A novel missense mutation of Wilms' Tumor 1 causes autosomal dominant FSGS.

FSGS is a clinical disorder characterized by focal scarring of the glomerular capillary tuft, podocyte injury, and nephrotic syndrome. Although idiopathic forms of FSGS predominate, recent insights into the molecular and genetic causes of FSGS have enhanced our understanding of disease pathogenesis. Here, we report a novel missense mutation of the transcriptional regulator Wilms' Tumor 1 (WT1) as the cause of nonsyndromic, autosomal dominant FSGS in two Northern European kindreds from the United States. We performed sequential genome-wide linkage analysis and whole-exome sequencing to evaluate participants from family DUK6524. Subsequently, whole-exome sequencing and direct sequencing were performed on proband DNA from family DUK6975. We identified multiple suggestive loci on chromosomes 6, 11, and 13 in family DUK6524 and identified a segregating missense mutation (R458Q) in WT1 isoform D as the cause of FSGS in this family. The identical mutation was found in family DUK6975. The R458Q mutation was not found in 1600 control chromosomes and was predicted as damaging by in silico simulation. We depleted wt1a in zebrafish embryos and observed glomerular injury and filtration defects, both of which were rescued with wild-type but not mutant human WT1D mRNA. Finally, we explored the subcellular mechanism of the mutation in vitro. WT1(R458Q) overexpression significantly downregulated nephrin and synaptopodin expression, promoted apoptosis in HEK293 cells and impaired focal contact formation in podocytes. Taken together, these data suggest that the WT1(R458Q) mutation alters the regulation of podocyte homeostasis and causes nonsyndromic FSGS.

Phosphodiesterase 5 inhibition ameliorates angiontensin II-induced podocyte dysmotility via the protein kinase G-mediated downregulation of TRPC6 activity.

The emerging role of the transient receptor potential cation channel isotype 6 (TRPC6) as a central contributor to various pathological processes affecting podocytes has generated interest in the development of therapeutics to modulate its function. Recent insights into the regulation of TRPC6 have revealed PKG as a potent negative modulator of TRPC6 conductance and associated signaling via its phosphorylation at two highly conserved amino acid residues: Thr(69)/Thr(70) (Thr(69) in mice and Thr(70) in humans) and Ser(321)/Ser(322) (Ser(321) in mice and Ser(322) in humans). Here, we tested the role of PKG in modulating TRPC6-dependent responses in primary and conditionally immortalized mouse podocytes. TRPC6 was phosphorylated at Thr(69) in nonstimulated podocytes, but this declined upon ANG II stimulation or overexpression of constitutively active calcineurin phosphatase. ANG II induced podocyte motility in an in vitro wound assay, and this was reduced 30-60% in cells overexpressing a phosphomimetic mutant TRPC6 (TRPC6T70E/S322E) or activated PKG (P < 0.05). Pretreatment of podocytes with the PKG agonists S-nitroso-N-acetyl-dl-penicillamine (nitric oxide donor), 8-bromo-cGMP, Bay 41-2772 (soluble guanylate cyclase activator), or phosphodiesterase 5 (PDE5) inhibitor 4-{[3',4'-(methylenedioxy)benzyl]amino}[7]-6-methoxyquinazoline attenuated ANG II-induced Thr(69) dephosphorylation and also inhibited TRPC6-dependent podocyte motility by 30-60%. These data reveal that PKG activation strategies, including PDE5 inhibition, ameliorate ANG II-induced podocyte dysmotility by targeting TRPC6 in podocytes, highlighting the potential therapeutic utility of these approaches to treat hyperactive TRPC6-dependent glomerular disease.

Rare hereditary COL4A3/COL4A4 variants may be mistaken for familial focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a histological lesion with many causes, including inherited genetic defects, with significant proteinuria being the predominant clinical finding at presentation. Mutations in COL4A3 and COL4A4 are known to cause Alport syndrome (AS), thin basement membrane nephropathy, and to result in pathognomonic glomerular basement membrane (GBM) findings. Secondary FSGS is known to develop in classic AS at later stages of the disease. Here, we present seven families with rare or novel variants in COL4A3 or COL4A4 (six with single and one with two heterozygous variants) from a cohort of 70 families with a diagnosis of hereditary FSGS. The predominant clinical finding at diagnosis was proteinuria associated with hematuria. In all seven families, there were individuals with nephrotic-range proteinuria with histologic features of FSGS by light microscopy. In one family, electron microscopy showed thin GBM, but four other families had variable findings inconsistent with classical Alport nephritis. There was no recurrence of disease after kidney transplantation. Families with COL4A3 and COL4A4 variants that segregated with disease represent 10% of our cohort. Thus, COL4A3 and COL4A4 variants should be considered in the interpretation of next-generation sequencing data from such patients. Furthermore, this study illustrates the power of molecular genetic diagnostics in the clarification of renal phenotypes.

Application of inline variable pathlength technology for rapid determination of dynamic binding capacity in downstream process development of biopharmaceuticals.

Determination of dynamic binding capacity (DBC) for capture purification chromatographic step is usually the first experiment to be performed during downstream process development of biopharmaceuticals. In this work, we investigated the application of inline variable pathlength technology using FlowVPE for rapid determination of DBC on affinity resins for protein capture and proved its comparability with offline titer methods. This work also demonstrated that variable pathlength technology for DBC determination can be successfully applied to different classes of monoclonal antibodies and fusion proteins. This enabled rapid screening of affinity resins and optimization of the capture chromatography step. Hence, use of inline variable pathlength technology eliminated the dependency on offline titer data, traditionally used for DBC determination and accelerated overall process development timelines with less cost.

Iliac branch graft in the treatment of complex aortoiliac aneurysms: early results from a North American institution.

PURPOSE: To describe early experience with the use of iliac branch grafts (IBGs) in aortoiliac aneurysm repair. MATERIALS AND METHODS: From July 2007 to August 2009 (25 months), 14 patients (13 men, one woman) with a mean age of 70.1 years (range, 59.3-80.0 y) were treated with IBGs. Indications were abdominal aneurysm with common iliac artery (CIA) involvement (n = 11), juxtarenal aortic aneurysm with CIA involvement (n = 1), and bilateral CIA and internal iliac artery (IIA) aneurysms (n = 1). Postoperative endoleaks and patency rate were determined with computed tomography within 1 month of implantation and 1 year thereafter, with concurrent clinical evaluation for pelvic ischemia. Mean follow-up period was 18.7 months (range, 6-35 mo). RESULTS: Technical success rate, as defined by successful implantation of IBG with no intraprocedural type I or type III endoleak, was 86% (12 of 14). A total of 14 IBGs were successfully deployed in 12 patients. Two cases of technical failure were related to excessive iliac tortuosity. The mean hospitalization duration was 6.5 days (range, 3-14 d), with zero mortality at 30 days. There were two cases of type II endoleak treated conservatively and a single case of IBG-related type III endoleak that required repeat intervention. The rest of the stent-implanted aortic and iliac aneurysms remained stable in size, with no aneurysm rupture or death recorded. All stent-implanted iliac branches remained patent on follow-up. None of the patients who received IBGs had new symptoms of pelvic ischemia. CONCLUSIONS: Iliac branch graft placement is a feasible technique with excellent short-term results in the treatment of aortoiliac aneurysms.