A Human Integrin-α3 Mutation Confers Major Renal Developmental Defects

<div><p>The development of the mammalian kidney is a highly complex process dependent upon the interplay of various cell types, secreted morphogens, and the extra-cellular matrix (ECM). Although integrins are the most important receptors for ECM proteins and are ubiquitously expressed during kidney development, mice lacking expression of integrin α3 (Itga3) do not demonstrate a reduced number of nephrons, but mostly a disorganized GBM (glomerular basement membrane) leading to proteinuria. Thus, ITGA3 is considered mostly a passive GBM stabilizer and not an active player in nephrogenesis. Recently, mutations in the human <i>ITGA3</i> were shown to cause congenital nephrotic syndrome, epidermolysis bullosa and interstitial lung disease, otherwise termed NEP syndrome (<b>N</b>ephrotic syndrome, <b>E</b>pidermolysis bullosa and <b>P</b>ulmonary disease). Herein, we performed histological and molecular analysis on the kidneys of a single patient from the initial cohort harboring an <i>ITGA3</i> mutation, to illuminate the role of <i>ITGA3</i> in human renal development. We show the patient to harbor a unique phenotype at birth, including severe unilateral renal hypodysplasia. Interrogation of global gene expression in the hypodysplastic kidney versus three controls (fetal, child and adult kidneys) revealed perturbed expression in several renal developmental pathways implicated in hypodysplasia, including the Wnt, BMP (bone morphogenetic protein) and TGF (transforming growth factor) pathways. Moreover, the affected kidney showed upregulation of early embryonic genes (e.g. <i>OCT4</i> and <i>PAX8</i>) concomitant with downregulated kidney differentiation markers, implying a defect in proper renal differentiation. In conclusion, we show for the first time that ITGA3 is not merely a passive anchor for renal ECM proteins, as predicted by mouse models. Instead, our results may suggest it plays a central role in the interplay of cells, morphogens and ECM, required for proper nephrogenesis, thus adding <i>ITGA3</i> to the list of CAKUT (congenital anomalies of the kidney and urinary tract)-causing genes.</p></div

Clinical and histological features of the index patient.

<p>(A) A pedigree presenting the patient, born to healthy consanguineous parents, as the only affected child among nine siblings. (B) Renal ultrasound examination, demonstrating a small hyper-echogenic left kidney and an enlarged right kidney. (C) H&E staining of the patient's right kidney demonstrating a typical nephrotic syndrome phenotype including global sclerosis and mesangial proliferation. (D) The patient's left kidney presents histology consistent with renal hypodysplasia including the presence of cartilage, stroma and renal lesions of nephrotic syndrome similar to those observed in the right kidney.</p

Immuno-localization and interrogation of global gene expression of the patient's kidney.

<p>(A) Immunohistochemical staining for integrin α3 reveals a widespread expression pattern in the developing human fetal kidney (hFK), with localization to early duct precursors, ureteric buds and their differentiated derivatives and basement membrane of assembled fetal glomeruli. Integrin α3 expression was absent in the patient's kidneys. (B) Heat-map comparison of gene expression profile between the patient's kidney (PK) and an age matched control (CK) kidney. Unsupervised hierarchical clustering demonstrates that the PK is more similar genetically to human fetal kidney (hFK) than to the human adult kidney counterpart (hAK). (C) Microarray expression analysis of selected genes demonstrated altered expression in the PK of genes crucial for normal nephron formation, including the Wnt and TGFβ signaling pathways, early developmental genes and renal differentiation genes.</p

Reduced secretion and intracellular and extracellular aggregation of Arg446Cys VWA2.

<p><b>(a)</b> Cell culture supernatants and cell lysates from wild type (wt) and Arg446Cys VWA2 (R446C) expressing 293EBNA cells were separated by SDS-PAGE under reducing and non-reducing conditions and detected with an antibody against the One-STrEP-tag. Arrowheads indicate monomeric VWA2. On the right, equal loading is demonstrated by Ponceau staining of the membranes. Asterisks indicate artefact bands. <b>(b)</b> cDNA from non transfected (nt), non-transfected ER stress induced (nt+DTT), wt VWA2 transfected (wt) and Arg446Cys VWA2 (R446C) transfected 293EBNA cells was submitted to RT-PCR and the PCR products separated by agarose gel electrophoresis. Arrows indicate the bands for XBP-1 and ER stress induced XBP-1s. Equal loading is demonstrated by actin control RT-PCR shown below. <b>(c)</b> Equal amounts (0.2 μg) of affinity purified wild type (wt) and Arg446Cys VWA2 (R446C) were separated by SDS-PAGE under reducing and non-reducing conditions and detected with an antibody against the C-terminal fragment (P3) of human VWA2. Under non-reducing conditions higher aggregates are seen. Arrows indicate the border between separation and stacking gel. (d) Equal amounts of cell culture supernatants and cell lysates from wild type (wt) and Arg446Cys VWA2 (R446C) expressing 293EBNA cells as in (a) and of affinity purified wild type (wt) and Arg446Cys VWA2 (R446C) as in (b) were separated by agarose-polyacrylamide composite gels under non-reducing conditions and detected with an antibody against the One-STrEP-tag.</p

