Inactivation of Tsc2 in Mesoderm-Derived Cells Causes Polycystic Kidney Lesions and Impairs Lung Alveolarization

Abstract

The tuberous sclerosis complex (TSC) proteins are critical negative regulators of the mammalian/mechanistic target of rapamycin complex 1 pathway. Germline mutations of TSC1 or TSC2 cause TSC, affecting multiple organs, including the kidney and lung, and causing substantial morbidity and mortality. The mechanisms of organ-specific disease in TSC remain incompletely understood, and the impact of TSC inactivation on mesenchymal lineage cells has not been specifically studied. We deleted Tsc2 specifically in mesoderm-derived mesenchymal cells of multiple organs in mice using the Dermo1-Cre driver. The Dermo1-Cre-driven Tsc2 conditional knockout mice had body growth retardation and died approximately 3 weeks after birth. Significant phenotypes were observed in the postnatal kidney and lung. Inactivation of Tsc2 in kidney mesenchyme caused polycystic lesions starting from the second week of age, with increased cell proliferation, tubular epithelial hyperplasia, and epithelial-mesenchymal transition. In contrast, Tsc2 deletion in lung mesenchyme led to decreased cell proliferation, reduced postnatal alveolarization, and decreased differentiation with reduced numbers of alveolar myofibroblast and type II alveolar epithelial cells. Two major findings thus result from this model: inactivation of Tsc2 in mesoderm-derived cells causes increased cell proliferation in the kidneys but reduced proliferation in the lungs, and inactivation of Tsc2 in mesoderm-derived cells causes epithelial-lined renal cysts. Therefore, Tsc2-mTOR signaling in mesenchyme is essential for the maintenance of renal structure and for lung alveolarization.

Figure 1

Deletion of Tsc2 in mesoderm-derived cells resulted in reduced body growth and lethality at early life. A: LacZ staining (blue) of embryonic day 14.5 Dermo1-Cre/Gt(ROSA)26Sor^tm1Sor reporter mice to visualize the pattern of Cre expression. B: Comparison of body sizes between Tsc2^fx/fx/Dermo1-Cre [Tsc2 conditional knockout (CKO)] mice and their wild-type (WT) littermate controls. C: Comparison of whole body weights between Tsc2 CKO and their littermate controls at different ages. D: Altered survival was detected in Tsc2 CKO mice compared to the WT controls. n = 4 per genotype (C); n = 13 (4 females and 9 males; D). ^∗P < 0.05. P, postnatal day.

Figure 2

Histological comparison of brain (A), liver (B), and heart (C) between Tsc2^fx/fx/Dermo1-Cre female mice and wild-type (WT) female control mice at postnatal day 14 is shown by hematoxylin and eosin–stained tissue sections. Scale bars: 100 μm (A and B); 1 mm (C). CKO, conditional knockout.

Figure 3

Dermo1-Cre driven Tsc2 deletion in mouse kidney. A:Dermo1-Cre targeted cells in the kidney were characterized in mT-mG fluorescence protein reporter mice at postnatal day 14 (P14), in which membrane green fluorescent protein (GFP) expression (green) replaced membrane Tomato expression (red) only in cells in which Cre-mediated loxP DNA recombination occurred. B: Dermo1-Cre–mediated loxP DNA recombination in mT-mG reporter mice (green) was not detected in P14 kidney epithelial cells (Cdh1⁺) or in endothelial cells [platelet endothelial cell adhesion molecule 1 (PECAM1)⁺]. C: Alterations of Tsc2 protein and phosphorylation of S6, a downstream target of mTOR pathway, were detected in P7 Tsc2 conditional knockout (CKO) kidneys by Western blot. D: Gross view of kidneys isolated from Tsc2 CKO mice and wild-type (WT) control mice at P25. Scale bars: 50 μm (A and B); 1 mm (D).

