Genetics of focal segmental glomerulosclerosis

The NPHS1 gene mutations cause congenital nephrotic syndrome of the Finnish type, an autosomal recessive disorder characterized by massive proteinuria in utero and nephrosis at birth. Although this disease is not pathologically characterized by FSGS, it was the first mutation of structural protein of podocyte causally linked to nephrotic syndrome [5]. Moreover, nephrin deficiency was detected in other forms of nephrotic syndrome, and an overlap in the genes encoding nephrin/podocin (NPHS1/NPHS2) mutation spectrum (a triallelic hit) has recently been documented in patients with congenital FSGS [6]. NPHS1 product nephrin has 1,241 amino acid residues and belongs to the immunoglobulin (Ig) family of cell adhesion molecules. It contains a transmembrane domain, eight Ig-like repeats, and one fibronectin III-like module. Nephrin was localized by immunogold labeling and electron tomography at the slit between podocyte foot processes in a network of 35-nm long, globular cross-strands lining lateral, elongated pores in a zipper-like model [7]. Those pores are the same as or smaller than albumin molecules. Patients with NPHS1 mutations and nephrin-knockout mice had only narrow filtration slits that lacked the slit diaphragm network and the 35-nm-long strands; these findings suggest that nephrin molecules are directly involved in constituting the macromolecule-retaining slit diaphragm and its pores [8]. Functionally, nephrin controls the podocyte cytoskeleton in vivo by interacting with other proteins, such as Src homology-2 (SH2)/SH3 domain-containing Nck adaptor proteins [7]. Originally described mutation in Finnish patients was a two-nucleotide deletion in exon 2 [5], but other mutations have been reported since. Those include deletions, insertions, nonsense, missense, splicing mutations, and common polymorphisms [9‐11].

Positional cloning identified NPHS2, encoding podocin, as a causative gene in autosomal recessive steroid-resistant nephrotic syndrome, including FSGS [12]. NPHS2 encodes a putative 383 amino acid protein of approximately 42 kDa. It belongs to the stomatin family proteins (band-7 proteins). NPHS2 expression is restricted to the podocytes, as shown by in situ hybridization studies, and the protein is an integral plasma membrane protein associated with specialized lipid raft microdomains [12]. Podocin is required for the recruitment of nephrin into lipid rafts; it interacts with nephrin and CD2AP [13] and facilitates nephrin signaling [14]. In podocin-null mice, nephrin expression is increased, whereas ZO1 (tight junction protein) and CD2AP expression is decreased. The mice die a few days after birth from renal failure caused by diffuse mesangial sclerosis, and they develop proteinuria during the antenatal period [15].

At present, at least 26 different NPHS2 mutations associated with FSGS have been described, with a mutation defined as a polymorphism that is present in homozygosity in at least one patient with FSGS [12, 16‐20]. Many patients are compound heterozygotes with two distinct mutations. The age of onset is generally in infancy or childhood, but a few cases of adult onset kidney disease have been described, with the oldest patient presenting at age 36. In populations of European ancestry, NPHS2 mutations are present in 26% of families with familial FSGS and 12–19% of sporadic pediatric FSGS [16, 18]. On the other hand, NPHS2 mutations associated with pediatric FSGS appear to be uncommon in children of Asian ancestry [21]; little is known about children of African ancestry. FSGS associated with NPHS2 mutations is uniformly steroid resistant and generally shows poor response to cyclosporine as well [18]. Although early reports suggested otherwise, the consensus is now that patients with NPHS2 mutations are at reduced risk for recurrent FSGS following renal transplant [18, 22]. The most common polymorphism is R229Q, with a heterozygote frequency in the general population ranging from 0.03 to 0.13 [23]. R229Q heterozygosity has been associated with microalbuminuria in a Brazilian population [24] and a modest increased risk for FSGS in individuals of European ancestry, but not individuals of African ancestry [23].

