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Sudipto Roy, Carol Wicking, Carsten Bergmann and colleagues report that mutations in DZIP1L cause autosomal recessive polycystic kidney disease (ARPKD). Through studies of mouse and zebrafish models of DZIP1L loss of function, the authors demonstrate that DZIP1L is required for proper function of the periciliary diffusion barrier. Autosomal recessive polycystic kidney disease (ARPKD), usually considered to be a genetically homogeneous disease caused by mutations in PKHD1 , has been associated with ciliary dysfunction. Here, we describe mutations in DZIP1L , which encodes DAZ interacting protein 1-like, in patients with ARPKD. We further validated these findings through loss-of-function studies in mice and zebrafish. DZIP1L localizes to centrioles and to the distal ends of basal bodies, and interacts with septin2, a protein implicated in maintenance of the periciliary diffusion barrier at the ciliary transition zone. In agreement with a defect in the diffusion barrier, we found that the ciliary-membrane translocation of the PKD proteins polycystin-1 and polycystin-2 is compromised in DZIP1L -mutant cells. Together, these data provide what is, to our knowledge, the first conclusive evidence that ARPKD is not a homogeneous disorder and further establish DZIP1L as a second gene involved in ARPKD pathogenesis.

The complement system is a major component of our innate immune system, in which the complement proteins C5a and C5a‐des Arg bind to two G‐protein‐coupled receptors: namely, the C5a receptor (C5a1) and C5a receptor like‐2 receptor (C5a2, formerly called C5L2). Recently, it has been demonstrated that C5a, but not C5a‐des Arg, upregulates heteromer formation between C5a1 and C5a2, leading to an increase in IL‐10 release from human monocyte‐derived macrophages (HMDMs). A bioluminescence resonance energy transfer (BRET) assay was used to assess the recruitment of β‐arrestins by C5a and C5a‐des Arg at the C5a1 and C5a2 receptors. C5a demonstrated elevated β‐arrestin 2 recruitment levels in comparison with C5a‐des Arg, whereas no significant difference was observed at C5a2. A constitutive complex that formed between β‐arrestin 2 and C5a2 accounted for half of the BRET signal observed. Interestingly, both C5a and C5a‐des Arg exhibited higher potency for β‐arrestin 2 recruitment via C5a2, indicating preference for C5a2 over C5a1. When C5a was tested in a functional ERK1/2 assay in HMDMs, inhibition of ERK1/2 was observed only at concentrations at or above the EC50 for heteromer formation. This suggested that increased recruitment of the β‐arrestin‐C5a2 complex at these C5a concentrations might have an inhibitory role on C5a1 signaling through ERK1/2. An improved understanding of C5a2 modulation of signaling in acute inflammation could be of benefit in the development of ligands for conditions such as sepsis.

The complement cascade is comprised of a highly sophisticated network of innate immune proteins that are activated in response to invading pathogens or tissue injury. The complement activation peptide, C5a, binds two seven transmembrane receptors, namely the C5a receptor 1 (C5aR1) and C5a receptor 2 (C5aR2, or C5L2). C5aR2 is a non‐G‐protein‐signalling receptor whose biological role remains controversial. Some of this controversy arises owing to the lack of selective ligands for C5aR2. In this study, a library of 61 peptides based on the C‐terminus of C5a was assayed for the ability to selectively modulate C5aR2 function. Two ligands (P32 and P59) were identified as functionally selective C5aR2 ligands, exhibiting selective recruitment of β‐arrestin 2 via C5aR2, partial inhibition of C5a‐induced ERK1/2 activation and lipopolysaccharide‐stimulated interleukin‐6 release from human monocyte‐derived macrophages. Importantly, neither ligand could induce ERK1/2 activation or inhibit C5a‐induced ERK1/2 activation via C5aR1 directly. Finally, P32 inhibited C5a‐mediated neutrophil mobilisation in wild‐type, but not C5aR2−/− mice. These functionally selective ligands for C5aR2 are novel tools that can selectively modulate C5a activity in vitro and in vivo, and thus will be valuable tools to interrogate C5aR2 function.

