Developmental origins for kidney disease due to Shroom3 deficiency

CKD is a significant health concern with an underlying genetic component. Multiple genome-wide association studies (GWASs) strongly associated CKD with the shroomfamilymember 3 (SHROOM3) gene, which encodes an actin-associated protein important in epithelial morphogenesis. However, the role of SHROOM3 in kidney development and function is virtually unknown. Studies in zebrafish and rat showed that alterations in Shroom3 can result in glomerular dysfunction. Furthermore, human SHROOM3 variants can induce impaired kidney function in animal models. Here, we examined the temporal and spatial expression of Shroom3 in the mammalian kidney. We detected Shroom3 expression in the condensing mesenchyme, Bowman\u27s capsule, and developing and mature podocytes in mice. Shroom3 null (Shroom3Gt/Gt) mice showed marked glomerular abnormalities, including cystic and collapsing/degenerating glomeruli, and marked disruptions in podocyte arrangement and morphology. These podocyte-specific abnormalities are associated with altered Rho-kinase/myosin II signaling and loss of apically distributed actin. Additionally, Shroom3 heterozygous (Shroom3Gt/+) mice showed developmental irregularities that manifested as adult-onset glomerulosclerosis and proteinuria. Taken together, our results establish the significance of Shroom3 in mammalian kidney development and progression of kidney disease. Specifically, Shroom3 maintains normal podocyte architecture in mice via modulation of the actomyosin network, which is essential for podocyte function. Furthermore, our findings strongly support the GWASs that suggest a role for SHROOM3 in human kidney disease

Stromally Expressed β-Catenin Modulates Wnt9b Signaling in the Ureteric Epithelium

<div><p>The mammalian kidney undergoes cell interactions between the epithelium and mesenchyme to form the essential filtration unit of the kidney, termed the nephron. A third cell type, the kidney stroma, is a population of fibroblasts located in the kidney capsule, cortex and medulla and is ideally located to affect kidney formation. We found β-catenin, a transcriptional co-activator, is strongly expressed in distinctive intracellular patterns in the capsular, cortical, and medullary renal stroma. We investigated β-catenin function in the renal stroma using a conditional knockout strategy that genetically deleted β-catenin specifically in the renal stroma cell lineage (β-cat^s-/-). <i>β-cat^s-/-</i> mutant mice demonstrate marked kidney abnormalities, and surprisingly we show β-catenin in the renal stroma is essential for regulating the condensing mesenchyme cell population. We show that the population of induced mesenchyme cells is significantly reduced in <i>β-cat^s-/-</i> mutants and exhibited decreased cell proliferation and a specific loss of Cited 1, while maintaining the expression of other essential nephron progenitor proteins. <i>Wnt9b</i>, the key signal for the induction of nephron progenitors, was markedly reduced in adjacent ureteric epithelial cells in <i>β-cat^s-/-</i>. Analysis of Wnt9b-dependent genes in the neighboring nephron progenitors was significantly reduced while Wnt9b-independent genes remained unchanged. In contrast mice overexpressing β-catenin exclusively in the renal stroma demonstrated massive increases in the condensing mesenchyme population and <i>Wnt9b</i> was markedly elevated. We propose that β-catenin in the renal stroma modulates a genetic program in ureteric epithelium that is required for the induction of nephron progenitors.</p></div

Investigation of the renal stroma in <i>β-cat</i>^<i>S-/</i>- mutant kidneys.

<p>(A-L) Analysis of the renal stroma using stromal markers; Meis1/2, Pbx1, Foxd1, and TN-C at E15.5. As compared to <i>WT</i>, no overt changes were observed in the cortical stroma with respect to Meis1/2 (A, B), Pbx1 (E, F), Foxd1 (I, J), and TN-C (K, L). However, a reduction of Meis1/2 (C, D) and Pbx1 (G, H) was observed in the medullary region in <i>β-cat</i>^<i>S-/</i>- kidneys. (M, N) TUNEL assay at E15.5 reveals an increase in apoptosis in the medullary stroma of <i>β-cat</i>^<i>S-/</i>- kidneys compared to <i>WT</i>. (rc = renal capsule, cs = cortical stroma, ms = medullary stroma, A = apoptosis). Scale Bar = 50μm</p

<i>β-cat</i>^<i>S-/</i>- mutants demonstrate altered Wnt9b signaling to the condensing mesenchyme.