Figure 4

Renal pathology in the Tsc2 conditional knockout (CKO) mice. A: Comparison of overall kidney anatomical structures between the Tsc2 CKO mice and their littermate controls at different postnatal ages. B: Hematoxylin and eosin–stained kidney tissue sections at high magnification. Black dotted line highlights a hyperplastic tubule. C: Kidney cortex histology shown by periodic acid-Schiff staining. Arrow indicates multiple layers of hyperplastic tubular epithelia lining a cystic structure; arrowhead, a single layer of epithelium lining a cystic structure. Scale bars: 1 mm (A); 50 μm (B); 100 μm (C). P, postnatal day.

Figure 5

Cell proliferation and cystic epithelial cells in Tsc2 conditional knockout (CKO) kidney. A and B: Proliferative cells were detected by 3-hour 5′-ethynyl-2′-deoxyuridine (EdU) labeling, and the percentage of EdU-positive nuclei (green) was analyzed based on DAPI-nuclear counterstaining (blue). C: Endothelial cells in the renal cortex were detected by platelet endothelial cell adhesion molecule 1 (PECAM1) immunostaining. Different tubular epithelial cells were also labeled with Lotus tetragonobolus lectin (LTL; green) or Dolichos biflorus agglutinin (DBA; red)-specific binding. DBA-positive cells in the cystic epithelia are indicated with an arrow. D: Coimmunostaining of Cdh1 (green), vimentin (red), and DBA (gray) for postnatal day 21 (P21) kidneys with indicated Tsc2 genotypes. Vimentin-positive cells in tubules are indicated with arrowheads. E: Increased vimentin in total kidney tissue lysate at P21 was detected by Western blot. β-Actin is a loading control. F: Activated caspase 3 immunostaining for apoptotic cells (brown; arrows) in P21 kidneys. G: The numbers of apoptotic cells per 100 mm² area of kidney tissue sections are compared. n = 3 in each genotype (B and G). ^∗P < 0.05. Scale bars = 50 μm (A, C, D, and F). WT, wild type.

Figure 6

Lung mesenchyme-specific knockout of Tsc2 inhibited postnatal alveolarization. A and B: Lung cells targeted by the Dermo1-Cre driver at postnatal day 7 (P7) were analyzed in mT-mG fluorescence reporter mice. C and D: Altered Tsc2 protein expression and mTOR activation in P7 Tsc2 conditional knockout (CKO) lungs were analyzed by Western blot and immunostaining. E: Lung histological changes were evaluated by comparing hematoxylin and eosin–stained lung tissue structures between Tsc2 CKO and wild-type (WT) control at different postnatal ages. F: Alveolar sizes were quantitatively analyzed by measuring mean linear intercept. n = 4 per genotype (F). ^∗P < 0.05. Scale bars: 50 μm (A–D); 200 μm (E). a, airway; GFP, green fluorescent protein; PECAM1, platelet endothelial cell adhesion molecule 1; v, vasculature.

Figure 7

Altered lung proliferation and differentiation in Tsc2 conditional knockout (CKO) mice. A and B: Proliferative cells were detected by 3-hour 5′-ethynyl-2′-deoxyuridine (EdU) labeling (green) in postnatal day 7 (P7) mice, and quantified based on cell nuclear counterstaining with DAPI (blue). C: Lung myofibroblasts/smooth muscle cells, endothelial cells, type II and type I alveolar epithelial cells, airway club cells, and ciliated cells were immunostained with the related protein markers. D: Comparison of elastin fibers (black) in lung tissues between Tsc2 CKO and wild-type (WT) control mice at P7 and P14. E: Semiquantification of elastin deposition in P14 lung tissues, measured by the percentage of stained elastin area over total lung tissue area of four tissue sections. ^∗P < 0.05. Scale bars = 50 μm (A–D). a, airway; PECAM1, platelet endothelial cell adhesion molecule 1; SMA, α-smooth muscle actin; v, vasculature.