Expression of active B-Raf proto-oncogene in kidney collecting ducts induces cyst formation in normal mice and accelerates cyst growth in mice with polycystic kidney disease

Abstract

Polycystic kidney disease (PKD) is characterized by the formation and progressive enlargement of fluid-filled cysts due to abnormal cell proliferation. Cyclic AMP agonists, including arginine vasopressin, stimulate ERK-dependent proliferation of cystic cells, but not normal kidney cells. Previously, B-Raf proto-oncogene (BRAF), a MAPK kinase kinase that activates MEK-ERK signaling, was shown to be a central intermediate in the cAMP mitogenic response. However, the role of BRAF on cyst formation and enlargement in vivo had not been demonstrated. To determine if active BRAF induces kidney cyst formation, we generated transgenic mice that conditionally express BRAF^V600E, a common activating mutation, and bred them with Pkhd1-Cre mice to express active BRAF in the collecting ducts, a predominant site for cyst formation. Collecting duct expression of BRAFV600E (BRaf^CD) caused kidney cyst formation as early as three weeks of age. There were increased levels of phosphorylated ERK (p-ERK) and proliferating cell nuclear antigen, a marker for cell proliferation. BRaf^CD mice developed extensive kidney fibrosis and elevated blood urea nitrogen, indicating a decline in kidney function, by ten weeks of age. BRAF^V600E transgenic mice were also bred to Pkd1^RC/RC and pcy/pcy mice, well-characterized slowly progressive PKD models. Collecting duct expression of active BRAF markedly increased kidney weight/body weight, cyst number and size, and total cystic area. There were increased p-ERK levels and proliferating cells, immune cell infiltration, interstitial fibrosis, and a decline in kidney function in both these models. Thus, our findings demonstrate that active BRAF is sufficient to induce kidney cyst formation in normal mice and accelerate cystic disease in PKD mice.

Keywords: ADPKD; ERK; MAPK; cell proliferation; mitogen-activated protein kinase; polycystic kidney disease.

Figure 1.. Collecting duct (CD)-specific expression of constitutively active BRAF induces cell proliferation and cyst formation in wildtype kidneys.

(a) Representative kidney sections from 3-, 5-, and 10-week-old BRAF^LSL-V600E; Pkhd1-Cre (BRaf^CD) and phenotypic normal (BRAF^LSL-V600E, No Cre; wildtype [WT]) mice. Scale bar = 1 mm. Bar graphs are mean ± SEM for (b) number of cysts and (c) the percentage of cystic area per total kidney cross-sectional surface area (cystic index) for N = 11 mice per group. ***P < 0.001, compared to WT kidneys. Representative fluorescent images of adjacent kidney sections (5 μm) from 10-week-old BRaf^CD mice stained with (d) CD marker Dolichos biflorus agglutinin (DBA; green) or (e) Lotus tetragonolobus (LTA; green). DAPI (blue) was used to visualize nuclei. Scale bar = 100 μm. Fluorescent images of (f) WT and (g) BRaf^CD kidneys stained with an antibody to PCNA (red) and DBA (green). Scale bar = 5 μm. (h) Bar graph represents the mean ± SEM for the percentage of PCNA-positive cells in 10-week-old kidney sections (N = 6 per group) normalized to total nuclei. *P < 0.05, compared to WT kidneys.

Figure 2.. CD-specific expression of active BRAF leads to renal fibrosis and a decline in kidney function in wildtype mice.

Representative images of kidney sections from 10-week-old (a) wildtype (WT) and (b) BRaf^CD mice stained with Masson’s trichrome to visualize pathogenic collagen deposits (blue) and pre-fibrotic interstitial edema (pale blue appearance, arrows). Scale bar = 100 μm. (c) Data are mean ± SEM for fibrotic index (percentage of interstitial fibrosis and edema per total area of the section). Slides containing tissue sections were coded to conceal the group assignment before being visually scored (N = 4 kidneys per group). (d) Data are mean ± SEM for levels of blood urea nitrogen (BUN), a marker of renal function, in WT and BRaf^CD mice at 10 weeks of age (N = 4). *P < 0.05 and ***P < 0.001, compared to WT.

Figure 3.. CD expression of active BRAF accelerates renal cystic disease in Pkd1^RC/RC mice.

(a) Representative images of kidneys of Pkd1^RC/RC (RC/RC) and Pkd1^RC/RC; BRAF^LSL-V600E;Pkhd1-Cre (RC/RC; BRaf^CD) mice at 10 weeks of age. Scale bar = 5 mm. Bar graphs are mean ± SEM for (b) body weight (BW) and (c) kidney weight, as a percentage of body weight [KW/BW (%)], for Pkd1^RC/RC (RC/RC; N = 11) and Pkd1^RC/RC; BRAF^LSL-V600E; Pkhd1-Cre (RC/RC; BRaf^CD, N = 10) mice at 10 weeks of age. (d) Images of tissue sections from RC/RC and RC/RC; BRaf^CD kidneys at 5 and 10 weeks of age, demonstrating an acceleration of cystic disease. Scale bar = 1 mm. Bar graphs are mean ± SEM for (e) the number of cysts (> 50 μm diameter) per section and (f) percentage of total surface area of the section (cystic index). ***P < 0.001, compared to RC/RC.

