Here, we tested whether combined genetic heterozygosity might play a role in the etiology of FSGS. Given the limited number of proteins implicated in podocyte-specific functions, a simple approach was to directly test whether combined heterozygosity of podocyte-relevant genes might cooperate together to cause podocyte dysfunction and, subsequently, glomerular disease. CD2AP is a scaffold protein that interacts with a variety of proteins, including slit diaphragm, signaling, and cytoskeletal proteins (24
). We speculated that mutations in Cd2ap
might sensitize the podocyte to mutations in a wide variety of different genes. We found that heterozygosity of Cd2ap
combined with heterozygosity of Synpo
led to a significant incidence of glomerular dysfunction as evidenced by proteinuria and pathological changes consistent with FSGS.
Recent progress has helped to illuminate the important role of the podocyte in the etiology of glomerular diseases like FSGS (2
). Mouse knockout models demonstrate that deficiencies of either podocyte-specific genes (encoding for podocin, ref. 8
; nephrin, ref. 25
; and Neph1, ref. 11
) or more ubiquitously expressed genes (encoding for α-actinin–4, ref. 9
; CD2AP, ref. 12
; and Fyn, ref. 15
) can result in podocyte dysfunction leading to a severe congenital nephrotic syndrome. Genetic studies in humans suggest that less severe mutations in some of these genes may be responsible for a more variable pattern of disease that may be responsible for some cases of familial and idiopathic FSGS (26
Podocin mutations were first described in an autosomal recessive form of FSGS that occurs with variable onset in children between 2 and 10 years of age (4
). Over 40 different mutations in podocin have now been described (27
). Most of the affected patients with autosomal recessive FSGS have homozygous missense mutations, suggesting that the mutation causes a weak loss of function that is only able to cause disease when mutations of both alleles are present (27
). Importantly, cases of increased disease susceptibility have been reported with mutations of only 1 podocin allele, suggesting involvement of another genetic locus (4
Mutations in the gene encoding α-actinin–4 are linked to an autosomal dominant form of FSGS in which the clinical presentation is also widely variable (5
). In these patients, the disease has a late onset (first occurring in early adulthood) and is incompletely penetrant (5
). Mice that are completely deficient for α-actinin–4
develop a severe congenital nephrotic syndrome (9
) while mice that are heterozygous for the dominant acting K228E allele do not display any overt disease (28
). These observations strongly suggest that environmental as well as other genetic factors may play important roles in determining whether disease occurs when a single copy of the disease-causing allele is present.
Here, we focused on bigenic heterozygosity of Cd2ap
. We previously showed that mice that are heterozygous for Cd2ap
develop pathological changes after 9 months of age but not proteinuria (13
heterozygous mice, however, have an increased sensitivity to nephrotoxic injury (13
). Given the mild phenotype of Cd2ap
heterozygous mice, we speculated that combined heterozygous mutations in Cd2ap
and other associated proteins might increase susceptibility to podocyte dysfunction.
We first focused on combining Cd2ap
heterozygosity with deficiency of the Src family tyrosine kinase Fyn. While Fyn+/–
mice are phenotypically and histologically normal, 2 groups have demonstrated that podocyte foot processes are abnormal in Fyn–/–
). The occurrence of proteinuria in these mice, however, is controversial, with DeFranco’s group (15
) reporting proteinuria in most mice by 6 months of age and Holzman’s group (14
) not reporting any proteinuria in these mice. In our study, we found about 31% of Fyn–/–
mice develop proteinuria by 12 months of age. This value, which is intermediate between the results of the 2 previous studies, suggests that other factors may be involved in the initiation and/or severity of proteinuria. However, the combination of CD2AP haploinsufficiency with Fyn deficiency clearly accentuated disease, as 100% of these animals developed proteinuria. A gene dosage effect was also apparent, since mice that were heterozygous for Cd2ap
and null for Fyn
developed proteinuria at a rate that was almost double the rate of combined Cd2ap+/–
mice. Lastly, it should be noted that bigenic heterozygosity resulted in pathological changes that were not present in the Fyn+/–
Our experiments with the Synpo-deficient mice implicate, for what we believe is the first time, a role for Synpo in the pathogenesis of FSGS. Synpo is an actin-bundling protein that is specifically expressed in podocytes and in certain neurons in the brain (19
). Synpo knockout mice exhibit neuronal defects but do not exhibit any spontaneous kidney abnormality (20
). Recently, the discovery of multiple Synpo isoforms and the fact that the shortest Synpo isoform (Synpo-T) is still expressed in the Synpo knockout suggest that this isoform might be compensating for the loss of the larger 2 isoforms in podocytes (20
). The in vivo functionality of Synpo-T is not entirely clear, but in any case, our work establishes that Synpo-T is unable to fully compensate in the presence of CD2AP haploinsufficiency.
