—  SPECIALTY CONFERENCE  —

Nephropathology

Case 2 - Congenital Nephrotic Syndrome of the Finnish Type

Laura Finn
Children's Hospital
Seattle, Washington


Click on each slide thumbnail image for an enlarged view
Clinical History:
This AGA male infant (XY karyotype) was delivered at 36 weeks to a 31-year-old G3P3 mother who received no prenatal care. The placenta was large and the infant had widely spaced cranial sutures, large anterior and posterior fontanelles, mild periorbital and moderate peripheral edema. He developed respiratory distress and an infectious etiology was excluded. Evaluation revealed low serum albumin (<1.0 g/dl) and protein (2.6 g/dl) with nephrotic-range proteinuria; BUN and Cr were 12 mg/dl and 0.2 mg/dl, respectively. Renal sonogram showed enlarged kidneys with diffuse bilateral echogenicity. An open biopsy was performed. The infant failed to thrive and several bouts of sepsis followed. His proteinuria was unresponsive to drug management but his renal function remained intact. A unilateral nephrectomy was performed at 4 months of age (slide); persistent heavy proteinuria prompted removal of the second kidney at 7.5 months.


Case 2 - Figure 1 - At 4 months, there is patchy interstitial fibrosis and moderate tubular dilatation. Subcapsular glomeruli resemble those in the deeper cortex.
Case 2 - Figure 2 - Representative glomeruli adjacent to dilated proximal tubules have minimal mesangial hypercellularity without mesangial sclerosis.


Case 2 - Figure 3 - A prior biopsy at 6 weeks of age showed minimal fibrosis, mildly thickened arteries and mild mesangial matrix and cell increase. Only a few tubules are dilated.
Case 2 - Figure 4 - Podocyte foot process fusion is obvious by electron microscopy. Electron dense deposits are not identified. The basement membrane is appropriately thin for age.

Pathology
Light microscopy: The kidney sample from the first nephrectomy has multifocal dilatation of proximal tubules more pronounced in the deep cortex with occasional microcysts containing eosinophilic hyalinized material. There is mild interstitial fibrosis and inflammation. Some glomeruli have mild mesangial hypercellularity and increased matrix; duplicated capillary basement membranes are not evident. Infrequent globally sclerotic glomeruli are present and scattered superficial glomeruli appear immature. Glomerulocystic change is minimal. Several arterial walls are thickened. At 7.5 months the kidney had more advanced tubular ectasia with scattered microcysts. Immunofluorescent microscopy: The glomeruli were negative for the following: IgG, IgA, IgM, C3, C1q, C4, fibrinogen and albumin. 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 had a few small clusters of immature hematopoietic cells but was not expanded by fibrosis. Infrequent proximal tubules were only minimally dilated. No nephrin immunoreactivity could be demonstrated.

Diagnosis: Congenital nephrotic syndrome of the Finnish type.

Differential Diagnosis and Discussion
Early onset nephrotic syndrome, whether congenital (traditionally, presentation <3 months) or infantile (4-months to 1 year) includes diseases of both idiopathic and secondary etiology 1. Nephrotic syndrome in the first year of life may be associated with lesions that occur in older patients such as minimal change disease, diffuse mesangial proliferation and focal segmental glomerulosclerosis (FSGS); these, however, are rarely present at birth. Secondary causes include such diverse entities as toxins and HUS, but typically, congenital infections (syphilis, toxoplasmosis, CMV) are the culprit. Integration of the clinical and pathologic data often distinguishes between these disorders, which is worthwhile as most patients with idiopathic nephrotic syndrome respond to steroid therapy. A subgroup, though, is treatment resistant and may represent one of the entities unique to this age group, diffuse mesangial sclerosis (DMS) or congenital nephrotic syndrome of the Finnish type (CNF).

Based solely on the light microscopic appearance of the glomeruli, the diagnosis of diffuse mesangial proliferation might be considered. However, unresponsive massive proteinuria is atypical in that idiopathic entity and tubular dilation is also uncommon, and likely the consequence of such proteinuria. Progressive ectasia was striking in this patient over the 6-month interval between the first biopsy and second nephrectomy.

