Clinical features and outcome of childhood minimal change nephrotic syndrome: is genetics involved?

The pathogenesis of minimal change nephrotic syndrome (MCNS) is still unknown. We performed a clinical and genetic evaluation of 104 adults (mean age 35 years) who presented with MCNS in childhood (mean follow-up 30 years). Clinical data and the present health status were evaluated. Also, the genes encoding the four major slit diaphragm proteins, nephrin, podocin, Neph1 and CD2-associated protein were sequenced in 38 patients with MCNS of varying severity. MCNS presented at the mean age of 5 years, and 80% of the patients relapsed 1-28 (median 3) times during childhood. The 14 subjects (14%) who had proteinuric episodes still in adulthood had a refractory disease already as children. The participants did not show a strong tendency for allergy or immune diseases, and no familial clustering of MCNS was observed. The genetic analyses revealed heterozygous amino acid changes in nephrin and podocin in 10 of the 38 patients studied. On the other hand, the genes coding for Neph1 and CD2AP were highly conserved and no amino acid substitutions were detected. In conclusion, MCNS is a multifactorial disease, in which genetics play a minor role. Allelic variants of the podocyte proteins may, however, modify the phenotype in occasional individuals.

Podocytes are firmly attached to glomerular basement membrane in kidneys with heavy proteinuria.

Glomerular epithelial cells (podocytes) play an important role in the pathogenesis of proteinuria. Podocyte foot process effacement is characteristic for proteinuric kidneys, and genetic defects in podocyte slit diaphragm proteins may cause nephrotic syndrome. In this work, a systematic electron microscopic analysis was performed of the structural changes of podocytes in two important nephrotic kidney diseases, congenital nephrotic syndrome of the Finnish type and minimal-change nephrotic syndrome (MCNS). The results showed that (1) podocyte foot process effacement was present not only in proteinuric glomeruli but also in nonproteinuric MCNS kidneys; (2) podocytes in proteinuric glomeruli did not show detachment from the basement membrane or cell membrane ruptures; (3) the number of pinocytic membrane invaginations in the basal and apical parts of the podocytes was comparable in proteinuric and control kidneys; (4) in proteinuric kidneys, the podocyte slit pore density was decreased by 69 to 80% and up to half of the slits were so "tight" that no visible space between foot processes was seen; thus, the filtration surface area between podocytes was dramatically reduced; and (5) in the narrow MCNS slit pores, nephrin was located in the apical part of the podocyte foot process, indicating vertical transfer of the slit diaphragm complex in proteinuria. In conclusion, these results suggest that protein leakage in the two nephrotic syndromes studied occurs through defective podocyte slits, and the other structural alterations commonly seen in electron microscopy are secondary to, not a prerequisite for, the development of proteinuria.

Nephrin gene (NPHS1) in patients with minimal change nephrotic syndrome (MCNS).

BACKGROUND: Minimal change nephrotic syndrome (MCNS) is a major problem in pediatric nephrology. While the pathogenesis of MCNS is not known, the latest discoveries in the genetic diseases indicate that glomerular epithelial cells (podocytes) and the slit diaphragm play a primary role in development of proteinuria. Because nephrin is known to be a major component of the slit diaphragm, we analyzed the structure of nephrin gene (NPHS1) in patients with MCNS of different severity. METHODS: Clinical data and DNA samples were collected from 25 adults who had biopsy-proven MCNS in childhood. A direct sequencing was performed to all 29 exons of the NPHS1 gene. The significance of the findings was evaluated by similar analysis of DNA samples from 25 healthy control patients. RESULTS: The analysis of NPHS1 revealed no specific MCNS-associated mutation. However, 5 of the 25 MCNS patients had heterozygous allelic variants leading to nonconservative amino acid substitutions not previously reported (G879R; R800C; T294I; A916S). One of the five patients also had the Fin-major mutation, and two had new, conservative amino acid substitutions (S786N; A342G). Three of the five patients were classified as steroid sensitive, one was an early nonresponder, and one patient showed clear resistance to steroid treatment. Six known polymorphic changes in NPHS1 were also found, three of them leading to amino acid changes. The number of allelic variants was high both in MCNS patients and control patients (mean 3.0 and 2.6). CONCLUSION: The results suggest that genetic changes in nephrin may have a pathogenetic role in some patients with MCNS.

Tissue expression of nephrin in human and pig.

Nephrin is a major component of the glomerular filtration barrier. Mutations in the nephrin gene (NPHS1) are responsible for congenital nephrotic syndrome of the Finnish type (NPHS1). Nephrin was at first thought to be podocyte specific, but recent studies have suggested that nephrin is also expressed in nonrenal tissues such as pancreas and CNS. We studied the expression of nephrin in human and porcine tissues at different stages of development and correlated these findings to clinical characteristics of NPHS1 children. Immunofluorescence staining and Western blotting were used to detect nephrin protein in frozen tissue samples. Polyclonal antibodies against the intracellular part of nephrin were used in these analyses. In situ hybridization was used to detect nephrin mRNA in specimens from normal human subjects and patients with NPHS1. Nephrin protein was not detected in nonrenal tissues obtained from human and porcine fetuses, newborns, and infants. Likewise, nephrin mRNA expression was not observed outside kidney glomerulus in normal or NPHS1 children. The phenotype analysis of NPHS1 children with severe nephrin gene mutations supported the findings in the tissue expression studies and revealed no impairment of the neurologic, testicular, or pancreatic function in a great majority of the patients. The studies suggest that nephrin has no major clinical significance outside the kidney.

The number of podocyte slit diaphragms is decreased in minimal change nephrotic syndrome.

The pathophysiology of proteinuria in acquired kidney diseases is mostly unknown. Recent findings in genetic renal diseases suggest that glomerular epithelial cells (podocytes) and the slit diaphragm connecting the podocyte foot processes play an important role in the development of proteinuria. In this work we systematically evaluated the podocyte slit pores by transmission electron microscopy in two important nephrotic diseases, minimal change nephrotic syndrome (MCNS) and membranous nephropathy (MN). As controls, we used kidneys with tubulointerstitial nephritis (TIN). Effacement of podocyte foot processes was evident in proteinuric kidneys. However, quite normal looking foot processes and slit pores with varying width were also observed. Careful analysis of slit pores revealed, that the proportion of the pores spanned by the linear image of slit diaphragm, was reduced by 39% in kidneys from MCNS patients (1265 pores analyzed) compared with TIN samples (902 pores analyzed, p = 0.0003). To enhance the detection rate of the slit diaphragms, the "empty" podocyte pores were further analyzed with tilting series from -45 to +45. This revealed the linear diaphragm image in 71% and 26% of the slits in TIN and MCNS kidneys, respectively (p = 0.0003). In contrast to findings in MCNS, no significant reduction of the slit diaphragms were seen in MN kidneys compared with the controls. The results suggest that MCNS is associated with disruption of glomerular slit diaphragms.