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. 2023 Jun 7:10:20543581231165716.
doi: 10.1177/20543581231165716. eCollection 2023.

Minimal Kidney Disease Phenotype in Shroom3 Heterozygous Null Mice

Allison Lawlor  1 , Kristina Cunanan  1 , Joanna Cunanan  2 , Amy Paul  1 , Hadiseh Khalili  2 , Doyun Ko  3 , Ahsan Khan  1 , Robert Gros  3 , Thomas Drysdale  3 , Darren Bridgewater  1
Affiliations

Minimal Kidney Disease Phenotype in Shroom3 Heterozygous Null Mice

Allison Lawlor et al. Can J Kidney Health Dis. .

Abstract
in English, French

Background: Shroom family member 3 (SHROOM3) encodes an actin-associated protein that regulates epithelial morphology during development. Several genome-wide association studies (GWAS) have identified genetic variances primarily in the 5' region of SHROOM3, associated with chronic kidney disease (CKD) and poor transplant outcomes. These genetic variants are associated with alterations in Shroom3 expression.

Objective: Characterize the phenotypic abnormalities associated with reduced Shroom3 expression in postnatal day 3-, 1-month and 3-month-old mice.

Methods: The Shroom3 protein expression pattern was determined by immunofluorescence. We generated Shroom3 heterozygous null mice (Shroom3Gt/+) and performed comparative analyses with wild type littermates based on somatic and kidney growth, gross renal anatomy, renal histology, renal function at postnatal day 3, 1 month, and 3 months.

Results: The Shroom3 protein expression localized to the apical regions of medullary and cortical tubular epithelium in postnatal wild type kidneys. Co-immunofluorescence studies confirmed protein expression localized to the apical side of the tubular epithelium in proximal convoluted tubules, distal convoluted tubules, and collecting ducts. While Shroom3 heterozygous null mice exhibited reduced Shroom3 protein expression, no differences in somatic and kidney growth were observed when compared to wild type mice. Although, rare cases of unilateral hypoplasia of the right kidney were observed at postnatal 1 month in Shroom3 heterozygotes. Yet renal histological analysis did not reveal any overt abnormalities in overall kidney structure or in glomerular and tubular organization in Shroom3 heterozygous null mice when compared to wild type mice. Analysis of the apical-basolateral orientation of the tubule epithelium demonstrated alterations in the proximal convoluted tubules and modest disorganization in the distal convoluted tubules at 3 months in Shroom3 heterozygotes. Additionally, these modest abnormalities were not accompanied by tubular injury or physiological defects in renal and cardiovascular function.

Conclusion: Taken together, our results describe a mild kidney disease phenotype in adult Shroom3 heterozygous null mice, suggesting that Shroom3 expression and function may be required for proper structure and maintenance of the various tubular epithelial parenchyma of the kidney.

Contexte: Le gène SHROOM3 (membre 3 de la famille Shroom) code pour une protéine associée à l’actine qui régule la morphologie épithéliale pendant le développement. Plusieurs études d’association à l’échelle du génome (GWAS — Genome-wide association studies) ont identifié des variations génétiques, principalement dans la région 5’ du gène SHROOM3, associées à l’insuffisance rénale chronique (IRC) et à de mauvais résultats de transplantation. Ces variations génétiques sont associées à des altérations dans l’expression de (Shroom3).

Objectif: Caractériser les anomalies phénotypiques associées à une diminution de l’expression de Shroom3 chez des souris à l’âge postnatal de 3 jours, 1 mois et 3 mois.

Méthodologie: Le profil d’expression des protéines Shroom3 a été déterminé par immunofluorescence. Nous avons généré des souris hétérozygotes Shroom3 (Shroom3Gt/+) et procédé à des analyses comparatives avec des congénères de type sauvage en ce qui concerne la croissance somatique et rénale, l’anatomie rénale, l’histologie rénale et la fonction rénale à l’âge postnatal de 3 jours, 1 mois et 3 mois.

Résultats: L’expression de la protéine Shroom3 est localisée dans les régions apicales de l’épithélium tubulaire médullaire et cortical des reins des souris de type sauvage après la naissance. Des études de co-immunofluorescence ont confirmé l’expression des protéines localisée sur le côté apical de l’épithélium tubulaire dans les tubules contournés proximaux, les tubules contournés distaux et les tubes collecteurs. Les souris hétérozygotes Shroom3 ont présenté une expression réduite de la protéine Shroom3, mais aucune différence dans la croissance somatique et rénale n’a été observée par rapport aux souris de type sauvage. Cependant, de rares cas d’hypoplasie unilatérale du rein droit ont été observés à l’âge postnatal de 1 mois chez les souris hétérozygotes Shroom3. L’analyze histologique rénale n’a révélé aucune anomalie manifeste dans la structure globale des reins ou dans l’organization des glomérules et des tubules chez les souris hétérozygotes Shroom3 par rapport aux souris de type sauvage. L’analyze de l’orientation apicale-basolatérale de l’épithélium tubulaire a montré des altérations dans les tubules contournés proximaux et une légère désorganisation dans les tubules contournés distaux à l’âge de 3 mois chez les souris hétérozygotes Shroom3. En outre, ces légères anomalies n’étaient pas accompagnées d’une lésion tubulaire ou d’anomalies physiologiques dans la fonction rénale et cardiovasculaire.

Conclusion: Pris dans leur ensemble, nos résultats décrivent un phénotype d’insuffisance rénale légère chez les souris hétérozygotes Shroom3 adultes, ce qui suggère que l’expression et la fonction de la protéine Shroom3 peuvent être nécessaires pour la structure et le maintien appropriés des différents parenchymes épithéliaux tubulaires du rein.