Detailed outline of the tasks undertaken during AcWriMo at Maynooth University Library

<div><p>Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause (40–50%) of chronic kidney disease (CKD) in children. About 40 monogenic causes of CAKUT have so far been discovered. To date less than 20% of CAKUT cases can be explained by mutations in these 40 genes. To identify additional monogenic causes of CAKUT, we performed whole exome sequencing (WES) and homozygosity mapping (HM) in a patient with CAKUT from Indian origin and consanguineous descent. We identified a homozygous missense mutation (c.1336C>T, p.Arg446Cys) in the gene <i>Von Willebrand factor A domain containing 2</i> (<i>VWA2</i>). With immunohistochemistry studies on kidneys of newborn (P1) mice, we show that Vwa2 and Fraser extracellular matrix complex subunit 1 (Fras1) co-localize in the nephrogenic zone of the renal cortex. We identified a pronounced expression of Vwa2 in the basement membrane of the ureteric bud (UB) and derivatives of the metanephric mesenchyme (MM). By applying <i>in vitro</i> assays, we demonstrate that the Arg446Cys mutation decreases translocation of monomeric VWA2 protein and increases translocation of aggregated VWA2 protein into the extracellular space. This is potentially due to the additional, unpaired cysteine residue in the mutated protein that is used for intermolecular disulfide bond formation. VWA2 is a known, direct interactor of FRAS1 of the Fraser-Complex (FC). FC-encoding genes and interacting proteins have previously been implicated in the pathogenesis of syndromic and/or isolated CAKUT phenotypes in humans. <i>VWA2</i> therefore constitutes a very strong candidate in the search for novel CAKUT-causing genes. Our results from <i>in vitro</i> experiments indicate a dose-dependent neomorphic effect of the Arg446Cys homozygous mutation in <i>VWA2</i>.</p></div

Vwa2 co-localizes with Fras1 in the nephrogenic zone of newborn mice.

<p>Immunohistochemistry on coronal sections of the nephrogenic zone of newborn (P1) mice. Figures display representative staining results obtained from kidney sections of two different animals. Experiments were performed independently and yielded similar results. Staining for Vwa2 (green) and Fras1 (red) demonstrates co-localization of the Vwa2 and Fras1 proteins in the in the renal cortex of newborn mice (ureteric bud (UB) as well as derivatives of the metanephric mesenchyme (MM), comma-shaped bodies (CS), and S-shaped bodies (SS)) as seen by the yellow-appearing structures in the “merge” panel on the very right. Vwa2 (green) hereby always co-localizes with Fras1 (red). Vwa2 expression (green) is particularly pronounced in the superior aspect of the S-shaped body, at the border between the developing distal tubule and the proximal tubule portion (indicated by arrowheads). Fras1 (red), however, appears to have a more abundant expression pattern and is partially expressed where Vwa2 is not present.</p

Reduced secretion and intracellular and extracellular aggregation of Arg446Cys VWA2.

<p><b>(a)</b> Cell culture supernatants and cell lysates from wild type (wt) and Arg446Cys VWA2 (R446C) expressing 293EBNA cells were separated by SDS-PAGE under reducing and non-reducing conditions and detected with an antibody against the One-STrEP-tag. Arrowheads indicate monomeric VWA2. On the right, equal loading is demonstrated by Ponceau staining of the membranes. Asterisks indicate artefact bands. <b>(b)</b> cDNA from non transfected (nt), non-transfected ER stress induced (nt+DTT), wt VWA2 transfected (wt) and Arg446Cys VWA2 (R446C) transfected 293EBNA cells was submitted to RT-PCR and the PCR products separated by agarose gel electrophoresis. Arrows indicate the bands for XBP-1 and ER stress induced XBP-1s. Equal loading is demonstrated by actin control RT-PCR shown below. <b>(c)</b> Equal amounts (0.2 μg) of affinity purified wild type (wt) and Arg446Cys VWA2 (R446C) were separated by SDS-PAGE under reducing and non-reducing conditions and detected with an antibody against the C-terminal fragment (P3) of human VWA2. Under non-reducing conditions higher aggregates are seen. Arrows indicate the border between separation and stacking gel. (d) Equal amounts of cell culture supernatants and cell lysates from wild type (wt) and Arg446Cys VWA2 (R446C) expressing 293EBNA cells as in (a) and of affinity purified wild type (wt) and Arg446Cys VWA2 (R446C) as in (b) were separated by agarose-polyacrylamide composite gels under non-reducing conditions and detected with an antibody against the One-STrEP-tag.</p

Mutations in genes encoding components and interactors of the basement-membrane associated Fraser-complex constitute known monogenic causes of isolated CAKUT.

<p>Mutations in genes encoding components and interactors of the basement-membrane associated Fraser-complex constitute known monogenic causes of isolated CAKUT.</p