Virtual-tissue computer simulations define the roles of cell adhesion and proliferation in the onset of kidney cystic disease

Abstract

In autosomal dominant polycystic kidney disease (ADPKD), cysts accumulate and progressively impair renal function. Mutations in PKD1 and PKD2 genes are causally linked to ADPKD, but how these mutations drive cell behaviors that underlie ADPKD pathogenesis is unknown. Human ADPKD cysts frequently express cadherin-8 (cad8), and expression of cad8 ectopically in vitro suffices to initiate cystogenesis. To explore cell behavioral mechanisms of cad8-driven cyst initiation, we developed a virtual-tissue computer model. Our simulations predicted that either reduced cell-cell adhesion or reduced contact inhibition of proliferation triggers cyst induction. To reproduce the full range of cyst morphologies observed in vivo, changes in both cell adhesion and proliferation are required. However, only loss-of-adhesion simulations produced morphologies matching in vitro cad8-induced cysts. Conversely, the saccular cysts described by others arise predominantly by decreased contact inhibition, that is, increased proliferation. In vitro experiments confirmed that cell-cell adhesion was reduced and proliferation was increased by ectopic cad8 expression. We conclude that adhesion loss due to cadherin type switching in ADPKD suffices to drive cystogenesis. Thus, control of cadherin type switching provides a new target for therapeutic intervention.

FIGURE 1:

Renal tubule segment and renal epithelial cell models. (A–C) Epithelial cells have four compartments representing basal (dark red), apical (green), lateral (red), and cytoplasmic (yellow) regions. Transparent black represents ECM. Light blue represents lumen in C. (A) 3D renal tubule segment model. Dashed white arrows represent transport across the epithelial layer, and solid blue arrows represent fluid flow through the tubule lumen. Lateral (red) compartments and lumenal fluid are not shown in the 3D tubule rendering. (B) Without external cell–substrate or cell–cell binding cues, surface compartments representing adhesion molecules randomly distribute near the cell surface. (C) When renal epithelial cells contact other cells, lumen, or ECM, the cell surface domains arrange themselves according to their adhesive affinities, creating ordered apical, basal, and lateral domains.

FIGURE 2:

Simulated cyst formation from single isolated cells. (A–D) Cyst formation simulations starting from a single isolated cell. (A) Snapshots of reference cyst formation corresponding to 12 (1), 19.1 (2), 47.6 (3), and 142.9 (4) h. (B–D) Snapshots taken corresponding to 235.8 h, producing a (B) small-cyst phenotype, (C) large-cyst phenotype, and (D) complex multiple-cyst phenotype. Supplemental Table S2 gives a complete list of simulation parameters. Supplemental Table S4 gives a list of parameter values that produce each morphology. Lumen shown in blue, basal compartment shown in dark red; all other cell compartments were made transparent to enhance the visibility of basal compartment and lumen.

FIGURE 3:

Simulations predict that cyst initiation after knock-in of cad8 is due to decreased cell–cell adhesion. (A, B) Apical compartment of WT (green), apical compartment of simulated cad8-positive TC (blue), and basal surface cells (red); other cell compartments invisible for visualization purposes. (A) Starting from a stable 3D tubular structure (1) we simulated cad8- induced cyst formation by changing the properties of a single TC (single cell with blue apical compartment; 2). Decreased adhesion between WT and simulated cad8-positive TC leads to cell protrusion into the simulated ECM (3), decreased contact inhibition, increased proliferation, and formation of an ectopic cyst from TCs only (4). (B) 3D view of a simulation run using identical parameters as in A but producing two cysts due to stochastic nature of the simulations. (C) In in vitro cultures of cysts from tubules, HK-2^+cad8 cells are predominantly found in the forming cysts. Phase contrast image (top) and immunofluorescence images of cysts (bottom, arrows) stained for cad8 (magenta) and nuclei (white).

FIGURE 4:

Cadherin-8 expression reduces cell–cell adhesion in HK-2 cells. Using hanging-drop assays, we compared cell–cell adhesion between HK-2 and HK-2^+cad8 cells. Equal numbers of cells were plated in each group. Cell clusters were counted, and their diameter and circularity were quantified using FIJI ImageJ. Single cells were excluded from cluster analysis. HK-2^+cad8 cells formed 1/10 as many clusters (HK-2, 10,758 clusters, vs. HK-2^+cad8, 1050 clusters). HK-2^+cad8 cell clusters that formed were smaller and less compact than those formed by HK-2 cells (top). Insets, representative phase contrast images of hanging-drop experiments from HK-2 WT and HK-2^+cad8 cells. Mean cluster diameter (bottom left) and circularity (bottom right) were both significantly decreased in HK-2^+cad8 cells (p < 0.001).

FIGURE 5:

Changes in adhesion and proliferation produce distinct ranges of cyst morphology. Two-dimensional transverse slice views of 3D simulations. (A) Reduced WT-TC adhesion results in loss of contact inhibition and increased proliferation of TCs to form a cyst that grows out of the WT tubule and connects to the tubule via a narrow neck where TC and WT cells touch. (B) Increased proliferation due to decreased contact inhibition (increased α_c) in TCs while holding WT-TC adhesion constant results in lateral spread of TCs within the surface of the tubule, followed by formation of cysts that remain spread across and closely apposed to the surface of the tubule.

FIGURE 6:

Cyst morphology in ADPKD human nephrons, HK-2^+cad8 in vitro cysts, and virtual- tissue simulations. From images of human nephrons (Baert and Steg, 1977), images of HK-2^+cad8 in vitro cyst cultures, and simulations, we quantified cyst morphology as the aspect ratio of cyst height to cyst neck diameter (Supplemental Movie S8). (A) Cysts in human nephrons formed two groups: 1) saccular dilations (red bars, inset 3) and 2) simple cysts (blue bars, insets 1 and 2). (B) In an in vitro cystogenesis culture system, HK-2^+cad8 cells produced simple cyst morphology only (blue bars). Inset, an HK-2^+cad8 cyst (red, actin; blue, nuclei). (A, B) One-way analysis of variance analysis shows a significant difference between morphology of saccular dilations (red bars in A) and simple cysts (blue bars in B) and between saccular dilations and HK-2^+cad8 in vitro cysts (blue bars in B; both p < 0.01). No significant difference in morphology exists between simple cysts (blue bars in A) and HK-2^+cad8 in vitro cysts (blue bars in B). (C) Decreased TC-WT adhesion (blue) primarily produces cysts that grow away from the tubule, as in HK-2^+cad8 in vitro cysts and human nephron simple cysts. Decreased TC-WT and TC-TC adhesion produces cysts that grow away from the tubule (green), overlapping the low end of the decreased TC-WT adhesion range. Decreased lateral adhesion compartment size (orange) produced cyst morphology approaching that produced by decreased contact inhibition. Decreased contact inhibition (red) produced cysts resembling saccular dilations. Dotted lines, mean of simulations of each type that produced cysts (Supplemental Figure S10). Shaded areas, 1 SD. (D) Three-dimensional isosurface representation of cyst initiation morphology parameter space. Axes are ∝_c, J(TC, WT), and J(TC, TC). Color indicates morphology metric of cyst height over cyst neck diameter. Color scale normalized to maximum morphology metric. Black regions reflect baseline parameters where cyst formation does not occur. Yellow-white regions produced stalk-type cysts.