Cyst formation and activation of the extracellular regulated kinase pathway after kidney specific inactivation of Pkd1

Abstract

Polycystic kidney disease (ADPKD) results from failure of the kidney to properly maintain three-dimensional structure after loss of either polycystin-1 or -2. Mice with kidney selective inactivation of Pkd1 during embryogenesis develop profound renal cystic disease and die from renal failure within 3 weeks of birth. In this model, cysts form exclusively from cells in which Cre recombinase is active, but the apparent pace of cyst expansion varies by segment and cell type. Intercalated cells do not participate in cyst expansion despite the presence of cilia up to at least postnatal day 21. Cystic segments show a persistent increase in proliferation as determined by bromodeoxyuridine (BrdU) incorporation; however, the absolute proliferative index is dependent on the underlying proliferative potential of kidney tubule cells. Components of the extracellular regulated kinase (MAPK/ERK) pathway from Ras through MEK1/2 and ERK1/2 to the effector P90(RSK) are activated in both perinatal Pkd1 and adult Pkd2 ortholgous gene disease models. The pattern of MAPK/ERK activation is focal and does not correlate with the pattern of active proliferation identified by BrdU uptake. The possibility of a causal relationship between ERK1/2 activation and cyst cell proliferation was assessed in vivo in the acute perinatal Pkd1 model of ADPKD using MEK1/2 inhibitor U0126. U0126 treatment had no effect on progression of cyst formation in this model at doses sufficient to reduce phospho-ERK1/2 in cystic kidneys. Cysts in ADPKD exhibit both increased proliferation and activation of MAPK/ERK, but cyst growth is not prevented by inhibition of ERK1/2 activation.

Figure 1.

Rapid progression of polycystic kidney disease after kidney selective inactivation of Pkd1 in Pkd1^flox/−:Ksp-Cre mice. (A) Genomic Southern digested with HindIII and hybridized with probe 1 (Supplementary Material, Fig. S1) showing germ line transmission of the targeted allele indicated by the 8.5 kb fragment. (B) Genomic Southern showing the 2.9 kb NheI fragment in all tissues resulting from deletion of the neo cassette by FLP deleter and the novel kidney-specific 1.9 kb NheI band (arrow) resulting from deletion of exons 2–4 by Ksp-Cre. (C) Gross appearance of a cystic kidney from a P14 Pkd1^flox/−:Ksp-Cre compared with control. Scale bar, 2 mm. Kidneys from P1 (D, G), P7 (E, H) and P14 (F, I) Pkd1^flox/flox:Ksp-Cre mice with periodic acid-Schiff (PAS) stain. (D–F) Low power view at the same magnification showing rapid evolution of cystic disease. (G, H) Higher power images showing normal appearing glomeruli (g) and proximal tubules (p) with brush borders stained by PAS at P1 and P7. Cysts (cy) form from PAS negative tubules. (I) Glomeruli and PAS positive proximal tubules are still present at P14 but are now distorted by expansive cyst formation. Scale bar: (D–F), 2 mm; (G–I) 50 µm. (J) Progressive kidney enlargement (increasing kidney weight to body weight ratio) and declining kidney function [rising blood urea nitrogen (BUN)] during 14 postnatal days. Data are shown as mean ± SEM; n = 10 for each time point; *P < 0.01.

Figure 2.

Analysis of Cre activity in cystic kidneys of Pkd1^flox/−:Ksp-Cre:R26R mice. Kidneys of P3 (A, B), P7 (C–E) and P12 (F–H) mice stained with β-gal and counter-stained with nuclear fast red. Cyst lining cells at all stages stained positive with β-gal indicating the presence of Cre activity (A–H). However, lacZ positive tubules in the inner medulla showed mild dilatation and ectasia at P3 (B) and P7 (E). A few cells in cyst linings were negative for lacZ expression (arrows; C, D). At later stages, ∼90% of the renal parenchyma is occupied by lacZ positive cysts (F). Almost all cyst epithelial cells are lacZ positive at P12 (G). The majority of cyst lining cells appear as flattened cells with evidence of surrounding tubular atrophy (asterisk), although cysts with lacZ positive cuboidal cells (arrow; H) are also observed. Size bars: (A,B,G) 500 µm; (C,D,E,H) 100 µm; (F) 1 mm.

Figure 3.