The pathologic hallmark of CNF is dilatation of the proximal tubules with microcyst formation. Tubular dilatation in CNF typically begins deep and sampling limited to the superficial cortex could result in a delayed or incorrect diagnosis. A variety of glomerular changes may be seen including sclerosis, mesangial hypercellularity or matrix increase and thickened capillary loops; glomeruli may be completely normal. The EM changes include podocyte foot process fusion, as in most cases of massive proteinuria, and subendothelial expansion1. Unfortunately, all of these findings are non-specific.

CNF, an autosomal recessive disorder, is the most frequent single cause of congenital nephrotic syndrome, and particularly common though not restricted to the Finnish population. Its characteristic clinical presentation includes the following: (1) 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; (2) infants are typically premature and have large placentas; (3) 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 1, 2.

This latter point helps to distinguish clinically CNF from DMS in which renal function rapidly deteriorates. Similarities abound and include early onset, familial occurrence and tubular ectasia. 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. The subcapsular region often contains only diminutive and simplified glomeruli amongst primitive and atrophic tubules. Mesangial hypercellularity has been described in DMS but is not a constant feature. Interstitial fibrosis and inflammation may be significant 1, 3. Glomerular changes of DMS are common in Denys-Drash syndrome (DDS; nephrotic syndrome, Wilms tumor and genital abnormalities) and may be seen in Galloway-Mowat syndrome (nephrotic syndrome and CNS abnormalities).

These classic descriptions expounded from observations made over 35 years ago 2,4 are well known to most pediatric and nephro-pathologists. 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 and in particular, the slit diaphragm that bridges the interdigitating podocyte foot processes. Molecular approaches have allowed recent identification of several key components of the slit diaphragm.

Abnormalities in the NPHS1 gene (19q.13.1) that encodes nephrin are responsible for CNF 5, 6, 7 . Nephrin is a transmembrane adhesion molecule produced by podocytes that localizes to the slit diaphragm and is involved in cell-cell or cell-matrix interactions. It is proposed that strands of nephrin project from opposing foot processes to form the interlocking "teeth" of the slit diaphragm, resembling a zipper 8. Over 50 mutations have been reported in both Finnish and non-Finnish patients and affect both extracellular and intracellular domains 9. Two common mutations (Fin major and Fin minor) account for almost 95% of the cases in Finland and result in truncated non-functional proteins 6. Antibodies directed against the intra- and extra-cellular components of nephrin are negative in this group.

It would seem that nephrin staining would be a simple diagnostic modality but barriers exist, the most significant being that the antibody is currently not commercially available. Likewise, little data exist about nephrin staining in non-Finnish patients, most of whom have '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 10. Interestingly, staining was positive in one patient, heterozygous for Fin major anda missense mutation 11 and other non-Finnish patients with compound heterozygous mutations 9.

Mutational analysis would therefore seem to be the better diagnostic approach but is unfortunately only available on a research basis. Non-Finnish patients with compound heterozygous mutations, likely de novo events, may have a milder disease but currently a phenotypic/genotypic correlation has not been established 9. Current data would suggest that some patients with congenital nephrotic syndrome without Fin major and Fin minor mutations and whose kidneys express nephrin might respond to ACE inhibitor and indomethacin treatment 11. 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 12. Likewise, the large number of missense mutations make their classification as pathogenic or polymorphism problematic.

This issue is operative in prenatal testing as well. Direct mutational analysis of Fin major and Fin minor for parental carrier status, yielded no false negative results in 1183 Finnish pregnancies 13. However, this relatively simple and inexpensive approach of screening for Fin major and Fin minor mutations is not applicable to other populations that harbor a diverse range of often unique gene abnormalities 13.

Even fetal renal pathology may not be diagnostic. Equally high maternal serum and amniotic fluid alpha-fetoprotein (AFP) concentrations as well as similar proteinuric features of renal tubular microcysts were demonstrated in both homozygous and heterozygous (carrier) fetuses, even though the latter are not destined to suffer disease 14. Reduced nephrin synthesis and transient dysfunction of the slit diaphragm with protein leakage were hypothesized.

