RESUMO
Mutations in Inverted Formin 2 (INF2), a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause focal segmental glomerulosclerosis (FSGS) and Charcot-Marie-Tooth Disease (CMT) in humans. In addition to directly remodeling actin filaments in vitro, we have shown that INF2 regulates intracellular actin dynamics and actin dependent cellular behavior by opposing Rhoa/Dia signaling. As a step towards a better understanding of the human kidney disease, we wanted to explore the relevance of these findings to the in vivo situation. We used dose dependent knockdown of INF2 to first define an in vivo model and establish an overt glomerular phenotype in zebrafish. This simple assay was validated by rescue with wild type INF2 confirming the specificity of the findings. The edema, podocyte dysfunction, and an altered glomerular filtration barrier observed in the zebrafish pronephros correlate with mistrafficking of glomerular slit diaphragm proteins, defective slit-diaphragm signaling, and disinhibited diaphanous formin (mDia) activity. In contrast to wild-type human INF2, INF2 mutants associated with kidney disease fail to rescue the zINF2 morphant phenotype. Of particular interest, this INF2 knockdown phenotype is also rescued by loss of either RhoA or Dia2. This simple assay allows the demonstration that INF2 functions, at least in part, to modulate Dia-mediated Rho signaling, and that disease causing mutations specifically impair this regulatory function. These data support a model in which disease-associated diaphanous inhibitory domain (DID) mutants in INF2 interfere with its binding to and inhibition of Dia, leading to uncontrolled Rho/Dia signaling and perturbed actin dynamics. Methods to fine tune Rho signaling in the glomerulus may lead to new approaches to therapy in humans.
RESUMO
BACKGROUND: Accurate diagnosis of the primary cause of an individual's kidney disease can be essential for proper management. Some kidney diseases have overlapping histopathologic features despite being caused by defects in different genes. In this report, we describe 2 consanguineous Saudi Arabian families in which individuals presented with kidney failure and mixed clinical and histologic features initially believed to be consistent with focal segmental glomerulosclerosis. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: We studied members of 2 apparently unrelated families from Saudi Arabia with kidney disease. MEASUREMENTS: Whole-genome single-nucleotide polymorphism analysis followed by targeted isolation and sequencing of exons using genomic DNA samples from affected members of these families, followed by additional focused genotyping and sequence analysis. RESULTS: The 2 apparently unrelated families shared a region of homozygosity on chromosome 2q13. Exome sequence from affected individuals lacked sequence reads from the NPHP1 gene, which is located within this homozygous region. Additional polymerase chain reaction-based genotyping confirmed that affected individuals had NPHP1 deletions, rather than defects in a known focal segmental glomerulosclerosis-associated gene. LIMITATIONS: The methods used here may not result in a clear genetic diagnosis in many cases of apparent familial kidney disease. CONCLUSIONS: This analysis shows the power of new high-throughput genotyping and sequencing technologies to aid in the rapid genetic diagnosis of individuals with an inherited form of kidney disease. We believe it is likely that such tools may become useful clinical genetic tools and alter the manner in which diagnoses are made in nephrology.