<p>(A-P) Analysis of Wnt9b dependent and independent gene targets by immunofluorescence and real-time quantitative PCR at E15.5 and E14.5 respectively. (A-F) In contrast to <i>WT</i> at E15.5, the number of Pax2 and Six2 positive cells in the condensing mesenchyme was reduced in <i>β-cat</i>^<i>S-/</i>- kidneys. No changes were observed in the <i>Pax2</i> and <i>Six2</i> mRNA expression levels at E14.5 by qRT-PCR. (G-I) Both the number of Cited 1 positive cells and <i>Cited 1</i> mRNA expression levels were reduced (1.00 versus 0.62, p = 0.003) in <i>β-cat</i>^<i>S-/</i>- kidneys. (J-L) The levels of Amphiphysin were significantly reduced in the condensing mesenchyme at both the protein and mRNA levels in <i>β-cat</i>^<i>S-/</i>- kidneys (1.01 versus 0.48, p = 0.025) (M-O) In situ hybridization and qRT-PCR analysis of <i>Wnt4</i> demonstrates a reduction in <i>Wnt4</i> mRNA levels in E14.5 <i>β-cat</i>^<i>S-/</i>- kidneys (1.00 versus 0.30, p = 0.0007). (P) QRT-PCR of Wnt9b-independent gene <i>Eya1</i> demonstrates no changes in mRNA expression levels in <i>β-cat</i>^<i>S-/</i>- kidneys. In contrast, Wnt9b-dependent gene <i>Tafa5</i> (1.00 versus 0.42, p = 0.003) was significantly decreased in <i>β-cat</i>^<i>S-/</i>- kidneys (scale bar = 50μm) (rv = renal vesicle, ub = ureteric bud).</p

<i>β-cat</i>^<i>S-/</i>- mutants demonstrate multiple kidney abnormalities.

<p>(A,B) Gross anatomy of PN0 <i>WT</i> and <i>β-cat</i>^<i>S-/</i>- kidneys show comparable size and shape. (C-J) In contrast to <i>WT</i>, histological analysis of <i>β-cat</i>^<i>S-/</i>- mutant kidneys demonstrate numerous kidney abnormalities. (C,D) As compared to <i>WT</i>, <i>β-cat</i>^<i>S-/</i>- mutant kidneys were lobular, lacked a distinct boarder, contained numerous cysts in the medulla and cortex (star), with an ill-defined cortical medullary axis and misplaced glomeruli (arrow). (E-J) In contrast to <i>WT</i>, high magnification of <i>β-cat</i>^<i>S-/</i>- kidneys at PN0 revealed a non-adherent sporadic renal capsule (F and J), misplaced tubules just under the renal capsule (F and J), glomeruli in the medulla (H) and a marked reduction in medullary stroma (H). (A, B scale bar = 1mm, C, D scale bar = 100μm, ad = adrenal gland, k = kidney, b = bladder, rc = renal capsule, cs = cortical stroma, ms = medullary stroma, g = glomerulus).</p

Intracellular localization of β-catenin in capsular, cortical, and medullary stroma.

<p>(A-L) Immunofluorescence showing β-catenin intracellular distribution in the capsular, cortical, and medullary stroma. (A-C) In the capsular stroma, β-catenin localizes in a membrane and cytoplasmic pattern and does not co-localize with nuclear stromal factor Pbx1. (D) Schematic diagram of the intracellular β-catenin localization and a suggested role in cell-cell adhesion via adherens junctions. (E-G) β-catenin weakly co-localizes with Pbx1 in the nuclear compartment but is primarily cytoplasmic. (H) Schematic diagram of the intracellular β-catenin localization showing possible roles in the cytoplasm and nucleus. (I-K) In the medulla, β-catenin co-localizes with Pbx1 primarily to the nuclear compartment but some cytoplasmic β-catenin expression is observed. (L) Schematic diagram of the intracellular β-catenin localization showing a more prominent role in the nucleus. (scale bar = 5μm, M = membrane, C = cytoplasm, N = nuclear, AJ = Adherens junctions).</p

Temporal analysis of the embryonic kidney phenotype in <i>β-cat</i>^<i>S-/</i>- mutants.

<p>(A, B) Histological analysis of <i>WT</i> and <i>β-cat</i>^<i>S-/</i>- embryonic kidneys at E13.5 demonstrates no abnormalities in the stromal population, developing nephrons, or kidney patterning. (C, D) In contrast to <i>WT</i> at E14.5, <i>β-cat</i>^<i>S-/</i>- kidneys demonstrate abnormally located glomeruli, and a non-adherent irregular patterned renal capsule. (E-J) In contrast to <i>WT</i> at E15.5, the non-adherent capsular phenotype persists in <i>β-cat</i>^<i>S-/</i>- kidneys (H) and the cortical stroma is reduced and loosely packed (H). Similarly, the medullary stroma in <i>β-cat</i>^<i>S-/</i>- kidneys is markedly reduced compared to <i>WT</i> (J) and glomeruli are also abnormally located within the medulla (rc = renal capsule, cs = cortical stroma, ms = medullary stroma, g = glomerulus, arrowhead = ureter). Scale Bar C-F = 100μm, G-J = 50μm</p

β-catenin in the renal stroma modulates Wnt9b expression in ureteric epithelial cells.