How does the practicing pathologist integrate this knowledge into his diagnoses? From a practical standpoint, the answer is that he must wait until molecular, immunoelectron microscopic and even immunohistochemical testing become available outside of a research setting. The classic three pronged light, immunofluorescent (or immunoperoxidase) and ultrastructural examination combined with a thorough clinical history remains the best approach to nephrotic syndrome.

As our understanding of the genetic basis of nephrotic syndrome grows, targeted therapeutic approaches may develop. Currently, however, the only cure for CNF is renal transplant and though function and survival is generally good, recurrent nephrotic syndrome has been documented 15. Analogous to the development of de novo anti GBM disease in transplant recipients with Alport's disease, anti-nephrin antibodies were detected in some patients transplanted for CNF who were thus primarily exposed to nephrin 16, 17. Recurrent nephrotic syndrome was only in patients with homozygous Fin major mutationsand notably, some responded to cyclophosphamide 17.

This raises the interesting notion that some patients with idiopathic nephrotic syndrome, like FSGS, may have anti-nephrin antibodies. This question remains to be answered, as does the definitive role of nephrin in other acquired proteinuric kidney disease. Investigations to date have varied results. Though no significant differences were observed in nephrin mRNA or protein expression in proteinuric pediatric patients studied by in situ hybridization or immunoperoxidase staining, respectively 18 , immunofluorescent staining showed reduced and redistributed nephrin in a group of adult patients with proteinuria 19. Immunoelectronmicroscopy, (perhaps a more suitable investigative method) in kidneys from patients with glomerulonephritis, demonstrated lower nephrin expression only in regions of foot process effacement 20. Whether this observation is the cause or consequence of podocyte disruption remains to be proven. What is becoming well established, however, is the importance of nephrin's interactions with other podocyte-associated proteins.

CD2AP (CD2-associated protein), a widely expressed adapter molecule first identified in T-cells and NK cells, is critical to podocyte function and also localizes to the slit diaphragm where it appears to interact with the cytoplasmic tail of nephrin 21. It was found that the mouse knockout of CD2AP dies at 6-7 weeks with proteinuria and foot process effacement 22. A renal disease resulting from the absence of CD2AP has not yet been identified in humans.

Podocin also localizes to the slit diaphragm and is predicted to be an integral membrane protein acing to organize the cytoskeleton 23 ,24. Mutations (nonsense, frameshift and missense) in the gene for podocin, NPHS2 (1q25-q31) are responsible for autosomal recessive steroid-resistant idiopathic nephrotic syndrome (SRNS) 23. Cellular activation through nephrin expression is greatly enhanced by podocin and together, nephrin and podocin may form a signaling complex that supports the functional and structural integrity of podocytes 25. Moreover, podocin not only binds to nephrin but it can directly bind to CD2AP, which may facilitate the interaction of the former proteins 24.

Alpha-actinins, proteins that crosslink actin filaments, are highly expressed in podocytes, and are involved in cytoskeleton organization. Mutations in the actin-binding region of the alpha-actinin-4 gene (ACTN4, 19q13) have been identified in patients with an autosomal dominant form of FSGS (FSGS1) 26. 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 impact that cytoskeleton perturbation can have on normal foot process function. This is underscored by recent demonstrations that nephrin, CD2AP and podocin are linked to the actin cytoskeleton and that depolymerization of actin filaments in cultured cells leads to the loss of cell membrane localization of these proteins 27, 28.

The discovery of the genetic defect in CNF has led to an explosion of knowledge about the glomerular filtration barrier. Implementing this new molecular data with pathology will hopefully lead to useful clinical applications.

