—  SPECIALTY CONFERENCE  —

PEDIATRIC PATHOLOGY

Case 4 - Congenital nephrotic syndrome, c/w Finnish type

Laura Finn, M.D.
University of Washington and Children's Hospital and Regional Medical Center
Seattle, Washington

Clinical History

This AGA female infant (XX karyotype) was delivered vaginally to a 19-year-old G1P1 mother at 34 weeks gestation with Apgars of 71 and 95. The placenta was large and the infant was noted to have widely spaced cranial sutures and large anterior and posterior fontanelles. She was discharged in good condition on day 12 but presented at 1 month of age with abdominal distension and respiratory distress. Peritoneal fluid and blood cultures grew Streptococcus pneumoniae. Evaluation revealed hypoalbunemia and nephrotic range proteinuria (3.37grams/24hr); complement levels were normal; BUN and Cr were 3mg/dl and 0.3mg/dl, respectively. The kidney size by ultrasound was the upper limit of normal and there was diffuse bilateral echogenicity. Multiple bouts of sepsis and peritonitis ensued. She was unresponsive to medical management and underwent a nephrectomy at 4 months and 7.5 months of age.

Pathology
Light microscopy: The wedge of kidney has ectatic and microcystically dilated proximal tubules surrounded by mildly fibrotic and minimally inflamed interstitium. Glomerulocystic change is minimal. Most glomeruli have at least mild mesangial hypercellularity and increased matrix; rare globally sclerotic glomeruli and immature glomeruli are present. Duplicated capillary basement membranes are not evident. Some arterial walls are thickened. Immunofluorescent microscopy: 2+ focal mesangial staining with IgM and C3. Electron microscopy: Foot process effacement was widespread. The mesangial matrix was focally increased but electron dense deposits were not seen.

A wedge biopsy performed at 6 weeks of age contained primarily superficial cortical tissue. The glomeruli were largely normocellular with prominent visceral epithelial cells as expected for age. The interstitium was not expanded and infrequent proximal tubules were only minimally dilated.

Slides  (Click to enlarge)


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A biopsy at 6 weeks of age showed patchy fibrosis, mildly thickened arteries and mild mesangial matrix and cell increase. Only a few tubules are dilated. At 7.5 months, there is more prominent fibrosis and more extensive tubular dilatation. Subcapsular glomeruli resemble those in the juxtacortex. A glomerulus has minimal mesangial hypercellularity without mesangial sclerosis. Podocyte foot process fusion is obvious by electron microscopy. Electron dense deposits are not identified. The basement membrane is appropriately thin for age.

Diagnosis
Congenital nephrotic syndrome, c/w Finnish type.

Differential Diagnosis and Discussion
The differential diagnosis of congenital (<3 months) and infantile (4-months to 1 year) nephrotic syndrome includes diseases of both idiopathic and secondary etiology. Secondary causes such as congenital infections, toxins and HUS are useful to exclude with a biopsy, as some of these entities may respond to treatment. Within the former category are congenital nephrotic syndrome (Finnish type) [CNF], diffuse mesangial sclerosis [DMS] (isolated or in association with malignancies and genital abnormalities [Denys-Drash syndrome, (DDS)]), minimal change disease, mesangial proliferative glomerulonephritis (GN) and FSGS. Integrating the clinical and pathologic findings usually allows differentiation among these entities (1).

Based on the light microscopic appearance of the glomeruli, this case could be diagnosed as mesangial proliferative GN; however unresponsive massive proteinuria is atypical. Tubular dilation is also uncommon and is possibly the consequence of such proteinuria. Progressive ectasia was evident in this patient.

CNF, an autosomal recessive disorder, is particularly common though not restricted to the Finnish population and has a characteristic clinical presentation: nephrotic syndrome with massive proteinuria is normally obvious within the first few days of life and almost always by 3 months, however renal function remains normal through the first six months; infants are typically premature and have large placentas; maternal and amniotic fluid alpha-fetoprotein (AFP) levels are often elevated reflecting fetal proteinuria. By 5 to 8 years of age there is end-stage renal failure but most usually die from sepsis, not renal failure, within the first year. The pathologic hallmark of CNF is dilatation of the proximal tubules, sometimes to the level of microcyst formation. A variety of glomerular changes may be seen including sclerosis, mesangial hypercellularity or matrix increase and thickened capillary loops; they may be completely normal. The EM changes are non-specific and include foot process fusion and subendothelial widening 1.

