of glomeruli from (A) normal portion of a nephrectomy for carcinoma and (B) one of the two siblings presenting R229Q associated with A297V podocin mutation.
Heterozygous Podocin Mutations Occurring in Relatives of Nephrotic Patients
Proteinuria and microalbuminuria were determined in parentsand siblings of three nephrotic patients who had been previouslycharacterized: one carrying a composite 460/8insT associatedwith V180M mutation, the other presenting R138Q in homozygosityand the mother of a child with the P20L. In the first case,the proband inherited 460/8insT from the mother, while the fatherand two siblings were carriers of the second mutation. All theobligatory carriers had a normal urinalysis, including levelsof albuminuria < 15 mg/L, and their renal function was normal.
Exclusion of Mutations at Exon 8 of -Actinin 4
Mutational analyses at two hot-spot mutations at exon 8 of -actinin4 (4) (uniquely reported in the literature) showed no relevantalterations in our patients.
The discovery of the podocyte structural components that determineglomerular permselectivity and their implication in familialNS (1–4) represented a fundamental event in the rapidlyevolving research area on the mechanisms of proteinuria. Nephrin(NPHS1), podocin (NPHS2), and -actinin are the three proteinsimplicated in familial forms of NS in humans, with recessiveand dominant inheritance. In familial cases, the clinical pictureranges from congenital and often malignant outcome in the caseof nephrin mutations to more benign, albeit variable, formsin the case of podocin mutations. These latter have also beenreported in sporadic patients with NS (11,12), with a clinicalpicture resembling idiopathic FSGS. The present study was designedto extend to patients with steroid sensitivity and steroid dependencethe molecular analyses of the above genes involved in familialforms of NS, trying to compare the clinical features with idiopathicFSGS. The results presented here confirm the occurrence of homozygouspodocin mutations in sporadic NS, with an incidence of 12% inour cohort of children with steroid resistance. The phenotypeconsisted in all cases of severe proteinuria occurring in earlychildhood and of progression to end-stage renal failure, generallyoccurring within the second decade of life, i.e., later thanin patients with idiopathic FSGS. Even the pathologic picturewas variable, with two children showing MCN and mesangial depositsof IgM, respectively, and one case in which renal lesions moreresembled an immunodeposit glomerulonephritis. These resultsshow a different incidence of homozygous podocin mutations thanreported in previous studies by our group (11) and others (12)and complete the clinical-pathologic description. The main reasonfor this discrepancy is that in previous studies only patientswith rapid deterioration of renal function had been enrolled,on the basis of assumption of a marked malignancy of the disease.By extending the enrollment to other patients with slower progression,we described five new patients. Besides this confirmatory findingand the description of clinical features in carriers of homozygouspodocin mutations (including atypical pathologic features),we observed an unexpected fact, that is a high incidence ofcarriers of single podocin mutations (8 of 165). Finally, weare also reporting a frequency of the R229Q variant higher thanin normal people, a finding that confirms recent data by Tsukaguchiet al. (19), who associated this variant with late-onset FSGS.The same authors demonstrated a biologic difference betweenthe R229Q and the wild-type peptide, characterized by an alteredbinding to nephrin of the former. This key observation givesfunctional support to the idea that podocin resulting from R229Qis biochemically altered. The meaning of all these data arethat we probably miss a second NPHS2 mutation in heterozygousand R229Q patients due to the methodological approach (e.g.,mutation in noncoding or regulatory regions). Tsukaguchi etal. (19) identified an associated podocin mutation in two patientswith late-onset FSGS, and we found a second NPHS2 mutation intwo siblings described anecdotally in this paper and could showmarkedly decreased expression of podocin in their glomeruli.A second possibility supporting a causative effect of heterozygouspodocin mutations is loss of heterozygosity in glomeruli, amechanism that has been documented in other renal diseases suchas polycystic kidney (20). In this light, we sequenced a transcriptcDNA purified from a bioptic renal specimen obtained from aheterozygous carrier of podocin mutation and found the expressionof the normal allele, thus excluding this possibility.