There has been intensive effort to identify in vivo biomarkers that can be used to monitor drug‐induced kidney damage and identify injury before significant impairment occurs. Kidney injury molecule‐1 (KIM‐1), neutrophil gelatinase‐associated lipocalin (NGAL), and human macrophage colony stimulating factor (M‐CSF) have been validated as urinary and plasma clinical biomarkers predictive of acute and chronic kidney injury and disease. Similar validation of a high throughput in vitro assay predictive of nephrotoxicity could potentially be implemented early in drug discovery lead optimization to reduce attrition at later stages of drug development. To assess these known in vivo biomarkers for their potential for in vitro screening of drug‐induced nephrotoxicity, we selected a panel of nephrotoxic agents and examined their effects on the overexpression of nephrotoxicity biomarkers in immortalized (HK‐2) and primary (commercially available and freshly in‐house produced) human renal proximal tubule epithelial cells. Traditional cytotoxicity was contrasted with expression levels of KIM‐1, NGAL, and M‐CSF assessed using ELISA and real‐time quantitative reverse transcription PCR. Traditional cytotoxicity assays and biomarker assays using HK‐2 cells were both unsuitable for prediction of nephrotoxicity. However, increases in protein levels of KIM‐1 and NGAL in primary cells were well correlated with dose levels of known nephrotoxic compounds, with limited correlation seen in M‐CSF protein and mRNA levels. These results suggest that profiling compounds against primary cells with monitoring of biomarker protein levels may have potential as in vitro predictive assays of drug‐induced nephrotoxicity.

Autosomal recessive polycystic kidney disease (ARPKD), usually considered to be a genetically homogeneous disease caused by mutations in PKHD1, has been associated with ciliary dysfunction. Here, we describe mutations in DZIP1L, which encodes DAZ interacting protein 1-like, in patients with ARPKD. We further validated these findings through loss-of-function studies in mice and zebrafish. DZIP1L localizes to centrioles and to the distal ends of basal bodies, and interacts with septin2, a protein implicated in maintenance of the periciliary diffusion barrier at the ciliary transition zone. In agreement with a defect in the diffusion barrier, we found that the ciliary-membrane translocation of the PKD proteins polycystin-1 and polycystin-2 is compromised in DZIP1L-mutant cells. Together, these data provide what is, to our knowledge, the first conclusive evidence that ARPKD is not a homogeneous disorder and further establish DZIP1L as a second gene involved in ARPKD pathogenesis.

The complement system is a major component of our innate immune system, in which the complement proteins C5a and C5a-des Arg bind to two G-protein-coupled receptors: namely, the C5a receptor (C5a1) and C5a receptor like-2 receptor (C5a2, formerly called C5L2). Recently, it has been demonstrated that C5a, but not C5a-des Arg, upregulates heteromer formation between C5a1 and C5a2, leading to an increase in IL-10 release from human monocyte-derived macrophages (HMDMs). A bioluminescence resonance energy transfer (BRET) assay was used to assess the recruitment of β-arrestins by C5a and C5a-des Arg at the C5a1 and C5a2 receptors. C5a demonstrated elevated β-arrestin 2 recruitment levels in comparison with C5a-des Arg, whereas no significant difference was observed at C5a2. A constitutive complex that formed between β-arrestin 2 and C5a2 accounted for half of the BRET signal observed. Interestingly, both C5a and C5a-des Arg exhibited higher potency for β-arrestin 2 recruitment via C5a2, indicating preference for C5a2 over C5a1. When C5a was tested in a functional ERK1/2 assay in HMDMs, inhibition of ERK1/2 was observed only at concentrations at or above the EC 50 for heteromer formation. This suggested that increased recruitment of the β-arrestin-C5a2 complex at these C5a concentrations might have an inhibitory role on C5a1 signaling through ERK1/2. An improved understanding of C5a2 modulation of signaling in acute inflammation could be of benefit in the development of ligands for conditions such as sepsis.