Diffuse mesangial sclerosis (DMS) may be confused with CNF by its early onset, familial occurrence and tubular ectasia but the rapid deterioration of renal function distinguishes the two clinically. The histology typically emphasized in DMS besides the obvious mesangial sclerosis, is podocyte hypertrophy and zonal differentiation, with the deepest glomeruli being the least affected. Mesangial hypercellularity has been described in DMS but is not a constant feature. Interstitial fibrosis and inflammation may be significant 2.

These classic descriptions expounded from observations made over 35 years ago 3,4 are well known to most pediatric pathologist. Today evidence suggests that the unresponsive, early-onset massive proteinuria of congenital nephrotic syndrome is primarily associated with defects in the structure and function of the podocyte foot process (Table 1), the outer most component of the three layered glomerular filtration barrier that also includes the glomerular basement membrane (GBM) and the fenestrated endothelial cells lining the inside of the glomerular capillary. A zipper-like membrane known as the slit diaphragm bridges the interdigitating podocyte foot processes (Figure 1).

Nephrin, a transmembrane protein produced by podocytes and localized to the slit diaphragm, is the product of the NPHS1 gene (19q.13.1); abnormalities in NPHS1 are responsible for CNF5,6,7. Over 50 mutations have been reported in both Finnish and non-Finnish patients 8. Two common mutations (Finmajor and Finminor) are present in almost 95% of the cases in Finland and result in truncated non-functional proteins 6; staining with antibodies directed against the intra- and extra-cellular components of nephrin is negative in this group. Most mutations found in non-Finnish patients are 'private' missense mutations that lead to misfolding of the mutant nephrin and its retention in the endoplasmic reticulum rather than transport to the cell membrane 9. Too little data is available to know if nephrin can be immunohistochemically demonstrated in these glomeruli; however staining was positive in one patient, heterozygous for Finmajor and a missense mutation 10. Non-Finnish patients with compound heterozygous mutations may have a milder disease but currently a phenotypic/genotypic correlation has not been established. Though accurate DNA testing is theoretically available, no NPHS1 mutations were identified in some cases of classic CNF implying that mutations may be present in regulatory elements of the gene (introns or flanking regions) or that proteins that interact with nephrin may be altered. Likewise, the large number of missense mutations make their classification as pathogenic or polymorphism problematic.

One protein that interacts with the intracellular component of nephrin is CD2AP (CD2-associated protein), a widely expressed protein first identified in T-cells and NK cells, which also localizes to the slit diaphragm. It was recently found that the mouse knockout of CD2AP dies at 6-7 weeks from proteinuria with foot process effacement 11. Nephrin (staining) and a slit diaphragm are initially present in these mice, but disappear in parallel, implying that other intracellular proteins compensate for CD2AP during development and that there are transitions in podocyte gene expression 12. A renal disease resulting from the absence of CD2AP has not yet been identified in humans.

Another recently discovered component of the glomerular filtration barrier is podocin, predicted to be an integral membrane protein though the exact subcellular location is currently unknown 13. Mutations (nonsense, frameshift and missense) in the gene responsible for podocin, NPHS2 (1q25-q31) were identified in families with steroid-resistant idiopathic nephrotic syndrome (SRNS), characterized by an autosomal recessive mode of inheritance, onset between three months and five years of age, biopsy showing FSGS, and rapid progression to end-stage renal failure; extrarenal disorders were absent 13,14. Podocin, as demonstrated by in-situ hybridization, appears to be confined to podocytes and is first expressed in the late S-phase. The structure of podocin suggests that it may act to organize the cytoskeleton and mediate podocyte signaling through indirect or direct interactions with nephrin and CD2AP 13. NPHS2 mutations were recently described in 9 of 44 children with clinically and pathologically characteristic non-familial FSGS 15. Though the authors advocate molecular testing, it is remains available only on a research basis.