CD2-associated protein was discovered as a binding ligand to the adhesion molecule CD2 during cell–cell interaction of lymphocyte T with antigen presenting cell. This interaction initiates the process of protein segregation, CD2 clustering, and cytoskeletal polarization. The CD2AP gene encodes a protein of 639 amino acids with a deduced molecular mass of approximately 70 kDa, and messenger ribonucleic acid (mRNA) is ubiquitously expressed in human tissues. Cd2ap knock-out mice manifest compromised immune function and they developed proteinuria by 2 weeks of age and died from renal failure at 6–7 weeks of age [25]. In the kidney, the lesion resembles FSGS, with mesangial cell hyperplasia and extracellular matrix deposition. Mice with Cd2ap haploinsufficiency (heterozygous deletion) develop glomerular changes at 9 months of age. They have increased susceptibility to glomerular injury by immune complexes, with likely impairment of the intracellular degradation pathways [26]. CD2AP is the human ortholog of mouse Cd2ap, with 86% identity at the amino acid level. CD2AP (originally named CMS) was identified in the yeast two-hybrid system as interacting partner of p130 (Cas), a cell cycle regulatory protein [27]. CD2AP is a multifunctional adapter molecule, which is localized to the cytoplasm, membrane ruffles, and leading edges of cells. It functions as scaffolding protein involved in the dynamic regulation of the actin cytoskeleton [27]. Two African Americans with primary idiopathic FSGS were found to have a mutation of CD2AP splice acceptor of exon 7 on one allele. This mutation was not seen in control subjects. Although these data are suggestive, family studies showing a consistent relationship between mutation and renal phenotype will be required to establish a pathogenic role. Data supporting a role for CD2AP haploinsufficiency comes from bigenic mouse models of FSGS involving pairwise interaction of CD2AP, Fyn, and synaptopodin [28].

ACTN4 mutations have been found to be the cause of autosomal dominant FSGS in five families [29, 30]. These patients tend to present in the teenage years or later and progress to end-stage renal disease (ESRD) slowly. The penetrance is variable, and some patients with mutations have a very mild phenotype. Alpha-actinin-4 is an actin-bundling protein associated with cytoskeleton, cell motility, and cancer invasion [31]. It is highly expressed in podocytes, and interacts with synaptopodin and with the tight junction protein membrane-associated guanylate kinase (MAGI)-1 in rat kidney epithelial cells [32]. Mutant alpha-actinin-4 binds actin more tightly that does wild-type alpha-actinin-4, indicating a gain of function mutation. Actn4-null mice develop proteinuria, progressive glomerular disease, and die by several months of age. Histological abnormalities are limited to the kidneys and include diffuse podocyte foot process effacement and globally disrupted morphology [33]. Cultured podocytes from these mice adhere poorly to basement membrane components, suggesting that podocyte loss may contribute to podocytopenia and FSGS [34].

TRPC6 belongs to a protein family whose members function as ion channels, mediating capacitative calcium entry into the cell. TRPC6 is a nonselective calcium channel that is activated by diacylglycerol in a membrane-delimited fashion, independently of protein kinase C [35]. TRPC6 is responsible for calcium entry during cell proliferation and is expressed primarily in the placenta, lung, spleen, ovary, and small intestine. In the kidney, TRPC6 is expressed in tubules and glomeruli, including podoctyes and glomerular endothelial cells.

TRPC6 mutations have been identified in six families of European and African geographic ancestry with autosomal dominant FSGS [36, 37]. Each family carried a distinct missense mutation. Age at renal disease presentation ranged from 17 to 52 years, and the duration from onset to ESRD averaged approximately 10 years. TRPC6 interacts with nephrin and podocin, thus localizing the protein to the slit diaphragm complex. Two mutant proteins are associated with increased calcium amplitudes, indicating an activating mutation. The discovery that abnormal TRPC6 disturbs podocyte function suggests that calcium signaling may play a critical role in facilitating podocyte regulation of intracellular function, including control of cytoskeletal and foot process architecture.

Recently, seven families with infantile or early onset proteinuric renal disease were found to have mutations in PLCE1, which encodes an isoform of phospholipase C, an enzyme that participates in intracellular signaling [4]. In affected individuals, proteinuria was detected between age 2 months and 9 years; renal histology included diffuse mesangial sclerosis and FSGS. Response to treatment included steroid resistance and complete remission to either steroids or cyclosporine. This is the first time a genetic podoctye disease has been associated with steroid-sensitive nephrotic syndrome [4].

The above genes have mutations associated with FSGS in the absence of extrarenal manifestations. To date, at least five genes have been associated with syndromes of which FSGS is often a part.

The recent advances in understanding the pathophysiology of FSGS and molecular function of glomerular filtration barrier come in large part from genetic linkage and positional cloning studies. Because a substantial percentage (up to 30%) of sporadic, steroid-resistant nephrotic syndrome patients carry the specific genetic mutation causally linked to FSGS, it may be advisable that all patients who have failed steroids should be offered genetic screening for mutations at the time of or prior to starting more aggressive treatments, as those patients have been shown to unfavorably respond to immunosuppressant therapy [18]. Genetic testing could be performed in several research laboratories and now is also being offered by a commercial laboratory. Genetic testing for familial forms of FSGS might also be clinically informative in situations where living-related donor transplant is considered.

The exact role and function of the newly discovered genes and proteins are being investigated by in vitro and in vivo mechanistic studies. Those genes and proteins interactions seem to change susceptibility to kidney disease progression. Better understanding of their exact role in the development of FSGS may influence future therapies and outcomes in this complex disease.