Developmental Origins for Kidney Disease Due to Shroom3 Deficiency

Abstract

CKD is a significant health concern with an underlying genetic component. Multiple genome-wide association studies (GWASs) strongly associated CKD with the shroom family member 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's capsule, and developing and mature podocytes in mice. Shroom3 null (Shroom3^Gt/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 (Shroom3^Gt/+) 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.

Keywords: Shroom3; chronic kidney disease; kidney development; kidney disease; podocyte.

Figure 1.

Shroom3 is expressed in the developing and mature kidney. (A–F) X-Gal staining of E13.5 and E18.5 kidneys representing Shroom3 endogenous gene expression. At E13.5 and E18.5, Shroom3 is expressed in medullary collecting duct, condensing mesenchyme adjacent to ureteric epithelium, developing nephrons, and maturing glomeruli in a pattern consistent with the podocyte cell layer. (G–I) Coimmunofluorescence for WT1 and Shroom3 at E18.5 confirms the X-Gal expression in condensing mesenchyme and developing and maturing podocyte cell layers. (J and K) X-Gal staining of a postnatal 3-month-old kidney showing Shroom3 expression in glomeruli and medullary collecting ducts. (L) At 3 months, Shroom3 expression is maintained in podocytes in an apical (arrowhead in inset) and cytoplasmic pattern (arrows in inset). cd, Collecting duct; cm, condensing mesenchyme; dn, developing nephron; g, glomerulus; mg, maturing glomerulus; p, podocyte; ub, ureteric bud; ue, ureteric epithelium.

Figure 2.

Disrupted glomerulogenesis and podocyte morphology in developing Shroom3 mutant kidneys. (A–F) Hematoxylin and eosin staining of E13.5, E14.5, and E18.5 Wild-type and Shroom3^Gt/Gt kidney sections. (D, inset) The Shroom3^Gt/Gt mutants show collapsing and degenerating glomeruli at E13.5 and cystic glomeruli, with a dilated Bowman’s capsules at E14.5 and E18.5. (G) Glomerular counting showing a dose-dependent reduction in glomerular number in Shroom3^Gt/+ and Shroom3^Gt/Gt compared with Wild-type. (H–O) Scanning electron microscopy and TEM of comparably aged glomeruli in Wild-type and Shroom3^Gt/Gt mice. (H, I, L, and M) In contrast to Wild-type, Shroom3^Gt/Gt scanning electron microscopy shows small rounded podocyte cell bodies with sporadically arranged podocytes in the glomerular tuft, marked microvillus transformation, and poorly organized foot process interdigitation. Original magnification, ×6000 in H and L; ×20,000 in I and M. (Original magnification, x5000 in J and N; x15,000 in K and O) TEM of Shroom3^Gt/Gt glomeruli reveals sporadic podocyte arrangement within the glomerulus. Original magnification, ×5000; ×15,000. (P–U) Immunofluorescence of podocyte proteins in embryonic Wild-type and Shroom3^Gt/Gt mutants. (P and S) In contrast to Wild-type, nephrin is more diffusely expressed in Shroom3^Gt/Gt mutant glomeruli. (Q, R, T, and U) Podocin and synaptopodin expression in Shroom3^Gt/Gt mutant glomeruli shows an irregular thin pattern in the podocyte layer compared with that in Wild-type. fp, Foot process; g, glomerulus; SEM, scanning electron microscopy.

Figure 3.

Shroom3 is required for apical actin distribution via Rock/MyoII signaling in podocytes. (A and A′) Immunofluorescence of actin and WT1 on E18.5 serial sections showing an apical crescent pattern of actin expression in Wild-type podocytes (arrows in inset). (B and B′) In contrast to Wild-type, there is a virtual absence of the apical actin expression in Shroom3^Gt/Gt podocytes (arrows in inset). (C) A cartoon diagram depicting actin distribution in Wild-type and Shroom3^Gt/Gt podocytes. (D) Schematic model of the actomyosin signaling pathway. (E and E′) E18.5 immunohistochemistry for Rock and WT1 on serial sections showing apical localization of Rock in Wild-type podocytes (arrow in inset). (F and F′) In Shroom3^Gt/Gt mice, there is a loss of apical Rock expression in podocytes (arrows in inset). (G and G′) p-Mlc and WT1 immunohistochemistry on E18.5 serial sections showing apical p-Mlc distribution in Wild-type podocytes (arrows in inset). (H and H′) Shroom3^Gt/Gt mutant podocytes show a loss of apical p-Mlc expression (arrows in inset). MLC, myosin light chain; p-Mlc, phosphomyosin light chain.

Figure 4.

Adult Shroom3^Gt/+ mice exhibit glomerular disease. (A–D) Histopathologic characterization of 3-month-old Wild-type and Shroom3^Gt/+ kidneys showed no overt glomerular abnormalities using periodic acid–Schiff (PAS) and Jones Methamine Silver (JMS) staining. (E–H) Histopathologic characterization of 1-year-old kidney tissue shows sclerotic glomeruli and a thickening of the Bowman’s capsule in Shroom3^Gt/+ kidneys compared with Wild-type kidneys. (I–L) Scanning electron microscopy and TEM of 1-year-old Wild-type and Shroom3^G/+ glomeruli show sporadic foot processes flattening and effacement in Shroom3^G/+ mutant kidneys (arrows). (M) Coomassie–stained SDS-PAGE of urine shows albuminuria (a 70-kD albumin band is shown as a positive control) in 1-year-old Shroom3^Gt/+ mice. (N) Western blot analysis for nephrin (a 185-kD nephrin band is shown as a positive control). (O) Analyses of the urinary protein-to-creatinine ratio from 1-year-old Wild-type and Shroom3^Gt/+ mice show a significant increase in urinary protein (n=10; P=0.03). (P) Model of the function of Shroom3 in the podocyte. FP, foot process; GBM, glomerular basement membrane; SD, slit diaphragm; SEM, scanning electron microscopy.