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

FACS analysis of single putative stem cell markers in HAK cells.

<p>(a) Summarizing bar graph of single marker staining in HAK cells. Data were calculated as average % of expressing cell±SD. Each marker was tested in 3 HAK. (b) Representative dot plot graphs of CD24 and CD133 co-staining demonstrate a large fraction of CD24⁺CD133⁺ cells in HAK. Quadrates were placed according to the isotype control confiding the negative staining to the lower left quadrant. Percentage of cells for each marker combination appears in the quadrant.</p

Antibodies used in the flow cytometry assays.

<p>Antibodies used in the flow cytometry assays.</p

FACS analysis of putative stem cell markers in EpCAM subpopulations.

<p>(a) Representative zebra graph of EpCAM staining and the subpopulation gating. EpCAM subpopulations were gated according to EpCAM staining intensity (negative, dim or bright) versus FSC. (b) Representative dot plot graphs of NCAM1, PSA-NCAM, FZD7, NTRK2, CD24 and CD133 expression levels in EpCAM subpopulations of HFK. Quadrates were placed according to isotype control confiding the negative staining to the lower left quadrant. Percentage of cells in each subgroup appears on the lower right quadrant. (c) Summarizing bar graphs of NCAM1, PSA- NCAM, NTRK2, FZD7, CD24 and CD133 expression levels in EpCAM subpopulations. Data are average % of cells in each subgroup±SD. Analysis of each marker was performed at least three times.</p

Secondary Abs or S/A conjugated enzymes used in flow cytometry assays.

<p>Secondary Abs or S/A conjugated enzymes used in flow cytometry assays.</p

Immunostaining of putative stem cells markers.

<p>(a–l) Representative immunostaining of FZD2, GPR39, DLK1, CD34, CD90 and CD24 in paraffin embedded sections of HFK (12, 13 or 18 weeks of gestation); (a–b) FZD2 immunostaining demonstrates widespread staining of renal tubules. (c–d) GPR39 immunostaining demonstrates ubiquitous expression in differentiated renal tubular and to a lesser extent in componenets of the nephrogenic cortex. (e–f) Dlk1 immunostaining demonstrates ubiquitous expression in differentiated renal tubular but not in MM and its derivatives renal, UBs or stroma. (g–h) CD34 immunostaining demonstrates exclusive localization to endothelial cells (glomerular and peri-tubular) in all parts of the HFK, including in the nephrogenic cortex. (i–j) CD90 immunostaining demonstrates predominant staining in renal tubular cells but not in MM and its derivatives, UBs or stroma. (k–l) CD24 immunostaining demonstrates widespread expression in mature tubules. Figure (c) is shown in low magnification (original×4), Figures a, e, g, i, k and b, d, f, h, j, l are shown in higher magnifications (original×20 and×40, respectively).</p

Immunostaining of putative stem cell markers.

<p>(a–j) Representative immunostaining of SIX2, NCAM1, FZD7, ACVR2B and NTRK2 in paraffin embedded sections of HFK (12, 13 and 18 weeks of human gestation); (a–b) localization of SIX2 to the MM, predominantly to the CM. (c–d) predominant staining of NCAM1 in the MM (including CM) and its derivatives (S- and comma- shaped bodies) and renal stroma, but not mature tubules or UBs. (e–f) FZD7 demonstrates preferential localization to the nephrogenic zone including MM and its derivatives, UBs, and newly forming tubules but not the stroma. (g–h) ACVRIIB immunostaining demonstrates predominant expression in the nephrogenic cortex; MM and its derivatives (S and comma shaped bodies), UBs, parietal epithelium of fetal glomeruli but not in the stroma. (i–j) NTRK2 is detected in the MM (including condensates) and its derivatives, UBs and some differentiated tubules. Figures c, e and g are shown in low magnification (original×4), Figures a, b, d, f and h–j are shown in higher magnifications (original×40; I, original×20).</p

Gene expression analysis in sorted PSA-NCAM subpopulations.

<p>Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of (a) renal stem/progenitor genes (<i>Six2, Cited1, Sall1, Wt1</i> and <i>Pax2</i>), (b) vimentin and E-cadherin genes expression in PSA-NCAM magnetically separated cells from HFK (15–19 weeks of gestation). Normalization was performed against control HPRT expression and RQ calculated relative to the PSA-NCAM⁻ fraction. Data were calculated as average±SD of at least 3 independent samples. ***P<0.001, *P<0.05 versus PSA-NCAM⁻. <i>Sall1</i> expression in NCAM⁺ EpCAM⁺ cells was near significance (p<0.059).</p