Additional file 6: Figure S5. of Impairment of Wnt11 function leads to kidney tubular abnormalities and secondary glomerular cystogenesis

Wnt11 deficiency influences kidney function. Creatinine clearance (A) is significantly reduced in the Wnt11 -/- mice when compared to WT, indicating reduced glomerular filtration rate (GFR). This situation corresponds to a diagnosis of mild to moderate renal failure in a human and is in line with the observed increase in blood urea nitrogen (BUN) (B) and reduced daily urine excretion (C). n = 8-10, p < 0.05. (JPG 146 kb

Additional file 5: Figure S4. of Impairment of Wnt11 function leads to kidney tubular abnormalities and secondary glomerular cystogenesis

OPT-based kidney morphometric. Wnt11 knockout (C, C´) alters the volumetric measures such as the relative volumes of the kidney and pelvis relative to WT (B, B´). (A) OPT volumetry data (n = 6-8). Drishti reconstruction of the pelvis (B, C) and highlighted regions depicted as B´ and C´ (boxed areas in B, C) revealing the sagittal/rostro-caudal tubular view. Note that the Wnt11 deficiency led to considerable changes in the overall 3D arrangement of the tubules as well as the degree of their convolution when compared to control (compare C to B and C’ to B´). Bars: B, C 800 μm; B’, C’ 300 μm. (JPG 379 kb

Additional file 7: Table S2. of Impairment of Wnt11 function leads to kidney tubular abnormalities and secondary glomerular cystogenesis

Urine biochemistry test in Wnt11 -/- and WT mice. Creatinine clearance (ml/min) was calculated from the formula UV/P ×1/1440. U; urinary concentration of the substance (mmol/l), V; urinary volume (ml), P; plasma creatinine concentration (μmol), 1440: minutes in 24 h. Creatinine clearance and urine volume were scaled to body weight. (DOCX 18 kb

Additional file 2: Figure S1. of Impairment of Wnt11 function leads to kidney tubular abnormalities and secondary glomerular cystogenesis

Formation of glomerular cysts and anomalies of papillary duct due to Wnt11 deficiency. A and D) Note discrete glomerular changes in E16.5 as the Bowman’s capsule of the glomerulus is enlarged and contains parietal podocytes (compare D to A, red arrows). B and E) Advanced cystic formation is found in NB, the glomerular tuft has an abnormal architecture when compared to the WT (arrow) and it is microcystic (star). C and F) Typical findings in all adult Wnt11 -/- are cortical glomerular microcysts that contain rudiments of the tuft (F, G stars) and hypertrophied glomeruli (compare F to C, black arrows). In the severe kidney tubular anomalies of Wnt11 deficient mice interstitial fibrosis is noted (F, empty arrow). G) High magnification of a cortical glomerular cyst (star) with dysmorphic tuft (arrow) in the kidney of Wnt11 -/- mouse. H, I) TEM shows normal structure of non-cystic glomeruli in Wnt11 -/- mice (RBC: red blood cell; US: urinary space; M: mesangial cell; GBM: glomerular basement membrane). J-M) Immunohistochemistry with AQP2 in kidney indicates that AQP2 staining is more intense in Wnt11 deficient papillary ducts compared to controls (arrows). In contrast, the cell morphology of the cortical collecting duct was normal and expression of AQP2 is not different from controls (arrows). Bars: J, L 200 μm, A, D, C-G 100 μm, B, E, K, M 50 μm, H, I 2 μm. (JPG 1128 kb

Wnt5a Deficiency Leads to Anomalies in Ureteric Tree Development, Tubular Epithelial Cell Organization and Basement Membrane Integrity Pointing to a Role in Kidney Collecting Duct Patterning

<div><p>The Wnts can be considered as candidates for the Congenital Anomaly of Kidney and Urinary Tract, CAKUT diseases since they take part in the control of kidney organogenesis. Of them <i>Wnt5a</i> is expressed in ureteric bud (UB) and its deficiency leads to duplex collecting system (13/90) uni- or bilateral kidney agenesis (10/90), hypoplasia with altered pattern of ureteric tree organization (42/90) and lobularization defects with partly fused ureter trunks (25/90) unlike in controls. The UB had also notably less tips due to <i>Wnt5a</i> deficiency being at E15.5 306 and at E16.5 765 corresponding to 428 and 1022 in control (p<0.02; p<0.03) respectively. These changes due to <i>Wnt5a</i> knock out associated with anomalies in the ultrastructure of the UB daughter epithelial cells. The basement membrane (BM) was malformed so that the BM thickness increased from 46.3 nm to 71.2 nm (p<0.01) at E16.5 in the <i>Wnt5a</i> knock out when compared to control. Expression of a panel of BM components such as <i>laminin</i> and of <i>type IV collagen</i> was also reduced due to the <i>Wnt5a</i> knock out. The <i>P4ha1</i> gene that encodes a catalytic subunit of collagen prolyl 4-hydroxylase I (C-P4H-I) in collagen synthesis expression and the overall C-P4H enzyme activity were elevated by around 26% due to impairment in <i>Wnt5a</i> function from control. The compound <i>Wnt5a</i>^+/-;<i>P4ha1</i>^+/- embryos demonstrated <i>Wnt5a</i>^-/- related defects, for example local hyperplasia in the UB tree. A R260H WNT5A variant was identified from renal human disease cohort. Functional studies of the consequence of the corresponding mouse variant in comparison to normal ligand reduced Wnt5a-signalling <i>in vitro</i>. Together Wnt5a has a novel function in kidney organogenesis by contributing to patterning of UB derived collecting duct development contributing putatively to congenital disease.</p></div

A human WNT5A R260H variant from a CAKUT cohort reduces signalling possibly via the frizzled receptor binding pocket widening as judged by simulation.