Another genetic cause of FSGS has been identified in families with a mild type of nephrotic syndrome (FSGS1); once again a defect in the podocyte is suspect. Patients presented in their teens or early adulthood and progressed slowly to end-stage renal disease. Dominant mutations were identified in the actin-binding region of alpha-actinin (ACTN4, 19q13), which is highly expressed in podocytes and functions by crosslinking actin filaments 16. The mutant protein was shown in vitro, to have an increased affinity for actin; this interaction may be altered in vivo rendering the podocyte susceptible to damage. While the pathogenesis of this variety of FSGS requires further elucidation, the discovery reinforces the significance of a properly organized cytoskeleton to the maintenance of normal foot process function.

The podocyte too may be pivotal in the glomerular injury of DMS/DDS, which are associated with defects in the Wilms' tumor suppressor gene (WT1; 11p13). WT1 encodes a zinc-finger transcription factor involved in kidney and gonadal differentiation by modulating (usually repressing) the activity of other proteins such as PAX2, IGF2, PDGF, EGFR, etc. 17. More than 60 constitutional de novo mutations in WT1 have been identified in patients with the complete or incomplete form of DDS and a small percentage of patients, usually female, with isolated DMS18-20. Most are missense changes in either exon 8 or 9, encoding zinc-fingers 2 and 3, respectively, that interfere with DNA binding. DNA analysis for mutations in WT1 is currently performed in this country on a research basis only.

The podocytes are responsible for basement membrane production and are the cells morphologically altered in DMS. The PAX2 transcription factor and WT1 are inversely expressed in early kidney development, as WT1 represses PAX221. In the mature glomerulus, WT1 persists only in the podocytes and PAX2, only in the parietal epithelium. Abnormal PAX2 expression, in conjunction with diminished WT1, has been identified in the podocytes of children with DDS and isolated DMS whereas none of the nephrotic control subjects had altered expression. Though its role has not been thoroughly dissected, WT1 is directly involved in nephrogenesis and mutations, through a variety of cascades, may lead to defective glomerular structures.

And so the saga continues...