So far, the association of steroid-resistant NS and heterozygousmutations of podocin has been reported in one family and twosporadic patients by Karle et al. (12). Moreover, five patientswith heterozygous nephrin mutations and congenital nephroticsyndrome have also been described by Lenkkeri et al. (2). Ninefetuses with heterozygous nephrin mutations diagnosed in uteroand with histopathologic features of Finnish NS have been recentlyreported by Patrakka (8).
The novelty of our findings is that they describe the associationof single mutations of podocin with less severe phenotypes thanthose already reported for homozygous patients. On the otherhand, the mutations they display are different from those associatedwith the poor phenotypes and do not include putative severeloss of function defects. This raises the possibility that theencoded peptide may retain some functional aspects and determinea mild phenotype. On the other hand, one could also speculatethat heterozygous coding podocin mutations contribute to proteinuriain association with other factors rather than directly determiningit. Possible candidates are circulating plasma factors, thenature of which we (21–23) and others (24,25) are currentlyinvestigating; in this light, molecular defects of podocin wouldact as modifiers of the phenotype. Recent data by Le Berre etal. (26) on spontaneously proteinuric Buffalo/Mna rats supportthis possibility. Genetic analysis in Buffalo/Mna rats demonstratedthat two recessive genes are implicated in the development ofproteinuria and that one of them (Pur 1 on chromosome 13) issyntenic to the podocin-containing region of chromosome 1 inhumans. Proteinuria and FSGS develop spontaneously in this strainof rats at the age of 2 mo, suggesting the implication of inheritedcauses. Le Berre et al. (26) showed that proteinuria recurredwhen normal LEW.1W kidneys were transplanted in Buffalo/Mnarats, whereas it relapsed when Buffalo/Mna kidneys were transplantedinto LEW.1W, suggesting the existence of cofactors in spontaneouslyproteinuric rats. It must be noted that this is highly reminiscentof what happens in humans with idiopathic FSGS who develop rapidrecurrence after renal transplant. Studies on this topic arecurrently in progress. Preliminary data on recurrence of proteinuriain patients with homozygous podocin mutations who underwentrenal graft support its possible relationship with high serumpermeability activity (27) and extends to humans the conceptthat podocin mutation may act in synergy with other actors ofa multifactorial system.
In conclusion, the data reported here confirm the high incidenceof podocin mutations in children with sporadic NS and contributeto outline the clinical and pathologic features in these patients.They also demonstrate a considerable incidence of heterozygousmutations of podocin in a subset of proteinuric patients witha favorable outcome and confirm a slightly higher incidenceof R229Q in patients with a milder phenotype. The pathogeneticimplication of heterozygous podocin defects per se in proteinuriamust be further investigated in view of the possibility thatdetection of a second mutation could have been missed. A suggestivealternative is the involvement of other gene(s) or factor(s).
Acknowledgments
This work was supported by a grant from the Italian Ministryof health (Progetto Finalizzato ICS 070.2/RF00.167 and Progettofinalizzato 2002). Financial support was given by the KIDneyFund, and the Authors are indebted to Prof. Rosanna Gusmano.
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Received for publication September 17, 2002.
Accepted for publication January 17, 2003.
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Top
Abstract
Introduction
-actinin 4 was the most recently recognized structural component of the podocyte causing proteinuria in rare cases of dominant NS in humans (4). Recent data on nephrin/podocin/
-hexyl-glucopyranoside. Two-dimensional electrophoresis, preparation, and rehydration of immobilized gradients (IPG) and polyacrylamide gels have been described in detail elsewhere (17). Briefly, the IPG strips were rehydrated overnight at 4°C in 9 M urea, 2% wt/vol CHAPS, 0.6% wt/vol carrier ampholytes (IPG; Amersham Pharmacia Biotech), and a trace of bromophenol blue. Proteins, 30 µg, were solubilized with a solution containing 9 M urea, 4% wt/vol CHAPS, and 40 mM Tris. 
2 test. Data are given as mean ± SEM. 