Cell composition and ultrastructural analysis of cysts in Pkd1^flox/−:Ksp-Cre kidneys. (A, B) The largest and most common cysts (cy) stained positive for lectin Dolichos biflorus (DBA) indicating collecting duct origin (red). Cysts from distal convoluted tubules [B; parvalbumin, green (asterisk)] and medullary thick ascending limbs [C; Tamm-Horsfall protein, red (double asterisk)] were less common and smaller. Proximal tubules (A; L. tetragonolobus, green) did not form cysts. Intercalated cells marked by the E-subunit of vacuolar H⁺-ATPase (D, F; green) or pendrin (H; green) are interspersed as single cells in cyst linings (D; DBA, red), indicating that they do not participate in the cyst growth. This is not due to the lack of cilia as both H⁺-ATPase (E, F; green) and pendrin (G, H; green) positive intercalated cells have cilia marked by anti-acetylated α-tubulin (red, indicated by arrows) in wild-type (E, G) and cystic (F, H) kidneys. (I) IMCD3 segments in adult kidney have apical cilia (aquaporin-2, red; acetylated α-tubulin, green). (J) Transmission electron micrograph (TEM) of principal cells in adjacent cysts show that these cells are more squamoid appearing than normal cortical collecting duct cells, but still have normal appearing lateral junctions, mitochondria, and basement membranes. (K) Scanning electron micrograph (SEM) showing an intercalated cell from a cortical cyst with apical microplicae or microvilli strongly suggesting that this is a type A intercalated cell. (L) TEM of the predominant intercalated cell type seen in cortical cysts. The low density of the cytoplasm, the low number of mitochondria, and the degree of development of the tubulovesicular network suggest that this intercalated cell is relatively immature; lack of apical microplicae or microvilli strongly suggest it is a non-type A intercalated cell. Intercalated cells had normal appearing lateral junctions and basement membranes. (M) SEM of a non-type A intercalated cell from a cortical cyst. Arrows indicate primary cilium. Ages: (A, B, D–F) P14; (C, H) P7; (G) P21. Size bars: (A) 35 µm; (B, C, G, I) 15 µm; (D) 20 µm; (E, F) 10 µm; (H) 5 µm. Digital magnification of inserts: (F) 1×; (G) 4×; (H) 1.5×; (I) 2×.

Figure 4.

Proliferation in cyst lining cells during kidney development. Nuclear BrdU (green) incorporation 3 h after a single intraperitoneal injection shows relative proliferation in kidney tissue from P7 (A) and P14 (C) Pkd1^flox/−:Ksp-Cre and P24 (E) Pkd1^flox/−:Pkhd1-Cre cystic kidneys. (B, D, F) Respective littermate non-cystic controls. Green, BrdU positive nuclei; red, DBA; blue, DAPI. Size bars: (A, B) 50 µm; (C–F) 10 µm. (G) Quantitation of BrdU positive nuclei in DBA-positive segments in cystic and control kidney tissues. BrdU incorporation decreases in control kidneys and is undetectable by P24. BrdU incorporation in cyst lining cells is significantly increased over control and persists in the P24 cystic kidney.

Figure 5.

In vivo activation of the MAPK/ERK pathway in Pkd1 and Pkd2 cystic kidneys. P7 Pkd1^flox/−:Ksp-Cre (A,B) and 3-month old Pkd2^WS25/− (C,D) Cystic kidneys show activation of the MAPK/ERK pathway compared with the respective Pkd1^flox/+:Ksp-Cre and Pkd2^+/− non-cystic littermate controls. (A,C) Representative immunoblots for active and total components of the MAPK/ERK pathway from kidney tissue. (B,D) Densitometric quantitation of three independent immunoblots for each step in the pathway. Results are expressed as a ratio of the density of active to total forms for each pathway component. Black bars, cystic kidneys; gray bars, control kidneys. *P < 0.05; **P < 0.01. Increased pERK expression is confined to the cells lining a subset of cysts in P7 Pkd1^flox/−:Ksp-Cre (E, F, H, I) and P90 Pkd2^WS25/− (G, J) kidneys. pERK, green; DBA, red; DAPI, blue. (E–G) pERK and DAPI. (H–J) Include DBA singal; (H, J) Digitally magnified views of (E, G). Size bars: (E, G) 30 µm; (F, H–J) 15 µm. (K) Representative immunoblots for active and total components of the MAPK/ERK pathway in null and heterozygous cell lines for both Pkd1 and Pkd2. pERK activation was found in two independent cell lines for each genotype; data for only one cell line are shown. (L) Densitometric quantitation of three independent immunoblots. Results are expressed as a ratio of the density of active to total forms for each pathway component. Black bars, null cell lines; gray bars, heterozygous cell lines. P < 0.05.

Figure 6.

MAPK/ERK pathway inhibition does not alter progression of cyst formation in ADPKD. (A) Representative immunoblots of pERK1/2 activity in kidneys from vehicle-treated non-cystic (C) and cystic Pkd1^flox/−:Ksp-Cre (Cy) mice and from U0126-treated control (C-U) and cystic (Cy-U) mice. Mice received 32 mg/kg U0126 by intraperitoneal injections on P4 and P7 and kidneys were harvested 24 h after the second dose. (B) Densitometric quantitation of immunoblots from three independent experiments as described in (A) showing increased activation of pERK in cystic kidneys (Cy) and normalization of pERK after treatment with 32 mg/kg U0126 (Cy-U); *P < 0.05. (C) Representative kidney sections from P14 Pkd1^flox/−:Ksp-Cre mice after treatment with vehicle (control) or with 32 mg/kg U0126. (D) Percent fractional cyst area as a measure of severity of polycystic kidney disease in P14 Pkd1^flox/−:Ksp-Cre mice treated with vehicle (Cy) or 32 mg/kg U0126 (Cy-U); (C) non-cystic kidney; n = 3 mice, 6 kidneys examined for each group. U0126 treatment had no effect on cystic disease progression in this model.