References

  1. Habib R. Nephrotic syndrome in the 1st year of life. Pediatr Nephrol 1993;7:347-353.
  2. Habib R., Gubler M, Antignac C, Gagnadoux M. Diffuse Mesangial Sclerosis: A congenital glomerulopathy with nephrotic syndrome. Adv Nephrol 1993;22:43-57.
  3. Hallman N, Hjelt L, Ahvenainen EK. Nephrotic syndrome in newborn and young infants. Ann Pediatr Fenn 1956;54:227-241.
  4. Denys P, Malvaux P, Van den Berghe H, Tanghe W, Proesmans W. Association d'un syndrome anatomopathologique de pseudohermaphrodisme masculin, d'une tumeur de Wilms, d'une nephropathie parenchymateuse et d'un mosaicisme XX/XY. Arch Fr Pediatr 1967;24:729.
  5. Mannikko M, Kestila M, Holmberg C, Norio R, Ryynanen M, Olsen A, Peltonen L, Tryggvason K. Fine mapping and haplotype analysis of the locus for congenital nephrotic syndrome on chromosome 19q13.1. Am J Hum Genet 1995;57:1377-1383.
  6. Kestila M, Lenkkeri U, Mannikko M, Lamerdin J, McCready P, Putaala H, RuotsalainenV, Morita T, Nissinen M, Herva R, Kashtan CE, Peltonen L, Holmberg C, Olsen A Tryggvason K. Positionally cloned gene for a novel glomerular protein-nephrin -is mutated in congenital nephrotic syndrome. Mol Cell;1998:1:575-582.
  7. Ruotsalainen V, Ljungberg P, Wartiovaara J, Lenkkeri U, Kestila M, Jalanko H, Holmberg C, Tryggvason K. Nephrin is specifically located at the slit diaphragm of glomerular podocytes. Proc Natl Acad Sci USA. 1999;96:7962-7967.
  8. Beltcheva O, Maritn P, Lenkkeri U, Tryggvason K. Mutation spectrum in the nephrin gene (NPHS1) in congenital nephrotic syndrome. Hum Mutat 2001;17:368-373.
  9. Liu L, Done SC, Khoshnoodi J, Bertorello A, Wartiovaara J, Berggren P-O, Tryggvason K. Defective nephrin trafficking caused by missense mutations in the NPHS1 gene: insight into the mechanisms of congenital nephrotic syndrome. Hum Mol Genet 2001;10:2637-2644.
  10. Patrakka J, Kestila M, Wartiovaara J, Ruotsalainen V, Tissari P, Lenkkeri U, Mannikko M, Visapaa I, Holmberg C, Rapola J, Tryggvason K, Jalanko H. Congenital nephrotic syndrome (NPHS1): features resulting form different mutations in Finnish patients. Kidney Int 2000;58:972-980.
  11. Shih N-Y, Li J, Karpitskii V, Nguyen A, Dustin ML, Kanagawa O, Miner JH, Shaw AS. Congenital nephrotic syndrome in mice lacking CD2-associated protein. Science 1999;286:312-315.
  12. Li C, Ruotsalainen V, Tryggvason K, Shaw AS, Miner JH. CD2AP is expressed with nephrin in developing podocytes and is found widely in mature kidney and elsewhere. Am J Physiol Renal Physiol 2000;279:F785-792.
  13. Boute N, Gribouval O, Roselli S, Benessy F, Lee H, Fuchshuber A, Dahan K, Gubler M-C, Niaduet P, Antignac C. NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome. Nat Genet 2000;24:349-354.
  14. Fuchshuber A, Jean G, Gribouval O, Gubler M-C, Broyer M, Beckmann JS, Niaudet P, Antignac C. Mapping a gene (SRN1) to chromosome 1q25-q31 in idiopathic nephrotic syndrome confirms a distinct entity of autosomal recessive nephrosis. Hum Mol Genet 1995;4:2155-2158.
  15. Caridi G, Bertelli R, Carrea A, Di Duca M, Catarsi P, Artero M, Carraro M, Zennaro C, Candiano G, Musante L, Seri M, Ginevri F, Perfumo F, Ghiggeri GM. Prevalence, genetics, and clinical features of patients carrying podocin mutations in steroid-resistant nonfamilial focal segmental glomerulosclerosis. J Am Soc Nephrol 2001;12:2742-2746.
  16. Kaplan JM, Kim SH, North KN, Rennke H, Correia LA, Tong H-Q, Mathis BJ, Rodriguez-Perez J-C, Allen PG, Beggs AH, Pollak MR. Mutations in ACTN4, encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis. Nat Genet 2000;24:251-256.
  17. Pritchard-Jones K, Fleming S, Davidson D, Bickmore W, Porteous D,Gosden C, Bard J, Buckler A, Pelletier J, Housman D, van Heyningen V, Hastie N. The candidate Wilms' tumour gene is involved in genitourinary development. Nature 1990;346:194-197.
  18. Little M, Wells C. A clinical overview of WT1 gene mutations. Hum Mutat 1997;9:209-225.
  19. Jeanpierre C, Denamure E, Henry I, Cabanis M-O, Luce S, Cecille A, Elion J, Peuchmaur M, Loirat C, Niaudet P, Gubler M-C, Junien C. Identification of constitutional WT1 mutations, in patients with isolated diffuse mesangial sclerosis, and analysis of genotype/phenotype correlations by use of a computerized mutation database. Am J Hum Genet 1998;62:824-833.
  20. Schumacher V, Scharer K, Wuhl E, Altrogge H, Bonzel K-E, Guschmann M, Neuhaus TJ, Pollastro RM, Kuwertz-Broking E, Bulla M, Tondera A-M, Mundel P, Helmchen U, Waldherr R, Weirich A, Royer-Pokora B. Spectrum of early onset nephrotic syndrome associated with WT1 missense mutations. Kidney Int 1998;53:1594-1600.
  21. Yang Y, Jeanpierre C, Dressler GR, Lacoste M, Niaudet P, Gubler M-C. WT1 and PAX-2 podocyte expression in Denys-Drash Syndrome and isolated diffuse mesangial sclerosis. Am J Pathol 1999;154:181-192.
DiseaseGene locusGene Product InheritanceOnset
CNF 19q12-13 NPHS1 Nephrin AR <0 - 3m
DMS/DDS11p13 WT1 WT1 AR/Sporadic 0 - 6m
          
SRNS 1q25-31 NPHS2 podicin AR 3m-5y
FSGS1 19q13 ACTN4 a-actinin-4AD early adult

Table 1 - Congenital, early and late onset nephrotic syndromes