Epithelial tubule interconnection driven by HGF-Met signaling in the kidney

Abstract

The formation of functional epithelial tubules is critical for the development and maintenance of many organ systems. While the mechanisms of tubule formation by epithelial cells are well studied, the process of tubule anastomosis-where tubules connect to form a continuous network-remains poorly understood. In this study, we utilized single-cell RNA sequencing to analyze embryonic mouse kidney tubules undergoing anastomosis. Our analysis identified hepatocyte growth factor (HGF) as a key potential mediator of this process. To investigate this further, we developed an assay using epithelial spheroids with fluorescently tagged apical surfaces, allowing us to visualize and quantify tubule-tubule connections. Our results demonstrate that HGF promotes tubule anastomosis, and it does so through the MAPK signaling pathway and MMPs, independently of cell proliferation. Remarkably, treatment with HGF and collagenase was sufficient to induce tubule anastomosis in embryonic mouse kidneys. These findings provide a foundational understanding of how to enhance the formation of functional tubular networks. This has significant clinical implications for the use of in vitro-grown kidney tissues in transplant medicine, potentially improving the success and integration of transplanted tissues.

Keywords: epithelial tubule; hepatocyte growth factor; tubule anastomosis.

Fig. 1.

Ligand–receptor analysis identifies tubule anastomosis-promoting candidate genes. (A) Schematic of tubule anastomosis in developing mammalian kidney. The distal RV (black) connects with tips of the ureteric tubule (green). Nephrogenic stromal cells (blue) surround the site of anastomosis. (B) UMAP indicating the distal RV, UB tips, and progenitor stromal cell populations PS1, PS2, and PS3 in E18.5 mouse kidneys. (C) Ligand–receptor (L-R) ladder plots are shown for SP2-UB and SP3-UB. For a given pair of interacting cell types, ligands are ranked in descending order by expression specificity on the left-hand side and receptors ranked in descending order of expression specificity on the right-hand side. A line segment connects the ligands to receptors. The higher the “rung of the ladder,” the more specific the ligand–receptor interaction. The top 25 L-R pairs for each analysis are indicated. HGF and its two receptors, c-MET and ST14, are highly ranked in both. See SI Appendix, Table S1 for top 100 L-R interactions between all interacting cell types.

Fig. 2.

Development of a quantitative assay of spheroid interconnection. (A) Brightfield imaging of 2-wk-old spheroids depicts nonspherical structures consistent with anastomosis. (B) Brightfield and fluorescent images of GFP-Podxl⁺ spheroids show asymmetrical protrusions (a), adjacent spheroids with a common basal surface (b), and a dumbbell-shaped structure with a single lumen (c). Images are representative of five independent experiments. (Scale bars, 50 µm.) (C) Fluorescent images from experiments in which RFP-Podxl⁺ and GFP-Podxl⁺ cells were mixed prior to spheroid formation (a) show single (b), double (c), and intercalated (d and e) fluorescently labeled lumens. (D) Schematic of the assay in which RFP-Podxl⁺ and GFP-Podxl⁺ spheroids are grown separately, harvested, condensed, and replated. (E) Fluorescent images from the assay show single or dual-labeled lumens without intercalation. High-magnification fluorescent (b, c, and e) and brightfield (d) images of a dual-fluorescent structure are shown. Images are representative of at least 10 independent experiments. [Scale bars, 200 µm (C-a and E-a), 20 µm (C-b–e), 10 µm (E-b–e)]. (F) Schematic of the stages of anastomosis. (G–J) Spheroid anastomosis was quantified by four measures: (G) structures with dual GFP⁺ and RFP⁺ fluorescence (tubular anastomosis) as the percentage of total structures (a). Structures with lumenal, spectral overlap of GFP⁺ and RFP⁺ or “yellow” lumens as the percentage of total structures (b). Images represent at least 10 independent experiments. (Scale bars, 20 µm.) (H) Frequency distribution of structure cross-sectional area over time. (I) Structure number as the percentage of baseline count. This decreases over time due to anastomoses. Spheroid size (H) and number (I) can also be represented in a single graph, as in (J).

Fig. 3.