Figure 4.. CD expression of active BRAF increases ERK phosphorylation and cell proliferation in Pkd1^RC/RC kidneys.

Representative 10-week-old (a) RC/RC and (b) RC/RC; BRaf^CD kidney sections stained with an antibody to PCNA (red) and DBA (green). There were increased PCNA-positive cells in the cyst-lining epithelia as well as the interstitium (Supplemental Figure S5). Scale bar = 500 μm. (c) Percentage of PCNA-positive nuclei, normalized to total nuclei that were visualized with DAPI (blue). A minimum of 4 × 10³ cells per kidney section were counted. P < 0.01, compared to RC/RC (N = 6). (d) Representative immunoblots for P-ERK, ERK, and BRAF in 10-week-old kidneys without (−) and with (+) CD expression of the BRAF^V600E transgene. GAPDH was used as a loading control. The number above the blots are P-ERK/ERK and BRAF/GAPDH, respectively. Bar graphs are mean ± SEM for levels of (e) P-ERK/ERK and (f) BRAF/GAPDH for RC/RC and RC/RC; BRaf^CD kidneys. *P < 0.05, compared to RC/RC (N = 3).

Figure 5.. Effect of CD expression of active BRAF on renal fibrosis and function, and the survival of Pkd1^RC/RC mice.

Tissue sections from 10-week-old (a) RC/RC and (b) RC/RC; BRaf^CD were stained with Masson’s trichrome to visualize fibrosis. Scale bar = 50 μm. (c) The percentage of fibrosis to the surface area (SA) of the entire kidney section (fibrotic index) for RC/RC and RC/RC; BRaf^CD kidneys. (d) Bar graph represents the mean ± SEM for BUN levels from RC/RC and RC/RC; BRaf^CD mice. **P < 0.01 and ***P < 0.001, compared to RC/RC. (e) Gehan-Breslow survival curves for BRaf^CD (N = 14; 8 males and 6 females), RC/RC (N = 10; 7 males and 3 females), and RC/RC; BRaf^CD (N = 10; 7 males and 3 females) mice for 60 weeks. Wildtype mice lacking transgenic BRAF expression were not included since there is 100% survival during this period. Mice were given standard chow and water ad libitum and monitored regularly until a humane end point was reached. Average survival age for BRaf^CD mice (26 ± 1 weeks) and RC/RC; BRaf^CD mice (31 ± 1 weeks) were significantly different from that of the RC/RC mice (61 ± 4 weeks, P < 0.001).

Figure 6.. CD-selective expression of active BRAF increased renal cystic disease and fibrosis and accelerated the decline in kidney function in pcy/pcy mice.

(a) Representative kidney sections from 10-week-old pcy/pcy and pcy/pcy; BRAF^LSL-V600E; Pkhd1-Cre (pcy/pcy; BRaf^CD) mice. Scale bar = 1 mm. Bar graphs represent the mean ± SEM for (b) body weight (BW) and (c) kidney weight (KW), as a percentage of BW [KW/BW (%)], (d) cyst area, relative to total surface area [cystic index (% SA)], and (e) number of cysts in kidney sections from pcy/pcy and pcy/pcy; BRaf^CD mice. **P < 0.01 and ***P < 0.001, compared to pcy/pcy mice (N = 4–6). (f) Fluorescent images of kidney sections from pcy/pcy and pcy/pcy; BRaf^CD mice that were stained with PCNA (red), DBA (green) and DAPI (blue). (g) Bar graph displays the mean ± SEM for PCNA-positive cells as a % of total nuclei. *P < 0.05, compared to pcy/pcy kidneys (N = 4) (h) Representative immunoblots for P-ERK, ERK, BRAF, and GAPDH in kidney lysates from pcy/pcy mice without (−) or with (+) CD expression of active BRAF. The numbers above the bands are P-ERK/ERK and BRAF/GAPDH, respectively. (i) The percentage of fibrosis to the surface area (SA) of the entire kidney section (fibrotic index) for pcy/pcy and pcy/pcy; BRaf^CD kidneys. ***P < 0.001, compared to pcy/pcy (N = 6). (j) BUN levels in pcy/pcy and pcy/pcy; BRaf^CD mice at 10 weeks of age **P < 0.01, compared to pcy/pcy mice (N = 4).