BackgroundPrevious studies showed that Cdc42, a member of the prototypical Rho family of small GTPases and a regulator of the actin cytoskeleton, is critical for the normal development and health of podocytes. However, upstream regulatory mechanisms for Cdc42 activity in podocytes are largely unknown.MethodsWe used a proximity-based ligation assay, BioID, to identify guanine nucleotide exchange factors that activate Cdc42 in immortalized human podocytes. We generated podocyte-specific ARHGEF7 (commonly known as β-PIX) knockout mice by crossing β-PIX floxed mice with Podocin-Cre mice. Using shRNA, we established cultured mouse podocytes with β-PIX knockdown and their controls.ResultsWe identified β-PIX as a predominant guanine nucleotide exchange factor that interacts with Cdc42 in human podocytes. Podocyte-specific β-PIX knockout mice developed progressive proteinuria and kidney failure with global or segmental glomerulosclerosis in adulthood. Glomerular podocyte density gradually decreased in podocyte-specific β-PIX knockout mice, indicating podocyte loss. Compared with controls, glomeruli from podocyte-specific β-PIX knockout mice and cultured mouse podocytes with β-PIX knockdown exhibited significant reduction in Cdc42 activity. Loss of β-PIX promoted podocyte apoptosis, which was mediated by the reduced activity of the prosurvival transcriptional regulator Yes-associated protein.ConclusionsThese findings indicate that β-PIX is required for the maintenance of podocyte architecture and glomerular function via Cdc42 and its downstream Yes-associated protein activities. This appears to be the first evidence that a Rho–guanine nucleotide exchange factor plays a critical role in podocytes.
Nephrotic syndrome (NS) describes a group of kidney disorders in which there is injury to podocyte cells, specialized cells within the kidney's glomerular filtration barrier, allowing proteins to leak into the urine. Three mutations in ARHGDIA, which encodes Rho GDP dissociation inhibitor α (GDIα), have been reported in patients with heritable NS and encode the following amino acid changes: ΔD185, R120X, and G173V. To investigate the impact of these mutations on podocyte function, endogenous GDIα was knocked-down in cultured podocytes by shRNA and then the cells were re-transfected with wild-type or mutant GDIα constructs. Among the 3 prototypical Rho-GTPases, Rac1 was markedly hyperactivated in podocytes with any of the 3 mutant forms of GDIα while the activation of RhoA and Cdc42 was modest and variable. All three mutant GDIα proteins resulted in slow podocyte motility, suggesting that podocytes are sensitive to the relative balance of Rho-GTPase activity. In ΔD185 podocytes, both random and directional movements were impaired and kymograph analysis of the leading edge showed increased protrusion and retraction of leading edge (phase switching). The mutant podocytes also showed impaired actin polymerization, smaller cell size, and increased cellular projections. In the developing kidney, GDIα expression increased as podocytes matured. Conversely, active Rac1 was detected only in immature, but not in mature, podocytes. The results indicate that GDIα has a critical role in suppressing Rac1 activity in mature podocytes, to prevent podocyte injury and nephrotic syndrome.
Nephrotic syndrome is a kidney disease featured by heavy proteinuria. It is caused by injury to the specialized epithelial cells called “podocytes” within the filtration unit of the kidney, glomerulus. Previous studies showed that hyperactivation of the RhoGTPase, Rac1, in podocytes causes podocyte injury and glomerulosclerosis (accumulation of extracellular matrix in the glomerulus). However, the mechanism by which Rac1 is activated during podocyte injury is unknown. Trio is a guanine nucleotide exchange factor (GEF) known to activate Rac1. By RNA-sequencing, we found that Trio mRNA is abundantly expressed in cultured human podocytes. Trio mRNA was also significantly upregulated in humans with minimal change disease and focal segmental glomerulosclerosis, two representative causes of nephrotic syndrome. Reduced expression of Trio in cultured human podocytes decreased basal Rac1 activity, cell size, attachment to laminin, and motility. Furthermore, while the pro-fibrotic cytokine, transforming growth factor β1 increased Rac1 activity in control cells, it decreases Rac1 activity in cells with reduced Trio expression. This was likely due to simultaneous activation of the Rac1-GTPase activation protein, CdGAP. Thus, Trio is important in the basal functions of podocytes and may also contribute to glomerular pathology, such as sclerosis, via Rac1 activation.
Purpose of review:P4 medicine denotes an evolving field of medicine encompassing predictive, preventive, personalized, and participatory medicine. Using the example of kidney allograft rejection because of donor-recipient incompatibility in human leukocyte antigens, this review outlines P4 medicine’s relevance to the various stages of the kidney transplant cycle.Sources of information:A search for English articles was conducted in Medline via OvidSP (up to August 18, 2016) using a combination of subject headings (MeSH) and free text in titles, abstracts, and author keywords for the concepts kidney transplantation and P4 medicine. The electronic database search was expanded further on particular subject headings.Findings:Available histocompatibility methods exemplify current applications of the predictive and preventive domains of P4 medicine in kidney transplant recipients’ care. Pharmacogenomics are discussed as means to facilitate personalized immunosuppression regimens and promotion of active patient participation as a means to improve adherence.Limitations:For simplicity, this review focuses on rejection. P4 medicine, however, should more broadly address health concerns in kidney transplant recipients, including competing outcomes such as infections, malignancies, and cardiovascular disease. This review highlights how biomarkers to evaluate these competing outcomes warrant validation and standardization prior to their incorporation into clinical practice.Implications:Consideration of all 4 domains of the P4 medicine framework when caring for and/or studying kidney transplant recipients has the potential of increasing therapeutic efficiency, minimizing adverse effects, decreasing health care costs, and maximizing wellness. Technologies to gauge immune competency, immunosuppression requirements, and early/reversible immune-mediated injuries are required to optimize kidney transplant care.
Rho GTPases are regulators of the actin cytoskeleton and their activity is modulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchanging factors (GEFs). Glomerular podocytes have numerous actin-based projections called foot processes and their alteration is characteristic of proteinuric kidney diseases. We reported previously that Rac1 hyperactivation in podocytes causes proteinuria and glomerulosclerosis in mice. However, which GAP and GEF modulate Rac1 activity in podocytes remains unknown. Here, using a proximity-based ligation assay, we identified CdGAP (ARHGAP31) and β-PIX (ARHGEF7) as the major regulatory proteins interacting with Rac1 in human podocytes. CdGAP interacted with β-PIX through its basic region, and upon EGF stimulation, they both translocated to the plasma membrane in podocytes. CdGAP-depleted podocytes had altered cell motility and increased basal Rac1 and Cdc42 activities. When stimulated with EGF, CdGAP-depleted podocytes showed impaired β-PIX membrane-translocation and tyrosine phosphorylation, and reduced activities of Src kinase, focal adhesion kinase, and paxillin. Systemic and podocyte-specific CdGAP-knockout mice developed mild but significant proteinuria, which was exacerbated by Adriamycin. Collectively, these findings show that CdGAP contributes to maintain podocyte function and protect them from injury.
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