References

  1. Habib R. Nephrotic syndrome in the 1st year of life. Pediatr Nephrol 1993;7:347-353.
  2. Hallman N, Hjelt L, Ahvenainen EK. Nephrotic syndrome in newborn and young infants. Ann Pediatr Fenn 1956;54:227-241.
  3. Habib R, Gubler M-C, Antignac C, Gagnadoux M-F. Diffuse mesangial sclerosis: A congenital glomerulopathy with nephrotic syndrome. Adv Nephrol 1993;22:43-57.
  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, et al. 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, et al. 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, et al. Nephrin is specifically located at the slit diaphragm of glomerular podocytes. Proc Natl Acad Sci USA. 1999;96:7962-7967.
  8. Tryggvason K. Unraveling the mechanisms of glomerular ultrafiltration. Nephrin, a key component of the slit diaphragm. J Am Soc Nephrol 1999;10:2440-2445.
  9. Koziell A, Grech V, Hussain S, et al. Genotype/phenotype correlation of NPHS1 and NPHS2 mutations in nephrotic syndrome advocate a functional inter-relationship in glomerular filtration. Hum Mol Genet 2002; 11:379-388.
  10. Liu L, Done SC, Khoshnoodi J, et al. 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.
  11. Patrakka J, Kestila M, Wartiovaara J, et al. Congenital nephrotic syndrome (NPHS1): features resulting from different mutations in Finnish patients. Kidney Int 2000;58:972-980.
  12. Beltcheva O, Martin P, Lenkkeri U, Tryggvason K. Mutation spectrum in the nephrin gene (NPHS1) in congenital nephrotic syndrome. Hum Mutat 2001;17:368-373.
  13. Kallinen J, Heinonen S, Ryynanen M, et al. Antenatal genetic screening for congenital nephrosis. Prena Diagn 2001;21:81-84.
  14. Patrakka J, Martin P, Salonen R, et al. Proteinuria and prenatal diagnosis of congenital nephrosis in fetal carriers of nephrin gene mutations. Lancet 2002; 359:1575-1577.
  15. Laine J, Jalanko H, Holthofer H, et al. Post-transplantation nephrosis in congenital nephrotic syndrome of the Finnish type. Kidney Int 1993;44:867-874.
  16. Wang S-X, Ahola H, Palmen T, et al. Recurrence of nephrotic syndrome after transplantation in CNF is due to autoantibodies to nephrin. Exp Nephrol 2001;9:327-331.
  17. Patrakka J, Ruotsalainen V, Reponen P, et al. Recurrence of nephrotic syndrome in kidney grafts of patients with congenital nephrotic syndrome of the Finnish type. Transplantation 2002;73:394-403.
  18. Patrakka J, Ruotsalainen V, Ketola I, et al. Expression of nephrin in pediatric kidney diseases. J Am Soc Nephrol 2001;12:289-296.
  19. Doublier S, Ruotsalainen V, Salvidio G, et al. Nephrin redistribution on podocytes is a potential mechanism for proteinuria in patients with primary acquired nephrotic syndrome. Am J Pathol 2001;158:1723-31.
  20. Huh W, Kim DJ, Kim M-K, et al. Expression of nephrin in acquired human glomerular disease. Nephrol Dial Transplant 2002;17:478-84.
  21. Palmen T, Lehtonen S, Ora A, et al. Interaction of endogenous nephrin and CD-2 associated protein in mouse epithelial M-1 cell line. J Am S Nephrol 2002;13:1766-1772.
  22. Shih N-Y, Li J, Karpitskii V, et al. Congenital nephrotic syndrome in mice lacking CD2-associated protein. Science 1999;286:312-315.
  23. Boute N, Gribouval O, Roselli S, et al. NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome. Nat Genet 2000;24:349-354.
  24. Schwarz K, Simons M, Reiser J, et al. Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin. J Clin Invest 2001; 108:1621-29.
  25. Huber TB, Kottgen M, Schilling B, Walz G, Benzing T. Interaction with podocin facilitates nephrin signaling. J Biol Chem 2001;276:41543-6.
  26. 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.
  27. Yuan H, Takeuchi E, Salant D. Podocyte slit-diaphragm protein nephrin is linked to the actin cytoskeleton. Am J Physiol-Renal Physiol. 2002;282:F585-91.
  28. Saleem MA, Ni L, Witherden I, et al. Co-localization of nephrin, podocin, and actin cytoskeleton. Am J Pathol 2002;161:1459-1466.