<p>The putative influence of the WNT5A R260H variant discovered in human CAKUT disease cohort was modelled by simulation by using the xWnt8 structure as the reference. A) The R260H variant in the mouse Wnt5a-GFP reduces the capacity of the Wnt5a to inhibit the Wnt3a induced <i>Top Flash</i> luciferase activity when compared to the potential of the native Wnt5a by 17% (p<0.05). B) The simulation suggests that the human R260H variant broadens the WNT5A frizzled receptor binding pocket from 3.06 nm (Wt, in red) to 3.74 nm (R260H WNT5A, in pale blue circle and dotted line). The spatial location of the residue 260 variant in the patient with CAKUT is highlighted as a green circle. CM, conditioned media. The data are shown as means ± SD, n = 7/group, * <i>p</i> < 0.05.</p

<i>Wnt5a</i> deficiency increases the collagen prolyl 4-hydroxylase I (P4ha1) expression and the compound +/- is characterized by severe kidney filter anomaly.

<p>The kidneys were prepared and subjected to <i>collagen prolyl 4-hydroxylase I (P4ha1)</i> gene expression (A), total Collagen P4H (C-P4H) enzyme activity assays (B) or electron microscopic inspection (C-J) at E16.5 or in the adult. <i>Wnt5a</i> deficiency has increased the <i>P4ha1</i> gene expression (A) and C-P4H enzyme activity (B) when compared to control. The ureteric bud derived collecting duct basement membrane (BM) remains normal at E16.5 in the <i>Wnt5a+/-</i> and <i>P4ha1+/-</i> (compare D, E with C) but the BM is malformed in the <i>P4ha1+/-; Wnt5a+/-</i> at E16.5 (compare F with C, D, E). However, the kidney filter, the glomerular BM and the foot processes are compromised in the kidney of the <i>P4ha1+/-</i> and the <i>Wnt5+/-</i> mice at the age of seven months (compare H, I with G) and this is even more severe in the kidneys derived from the <i>P4ha1+/-; Wnt5a+/-</i> compound heterozygous mice (compare J with G, H, I). GBM; glomerular BM, P, podocyte, E, endothelial cell. The data in A and B are shown as means ± SD, n = 4. Scale bars, C-J 1μm.</p

<i>Wnt5a</i> deficiency leads to severe metanephric kidney anomalies.

<p>Embryonic urogenital systems (UGS) (A-D) and their kidneys (E-L) were dissected at E15.5 and E16.5. The UGS were inspected either as unstained (A-D) or their kidneys we subjected to Troma-1 antibody staining as whole mount and analysed with the optical projection tomography (OPT) to identify the pattern of the ureteric bud (UB) tree and the terminal UB derived tree tip counts (M) or sectioned (I-L). A) A normal urogenital system. The kidney (K), the gonad (G) and the adrenal gland (A) are marked. The <i>Wnt5a</i> deficiency leads to three categories of phenotypes; B) a kidney with duplex UB highlighted in the boxed image (the stars in B´), kidney hypoplasia (compare B with A), bilateral (C) or unilateral (D) renal failure. The OPT reveals variation noted in the pattern of UB tree development in the <i>Wnt5a</i> deficient embryonic kidneys when compared to control (compare F—H with E). The altered UB tree pattern can be depicted in the sections of the <i>Wnt5a</i> deficient kidneys when compared to control (compare J with I, dotted line, arrows). <i>Wnt5a</i> deficiency enlarges also the Bowman´s capsule lumen (asterisk) from control one (compare L with K, stars). Counting of the UB terminal tips from the OPT data indicates reduction in their number due to <i>Wnt5a</i> deficiency from controls M). Data in M are shown as means ± SD, <i>n</i> = 4–5 kidneys/group. Scale bars, A-D 400 μm, E-H 200 μm and I-L 50 μm. * <i>P</i> <0.05.</p

<i>Wnt5a</i> deficiency associates to compromised basement membrane integrity and integrin <i>Itga1</i> expression.

<p>Wild-type (Wt) and <i>Wnt5a</i>^-/- kidneys at E11.5 and E16.5 were prepared and subjected to electron microscopic (EM) inspection (A-F). The noted phenotypes were quantified by morphometric (G-H). The basement membrane (BM) that forms normally an extracellular matrix (ECM) sheet between ureteric bud derived epithelial tree and adjacent mesenchyme (A, D) becomes severely compromised in the <i>Wnt5a</i> deficient embryonic kidney already at the initiation of organogenesis at E11.5 (compare B, C with A, arrows) and is prominent also at E16.5 (compare E, F with D, arrows). Please note that the BM of the <i>Wnt5a</i> deficient kidney loses it integrity and the BM is more extended when compared to control BM (compare B, C, E, F with A, D, arrows). G, H) Quantitation of the BM (A-F) in depicting that the width of the BM and the distance of the BM from the cell membrane is increased in the absence of <i>Wnt5a</i> function when compared to control. Data are presented as means ± SD. *p < 0.05, n = 4 mice/group. Scale bars, A-F 200 nm.</p