It should be emphasized that the relatively mild pathological changes seen here are partially related to the way the experiment was designed. We used proteinuria as the end-point and sacrificed mice as soon as proteinuria was confirmed. Since we screened mice only monthly, the variability of proteinuria and pathological changes may have been due to variable length of time proteinuria was present. When a few proteinuric animals were followed for several months, the proteinuria and pathological changes progressed rapidly, leading to renal dysfunction and a significant level of glomerulosclerosis.
These studies demonstrating genetic epistasis among CD2AP, Fyn, and Synpo imply that these proteins might interact with each other. We showed, for what we believe is the first time, endogenous associations of CD2AP with Synpo and Fyn. The association of Fyn with CD2AP is probably mediated by the SH3 domain of Fyn binding to proline sequences in CD2AP, as a previous report showed that the SH3 domains of Fyn, Src, and Yes could bind to CD2AP in vitro (29
). Our study also demonstrated that Synpo and CD2AP form a protein complex. Mapping studies revealed that CD2AP and Synpo can directly interact and that this interaction is mediated by the SH3 domains of CD2AP, presumably by binding to proline-rich segments of Synpo. The fact that CD2AP binds to the nonoverlapping isoforms Synpo-short and Synpo-T suggests that there are at least 2 different proline-rich segments that can bind to CD2AP.
These data suggest that CD2AP, Fyn, and Synpo act in the same genetic pathway to maintain podocyte integrity. While the specific pathway is unclear, clues have emerged from the wide variety of proteins that are known to associate with CD2AP. For instance, CD2AP has been shown to interact with the slit diaphragm proteins podocin (30
) and nephrin (31
). A putative role for CD2AP in linking the slit diaphragm to the cortical actin cytoskeleton is suggested by associations of CD2AP with actin-capping protein (32
), cortactin (33
), and the GTPase-activating protein for ADP-ribosylation factor (ARF) family G proteins, ASAP1 (35
). CD2AP is also implicated in intracellular trafficking by its known interactions with proteins, such as synaptojanin, Rab4, and AP-2 (36
). A role for CD2AP in mediating ubiquitination and degradation in the lysosome is indicated by its known association with the ubiquitin ligase Cbl (38
In this study, we identified 2 new interacting partners for CD2AP, Fyn, and Synpo. As Fyn is known to tyrosine phosphorylate nephrin, it is exciting to speculate that the slit diaphragm is a type of signaling receptor and that CD2AP functions to orchestrate such complex processes as endocytosis and turnover of slit diaphragm components, establishments of cell polarity, and antiapoptotic signaling. The interaction between CD2AP and Synpo reinforces the idea that CD2AP plays a central role in regulating the actin cytoskeleton. We speculate that combined heterozygosity of CD2AP and many other proteins in the slit diaphragm signalosome may contribute to susceptibility to FSGS.
It is somewhat surprising that there are only a few published models where bigenic heterozygosity has been shown to directly lead to disease. These include congenital nonsyndromic hearing impairment (40
), human insulin resistance (41
), inherited disorders of fatty acid β-oxidation (43
), and polycystic kidney disease (44
). To our knowledge, this is the first description of combined heterozygosity causing any kind of glomerular disease. Koziell et al. reported a “triallelic” phenotype of 4 patients with homozygous mutations in either Nphs1
(nephrin) or Nphs2
(podocin), with an additional mutation in 1 allele of either Nphs1
(nephrin) or Nphs2
). Genetic epistasis between nephrin and podocin is suggested by the combined effects of the 3 mutations (45
). It is not clear, however, how the presence of the heterozygous allele influences the phenotype or the course of disease. But this reinforces the idea that the presence of additional mutations may play a role in susceptibility to FSGS.
The initial therapy for most cases of FSGS involves steroids or other potent immunosuppressants; however, the FSGS seen in established disease-causing mutations has typically been poorly responsive to steroids (46
). Only a small fraction of the cases of steroid-resistant FSGS or familial FSGS have identified mutations (2
). Thus, many of the remainder are likely to have some combinations of heterozygous mutations, as demonstrated in our models. The identification of such genes and alleles involved in familial FSGS will therefore aid the clinician in choosing which patients are likely to benefit from potentially toxic immunosuppressants. The future focus of glomerular diseases, such as FSGS, should expand from the evaluation of individual genes to the examination of the impact of many genes in combination. The use of combined mutational mouse models may allow us to identify genes that may act in concert with other genetic mutations to increase disease susceptibility.