HGF promotes extensive interconnection in replated MDCK spheroids. Replated spheroids were treated with HGF (10 ng/mL) or vehicle for 4 d. Medium was replenished every 2 d. (A) Representative brightfield and fluorescent images at the study endpoint. [Scale bars, 200 µm; 50 µm (Insets).] Measures of interconnection were scored by a blind observer. (B) Percentage of GFP⁺-RFP⁺ structures. (C) Percentage of structures with yellow lumens. (D) Structure number as a percentage of baseline. (E) Frequency distributions of cross-sectional areas of all identified structures by treatment group. (F) Structure count versus size plot after 24 h of treatment. Note reduction in the structure count and increase in structure sizes, reflecting HGF-induced interconnection. Data are representative of at least 10 independent experiments. Measures of interconnection and number were compared by Student’s t test: P < 0.0001**** and P < 0.001***.

Fig. 4.

MAPK/ERK signaling is necessary for tubular interconnection. (A) Replated MDCK spheroid cells were treated with HGF (10 ng/mL) and lysed after 0.5, 2, and 6 h. Levels of p-Stat3, total Stat3, pERK1/2, and total ERK were measured by western blotting. GAPDH was a loading control. (B and C) Replated MDCK spheroids were treated with MEK1/2 inhibitor, U0126 (10 µM), and ERK inhibitor, SCH772984 (5 µM), 30 min prior to HGF addition, and cells were lysed at 0.5, 2, and 7 h. The protein level of p-ERK1/2 was measured by western blotting with GAPDH as a loading control. (D–H) Replated spheroids were treated with HGF (10 ng/mL), U0126 (10 µM), SCH772984 (5 µM), or a combination for 2 d. (D) Representative images of replated spheroids after U0126 treatment. (Scale bars, 200 µm.) (E and G) Percentage of GFP⁺-RFP⁺ structures induced by HGF was reduced by U0126 and SCH772984. (F and H) Percentage of yellow-lumen structures induced by HGF was reduced by U0126 and SCH772984. Data are representative of at least two independent experiments performed in triplicate. Measures of interconnection (E–H) were compared by two-way ANOVA, P < 0.0001****.

Fig. 5.

Broad-spectrum inhibition of MMPs reduces lumenal interconnection. (A) Replated 3D MDCK cells were treated with HGF (10 ng/mL). Conditioned medium was collected at 6, 24, and 48 h, and extracellular MMP9 and MMP2 were analyzed by the gelatin zymography assay. (B and C) Quantification of active MMP9/pro-MMP9 activity and active MMP2/pro-MMP2. Data are expressed in arbitrary units, with the value = 1 corresponding to the activity of 6 h in untreated cells. Measures of quantification were compared by two-way ANOVA, P < 0.0001**** and P < 0.001***. (D–F) Replated spheroids were treated with HGF (10 ng/mL), ilomastat (50 nM), or a combination, for 2 d. (D) Representative images of replated spheroids after treatment. (Scale bars, 200 µm.) The percentage of GFP⁺-RFP⁺ structures (E) and yellow-lumen structures (F) from HGF induction was reduced by ilomastat treatment. Data are representative of at least two independent experiments performed in triplicate. Measures of interconnection were compared by two-way ANOVA, P < 0.0001****, P < 0.001***, and P < 0.01**.

Fig. 6.

HGF promotes extrinsic UB–UB fusion in embryonic kidney explants. Two E12.5 kidney explants (Ksp-Cre^Tg/+;Rosa-tdTomato^f/⁺) were coplated and treated ± collagenase (2.5 mg/mL) for 1 min at 37 °C and then plated ± HGF (100 ng/mL) for 72 h. Immunofluorescence of UB (red) and α-Par6b (white), which marks the tubule lumens, are shown. (A–D) Kidney explants with and without HGF (A, C) and collagenase-treated explants with and without HGF (B, D). UB–UB connection is present in D. (E) The Inset image from D shows the lumen interconnection. Arrowheads indicate ureter (U1 and U2). UB: ureteric bud. [Scale bars, 100 µm (A–D); 50 µm (E).] Similar results obtained in 4 of 5 experiments.