Ex vivo modeling of chemical synergy in prenatal kidney cystogenesis

Abstract

Cyclic adenosine monophosphate (cAMP) drives genetic polycystic kidney disease (PKD) cystogenesis. Yet within certain PKD families, striking differences in disease severity exist between affected individuals, and genomic and/or environmental modifying factors have been evoked to explain these observations. We hypothesized that PKD cystogenesis is accentuated by an aberrant fetal milieu, specifically by glucocorticoids. The extent and nature of cystogenesis was assessed in explanted wild-type mouse embryonic metanephroi, using 8-Br-cAMP as a chemical to mimic genetic PKD and the glucocorticoid dexamethasone as the environmental modulator. Cysts and glomeruli were quantified by an observer blinded to culture conditions, and tubules were phenotyped using specific markers. Dexamethasone or 8-Br-cAMP applied on their own produced cysts predominantly arising in proximal tubules and descending limbs of loops of Henle. When applied together, however, dexamethasone over a wide concentration range synergized with 8-Br-cAMP to generate a more severe, glomerulocystic, phenotype; we note that prominent glomerular cysts have been reported in autosomal dominant PKD fetal kidneys. Our data support the idea that an adverse antenatal environment exacerbates renal cystogenesis.

Figure 1. Whole mount of E13 metanephroi cultured for three days.

A. Experimental schema showing the eight different conditions studied with, for each, whole mount images of a representative organ at Day 0 and the same organ at Day 3. Bar is 500 µm. B. Higher power whole-mount views of representative organs from control (Vehicle), 470 nM dexamethasone (Dex) alone, 100 µM 8-Br-cAMP (cAMP)-only, or co-treatment with both. In this illumination, cystic structures appear as pale circles or ovals, and some of these are arrowed. Bar is 500 µm. C. Percentage of total explant area occupied by cystic structures. Each ♦ represents the value for a separate organ (n = 21 to 23 for each condition), with bars indicating group medians. Above each group is a letter; those designated by the same letter (e.g. “a”) are not significantly different from each other. In contrast, groups designated by different letters are significantly (P<0.05) different from each other (e.g. those marked “a” are different from all the other groups designated “b”, “c” and “d”).

Figure 2. Cyst numbers and organ areas on day three of culture.

A. Total numbers of cysts at day three of culture. B. Areas of organs at day three of culture expressed relative to those exposed to vehicle-only. For both A. and B., each ♦ represents the value for a separate organ (n = 21 to 23 organs for each condition), with bars indicating group medians. Groups designated by the same letter (e.g. all those designated by “a”) are not significantly different from each other. In contrast, groups designated by different letters are significantly different (P<0.05).

Figure 3. Effects of forskolin on day three of culture.

Higher power views of typical organs from vehicle (A), 470 nM dexamethasone alone (B), 10 µM forskolin-only (C), or co-treatment with both (D). Cystic structures appear as pale circles or ovals, and some of these are arrowed. Bar is 500 µm.

Figure 4. Histology of explants cultured for three days.

Representative images are shown for 3–5 organs which have been examined in each condition. All sections counterstained with hematoxylin (blue nuclei). A–D. Note lack of cysts in control (Vehicle) and 470 nM dexamethasone (Dex)-only exposed organs, with plentiful dilated tubules and cysts (some of which are indicated by asterisks) in rudiments exposed to 100 µM 8-Br-cAMP (cAMP)-only, or co-treated with both chemicals. In these frames arrows point to glomeruli; note the “glomerulocystic” phenotype in explants exposed to both 8-Br-cAMP and glucocorticoid. E–H. Tubules and cysts reactive (brown) with antibody to aquaporin-1 are indicted by arrows. I–L. Brown colour indicates tubules immunoreactive with calbinin-28 antibody. Note that cyst epithelia are negative. Calbinin-28 was prominently detected in non-dilated tubules in all four conditions. M–P. Nuclei that are brown have reacted with phospho-histone antibody. Note the prominent proliferation within the stromal compartment in organs exposed to 470 nM dexamethasone, either alone (N) or with 8-Br-cAMP (P). In P, the arrow indicates a rare labeled nucleus in cyst epithelia. Bar is 50 µm.

Figure 5. Glomerular numbers in explants.

Each point represents the number of glomeruli in a metanephros after three days of culture, as measured using a modified version of the physical fractionator technique. Bars indicate the median values of the experimental groups (n = 5 organs for each condition). Although there tended to be fewer glomeruli in the 8-Br-cAMP-exposed groups, the culture conditions had no significant effect on glomerular numbers (Kruskal-Wallis test P = 0.065). Key: Dex = 470 nM dexamethasone and cAMP = 100 µM 8-Br-cAMP.