Keywords: Shroom3; apical-basolateral cell orientation; chronic kidney disease (CKD); kidney function; renal tubular epithelium.

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Conflict of interest statement

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Shroom3 is expressed in the kidney tubular epithelium. (A and B) Immunofluorescence staining of adult 3-month-old wild type kidneys represent Shroom3 protein expression in the proximal convoluted tubules, distal convoluted tubules, and collecting ducts. Shroom3 expression levels is observed in the cortical tubules and medulla. (C) Immunofluorescence staining of adult 3-month-old wild type kidneys demonstrate no specific background staining of the anti-rabbit secondary antibody in the absence of the Shroom3 primary antibody. (D-L) Co-immunofluorescence staining of Shroom3 with known markers of the proximal convoluted tubules (SGLT2), distal convoluted tubules (SLC12A3), and collecting ducts (AQP2) confirm expression in these tubules. (J-L) Shroom3 is apically expressed in all the tubular epithelium analyzed. Scale bars = 50 µm (A-C). Scale bars = 25 µm (D-L).
Figure 2.
Figure 2.
Heterozygous Shroom3 mutant kidney expression pattern. (A and B) Immunofluorescence staining in wild type and Shroom3Gt/+mice demonstrates apical Shroom3 expression. Although reduced Shroom3 expression levels are observed in Shroom3Gt/+mice expression remains apically localized. (C) Immunofluorescence staining of adult 3-month-old wild type kidneys demonstrates no specific background staining of the anti-rabbit secondary antibody in the absence of the Shroom3 primary antibody. (D-F) Total body weight at postnatal day 3, 1 month, and 3 months revealed no statistically significant differences indicating no abnormalities in somatic growth in Shroom3Gt/+mice. (G-I) Kidney resection of wild type and Shroom3Gt/+mice at postnatal day 3, 1 month, and 3 months. The kidney gross anatomy is similar in wild type and Shroom3Gt/+mice. (J) Although, there were rare occurrences of Shroom3Gt/+mice showing hypoplastic unilateral kidneys predominantly observed in the right kidney of heterozygous mice at 1 month. (H and I) Combined kidney weight analysis at postnatal 1 month and 3 months show no statistically significant differences. Scale bars = 10µm (A-B). Scale bars = 25µm (C).
Figure 3.
Figure 3.
Shroom3Gt/+mice do not exhibit overt histological abnormalities. (A-C) Hematoxylin and eosin staining of postnatal day 3, 1 month, and 3 months wild type and Shroom3Gt/+whole kidney, glomerulus, cortical tubule, and medullary tubule sections, revealed no overt significant differences. Scale bars = 200µm (H&E: A-C low power), Scale bars = 25µm (A-C Glomeruli, Cortical tubule, Medullary tubules: high power).
Figure 4.
Figure 4.
Shroom3Gt/+mice exhibit modest alterations in apical-basolateral orientation in 3-month-old mice. (A-C) Immunofluorescence of postnatal day 3, 1 month, and 3 months wild type and Shroom3Gt/+mice in the proximal convoluted tubules (SGLT2), distal convoluted tubules (SLC12A3), and collecting ducts (AQP2). (A) At postnatal day 3, SGLT2 and AQP2 show diffuse cytoplasmic expression. SLC12A3 shows strong apical localization in both wild type and Shroom3Gt/+mice. (B) At postnatal 1 month, all markers show strong apical localization in both wild type and Shroom3Gt/+mice. (C) At postnatal 3 months, all markers show strong apical localization in wild type mice. In Shroom3Gt/+ mice, SGLT2 localization is more consistent with cytoplasmic staining while SLC12A3 and AQP2 expression pattern is similar between wild type and Shroom3Gt/+mice. (D) Immunofluorescence staining of adult 3-month-old wild type kidneys demonstrate no specific background staining of the anti-rabbit secondary antibody in the absence of SLC12A3 as well as the anti-mouse secondary antibody in the absence of SGLT2 and AQP2. Scale bars = 25µm (A-D).
Figure 5.
Figure 5.
Shroom3Gt/+mice do not exhibit proximal tubular and glomerular injury. (A) Immunohistochemistry for KIM-1 at postnatal day 3, 1 month and 3 months in wild type mice demonstrating no significant KIM-1 protein expression. (B) Immunohistochemistry for KIM-1 at postnatal day 3, 1 month and 3 months in Shroom3Gt/+mice demonstrating no significant KIM-1 protein expression. (C) KIM-1 positive control for analysis. (D) Immunofluorescence staining of CD44 at postnatal 3 months in wild type and Shroom3Gt/+ mice demonstrate no significant differences in expression levels. Shroom3Gt/+ mice exhibit a thicker parietal epithelium than wild type mice. (E) Immunofluorescence staining of adult 3-month-old wild type kidneys demonstrate no specific background staining of the anti-mouse secondary antibody in the absence of the CD44 primary antibody. Scale bars = 50µm (A-E).
Figure 6.
Figure 6.
Shroom3Gt/+mice do not show renal or cardiovascular physiological abnormalities at postnatal 3 months. (A) Urine protein-creatinine ratio analysis in wild type and Shroom3Gt/+mice reveal slight increase in Shroom3Gt/+mice, however there is no statistically significant difference (P > .05). (B) Analysis of serum creatinine levels in wild type and Shroom3Gt/+mice reveal no statistically significant difference (P > .05). (C) Heart rate measurements are unchanged in wild type and Shroom3Gt/+mice. (D) Mean arterial blood pressure is unchanged in wild type and Shroom3Gt/+mice.
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