<p>(A-C) When compared to <i>WT</i>, In situ hybridization and real-time quantitative PCR for <i>Wnt9b</i> demonstrates <i>Wnt9b</i> mRNA expression is significantly reduced (1.00 versus 0.29, p=0.008) in E14.5 <i>β-cat^S-/-</i> kidneys. (D-I) In situ hybridization and Real-time quantitative PCR for <i>Ret</i> and <i>Wnt11</i> demonstrated no differences in mRNA expression between <i>WT</i> and <i>β-cat^S-/-</i> at E14.5. (J-K) Histological analysis of <i>β-cat^GOF-S</i> mutant kidneys demonstrate a marked increase in condensing mesenchyme population when compared to <i>WT</i> at E14.5. (L-N) In situ hybridization and quantitative PCR demonstrate <i>Wnt9b</i> expression in <i>β-cat^GOF-S</i> kidneys was significantly increased (1.02 versus 3.102, p=0.021) as compared to <i>WT</i> at E14.5. (scale bar = 50 μm, rc-renal capsule, cm= condensing mesenchyme, ub = ureteric epithelium).</p

β-catenin is expressed in distinctive patterns in the renal stroma.

<p>(A-I) Immunofluorescence demonstrating β-catenin spatial and temporal expression in stromal cells. (A) At E11.5 Pbx1 is expressed in the nucleus of stromal cells (arrow head-inset) surrounding the condensing mesenchyme. Some Pbx1 positive stromal cells locate within the condensing mesenchyme, directly adjacent to epithelial cells (arrows). (B,C) At E11.5 β-catenin is expressed in the condensing mesenchyme and ureteric epithelium, and co-localizes with Pbx1 demonstrating expression in the renal stroma. At E11.5, some Pbx1 cells co-localize with β-catenin in the nuclear compartment of stromal cells (arrowhead-inset). (D) At E13.5, Pbx1 is expressed in capsular and cortical stroma surrounding the condensing mesenchyme. (E, F) At E13.5, β-catenin co-localizes in the cytoplasm of capsular stromal cells. The stromal cells located between developing nephrons express β-catenin in the cytoplasmic (arrow-inset) and nuclear compartment (arrowhead-inset). (G) At E17.5, Pbx1 marks the capsular, cortical, and medullary stroma. (H-I) β-catenin is expressed in the medullary stroma and co-localizes strongly with Pbx1 in the nuclear compartment (arrowhead-inset). (scale bar = 100μm, s = stroma, cm = condensing mesenchyme, ub = ureteric epithelium, rc = renal capsule, ms = medullary stroma, rp = renal pelvis).</p

The condensing mesenchyme cell population is reduced in <i>β-cat</i>^<i>S-/</i>- mutant kidneys.

<p>(A,B) As compared to <i>WT</i>, which demonstrates 3–4 cell layers of aggregated condensing mesenchyme, <i>β-cat</i>^<i>S-/</i>- kidneys display a reduced, single cell layer of loosely aggregated condensing mesenchyme. (C-H) Analysis of cell proliferation in the condensing mesenchyme was performed using Brdu cell proliferation assay. (C-E) As compared to <i>WT</i>, <i>β-cat</i>^<i>S-/</i>- mutants demonstrated a 6.46% reduction in condensing mesenchyme cell proliferation at E14.5 (<i>WT</i>, 34.56%±1.45, n = 28 versus <i>β-cat</i>^<i>S-/</i>-, 28.02%±1.05, n = 27, p = 0.0006). (F-G) At E15.5 <i>β-cat</i>^<i>S-/</i>- mutants demonstrated a 7.35% reduction in condensing mesenchyme cell proliferation when compared to <i>WT</i> (<i>WT</i>, 34.09%±1.65, n = 17 versus <i>β-cat</i>^<i>S-/</i>-, 26.73%±2.21, n = 15, p = 0.01). (I,J) A TUNEL assay at E15.5 did not reveal any changes in apoptosis in the condensing mesenchyme between <i>WT</i> and <i>β-cat</i>^<i>S-/</i>-. Scale Bar = 50μm</p