Figure 6. Histology of day three rudiments exposed to 100 µM 8-Br-cAMP (cAMP)-only.

A and B. Adjacent sections counterstained with hematoxylin. In A, several non-dilated tubules are stained brown (arrows), having bound Dolichos biflorus agglutinin; they most likely represent collecting ducts. The centre of each frame is dominated by a tubule which has a “U-turn”. Uromodulin was immunolocalised (brown color) in the undilated limb (indicated by the arrows in B). In contrast, epithelia in the adjacent limb, which is dilated (asterisk), were unreactive with the uromodulin antibody. The simplest deduction is that the dilatation is present in the descending limb of the loop of Henle. Bar is 100 µm.

Figure 7. Whole mounts of E13 metanephroi cultured for six days.

A. A representative organ at Day 0 and the same organ at Day 6 for each condition. Bar is 500 µm. B. Enlarged images of a typical organ from control (Vehicle), 470 nM dexamethasone (Dex) alone, 100 µM 8-Br-cAMP (cAMP)-only, or co-treatment with both. Cysts appear in this dark field illumination as black circles or ovals. Bar is 500 µm. C. Percentages of total explant areas occupied by cysts. Each ♦ represents the value for a separate organ (n = 21 to 28 organs for each condition), with the bars indicating the group medians. Above each group is a letter; those designated by the same letter (e.g. “a”) are not significantly different from each other. In contrast, groups designated by different letters are significantly (P<0.05) different from each other (e.g. those marked “a” are different from all the other groups designated “b”, “c” and “d”).

Figure 8. Organ areas at day six of culture.

Areas of explants at day six expressed relative to those exposed to vehicle-only; key as for 2B.

Figure 9. Histology of explants cultured for six days.

Representative images for 3–5 organs in each condition. All sections counterstained with hematoxylin (blue nuclei). A–D. Note lack of cysts in control (Vehicle) explants and the progressively greater size (a typical cyst is boxed in each image) and extent of cysts per organs exposed to 470 nM dexamethasone (Dex)-alone, 100 µM 8-Br-cAMP (cAMP)-only, or both chemicals. E–H. Brown color indicates immunoreactivity to megalin antibody. Note patchy reactivity of cyst epithelia in organs exposed to 470 nM dexamethasone-alone, 100 µM 8-Br-cAMP-alone or both chemicals. I–L. Uromodulin immunoreactivity (brown); note that cysts are not labeled. M–P. Immunostaining with antibody to calbindin-28. Most cyst epithelia are unreactive (M, O and P) but a small subset of cysts in cAMP-exposed organs were positive and one such is depicted in O′. Q–T. These sections were probed with Dolichos biflorus lectin. Note that the lectin prominently labeled non-dilated tubules between cysts; in addition, the great majority of cysts did not bind the lectin. In (R), an asterisk indicates a rare, labeled cyst. Bar in A–D is 500 µm, and bar for other frames is 50 µm.

Figure 10. Apoptosis as assessed by TUNEL labeling.

Histology images of day six cultures probed to detect apoptotic nuclei (green) with all nuclei counterstained blue. The left hand frame (A) is an image of an organ exposed to 100 µM 8-Br-cAMP-alone, and the right hand image (B) is of an organ exposed to this chemical plus 470 nM of dexamethasone. Note apoptotic nuclei in cyst lumens together with rare apoptotic nuclei (arrow in B) in epithelia lining cysts.

Figure 11. Histology of rudiments exposed to low concentration of cystogenic chemicals.

A–D. After three days in culture, neither 47 nM dexamethasone (Dex) nor 2 µM Br-cAMP (cAMP) alone resulted in cyst formation; however, in combination, small cysts formed (asterisk in D). E–H. On day six of culture, neither 4.7 nM dexamethasone nor 1 µM Br-cAMP alone resulted in cyst formation; however, in combination, numerous cysts were noted (asterisks in H). Bar is 50 µm.

Figure 12. F4/80 immunostaining of macrophages in rudiments at day 6 of culture.

(A) Vehicle-only exposed organ. (B) Dexamethasone-only exposed organ. (C) 8-Br-cAMP-only exposed organ. (D) Organ exposed to both cystogens. Immunoreactive cells, presumed macrophages, are brown and some are indicted by arrows. Note that F4/80 macrophages were detected in all four conditions. In cystic explants, their numbers did not appear increased and, moreover, they were often located distant from cyst epithelia. Bar is 50 µm.