Sulfatase 2 Is Associated with Steroid Resistance in Childhood Nephrotic Syndrome

Glucocorticoid (GC) resistance complicates the treatment of ~10–20% of children with nephrotic syndrome (NS), yet the molecular basis for resistance remains unclear. We used RNAseq analysis and in silico algorithm-based approaches on peripheral blood leukocytes from 12 children both at initial NS presentation and after ~7 weeks of GC therapy to identify a 12-gene panel able to differentiate steroid resistant NS (SRNS) from steroid-sensitive NS (SSNS). Among this panel, subsequent validation and analyses of one biologically relevant candidate, sulfatase 2 (SULF2), in up to a total of 66 children, revealed that both SULF2 leukocyte expression and plasma arylsulfatase activity Post/Pre therapy ratios were greater in SSNS vs. SRNS. However, neither plasma SULF2 endosulfatase activity (measured by VEGF binding activity) nor plasma VEGF levels, distinguished SSNS from SRNS, despite VEGF’s reported role as a downstream mediator of SULF2’s effects in glomeruli. Experimental studies of NS-related injury in both rat glomeruli and cultured podocytes also revealed decreased SULF2 expression, which were partially reversible by GC treatment of podocytes. These findings together suggest that SULF2 levels and activity are associated with GC resistance in NS, and that SULF2 may play a protective role in NS via the modulation of downstream mediators distinct from VEGF.

Increasing thermal stability and improving biodistribution of VEGFR2-binding affibody molecules by a combination of in silico and directed evolution approaches

The family of vascular endothelial growth factor (VEGF) ligands and their interactions with VEGF receptors (VEGFRs) play important roles in both pathological and physiological angiogenesis. Hence, agonistic and antagonistic ligands targeting this signaling pathway have potential for both studies on fundamental biology and for development of therapies and diagnostics. Here, we engineer VEGFR2-binding affibody molecules for increased thermostability, refolding and improved biodistribution. We designed libraries based on the original monomeric binders with the intention of reducing hydrophobicity, while retaining high affinity for VEGFR2. Libraries were displayed on bacteria and binders were isolated by fluorescence-activated cell sorting (FACS). In parallel, we used an automated sequence- and structure-based in silico algorithm to identify potentially stabilizing mutations. Monomeric variants isolated from the screening and the in silico approach, respectively, were characterized by circular dichroism spectroscopy and biosensor assays. The most promising mutations were combined into new monomeric constructs which were finally fused into a dimeric construct, resulting in a 15 °C increase in melting temperature, complete refolding capability after heat-induced denaturation, retained low picomolar affinity and improved biodistribution profile in an in vivo mouse model. These VEGFR2-binding affibody molecules show promise as candidates for further in vivo studies to assess their suitability as molecular imaging and therapeutic agents.