Figure 1.
Col1a1(3.6)-Cre-mediated conditional deletion of Pkd1 from the floxed Pkd1 allele (Pkd1flox) in different tissues.
(A) Kaplan–Meier survival curve. Heterozygous Col1a1(3.6)-Cre; Pkd1flox/+ had normal survival identical to control (Pkd1flox/flox ) mice, homozygous Col1a1(3.6)-Cre; Pkd1flox/flox mice began to die 1 week after birth and only half of these mice survived beyond 6 weeks. (B) Body weight of Col1a1(3.6)-Cre;Pkd1flox/+ and control (Pkd1flox/flox) mice were not different, but the body weight of both male and female Col1a1(3.6)-Cre;Pkd1flox/flox mice was reduced by ∼30% and ∼36% compared with the other two genotypes. (C) Genotyping PCR analysis of different tissues harvested from 6-week-old homozygous Col1a1(3.6)-Cre; Pkd1flox/flox mice showed that both Pkd1flox and Pkd1Δflox alleles existed in all tested tissues including bone and nonskeletal tissues, indicating that Col1a1(3.6)-Cre promoter is not specific for bone. (D–E) Real-time RT-PCR analysis of total Pkd1 transcripts in calvaria from both Col1a1(3.6)-Cre;Pkd1flox/flox and Col1a1(3.6)-Cre;Pkd1flox/null models, and in kidney and liver from Col1a1(3.6)-Cre;Pkd1flox/flox model at 6 weeks of age. Total Pkd1 transcripts were expressed as the fold changes relative to the housekeeping gene β-actin subsequently normalized to control Pkd1flox/flox or Pkd1flox/+ mice. Data represent the mean ± SD from five or six individual mice. Values sharing the same superscript are not significantly different at P<0.05. *Significant difference from control (Pkd1flox/flox); #significant difference from heterozygous Col1a1(3.6)-Cre;Pkd1flox/+ mice at P<0.05, respectively.
Figure 2.
Col1a1(3.6)-Cre-mediated deletion of Pkd1 results in osteopenia in Col1a1(3.6)-Cre;Pkd1flox/null newborn mice.
The whole skeleton mineralization (A and B), full-length mineralized femurs (C and D), bone volume of metaphyseal region (E and F), and cortical thickness of cortical bone (G and H) of femurs from Col1a1(3.6)-Cre;Pkd1flox/flox and Col1a(3.6)-Cre; Pkd1flox/null newborn mice by μCT analysis. A Pkd1 gene dose effect was observed during skeletogenesis between Col1a1(3.6)-Cre;Pkd1flox/flox and Col1a(3.6)-Cre; Pkd1flox/null newborn mice. Data represent the mean ± SD from three to four individual samples. *Significant difference from control mice (Pkd1flox/flox or Pkd1flox/+) at P<0.05.
Figure 3.
Col1a1(3.6)-Cre-mediated conditional deletion of Pkd1 leads to severe osteopenia in Col1a1(3.6)-Cre;Pkd1flox/flox adult mice.
(A) Bone mineral density (BMD), (B) Bone structure of femurs, (C) Bone mineral apposition rate (MAR), and (D) Femur length at 6 weeks of age. There was a Pkd1 gene dose-dependent reduction in BMD in both male and female heterozygous Col1a1(3.6)-Cre; Pkd1flox/+ and homozygous Col1a1(3.6)-Cre; Pkd1flox/flox mice compared with age-matched control mice (Pkd1flox/flox). µCT analysis revealed that the lower bone mass in male Col1a1(3.6)-Cre-mediated mice with conditional deletion of Pkd1 resulted from reductions in both trabecular BV/TV and cortical CtTh that were proportionate to the reduction of Pkd1 gene dose. These reductions in bone mass and structure were associated with a 19% and 41% reduction in mineral apposition rate (MAR) in male heterozygous Col1a1(3.6)-Cre; Pkd1flox/+ and homozygous Col1a1(3.6)-Cre; Pkd1flox/flox mice compared with age-matched control mice, respectively. In addition, the femurs of homozygous Col1a1(3.6)-Cre; Pkd1flox/flox mice were 17% shorter in length, indicating a postnatal bone growth retardation. Data represent the mean ± S.D. from five to six individual mice. *Significant difference from control (Pkd1flox/flox) and #significant difference from Col1a1(3.6)-Cre; Pkd1flox/+ mice at P<0.05, respectively.
Table 1.
Gene-expression profiles in 6-week-old mice.
Table 2.
Biochemistry analysis of serum in 6-week-old mice.
Figure 4.
Col1a1(3.6)-Cre-mediated conditional deletion of Pkd1 results in enhanced adipogenesis in bone marrow and in bone stromal cell cultures.
(A) Histology of adipocytes in decalcified tibias. Oil Red O staining (upper panel) showed that the numbers of adipocytes and fat droplets in tibia bone marrow were greater in 6-week-old Col1a1(3.6)-Cre; Pkd1flox/flox mice compared with age-matched control Pkd1flox/flox mice. Osmium tetroxide (OsO4) staining by μCT analyses (lower panel) showed that adipocyte volume/marrow volume (Ad.V/Ma.V, %) and adipocyte number (Ad.N, mm−3) were much higher in the proximal tibia from 6-week-old Col1a1(3.6)-Cre; Pkd1flox/flox mice compared with age-matched control Pkd1flox/flox mice. (B) Adipocytic differentiation in BMSC cultures. An increase of adipogenesis potential was observed in 6-week-old Col1a1(3.6)-Cre; Pkd1flox/flox BMSC cultures, evidenced by a significant increase of Oil Red O staining in adipogenic cultures. (C and D) Expression of adipogenic markers by real-time RT-PCR. Significantly increased levels of PPARγ and aP2 mRNAs were observed in 6-week-old Col1a1(3.6)-Cre; Pkd1flox/flox BMSC cultures compared with control (Pkd1flox/flox) cultures. Data are expressed as the mean ± SD from three independent experiments. # Significant difference from control (Pkd1flox/flox) at P<0.05.
Figure 5.
Effects of Col1a1(3.6)-Cre-mediated conditional deletion of Pkd1 on osteoblastic proliferation and maturation, as well as gene expression profiles ex vivo.
(A) Total Pkd1 transcripts by real-time RT-PCR. All Pkd1 transcripts were dose-dependently reduced in primary osteoblast cultures from heterozygous Col1a1(3.6)-Cre; Pkd1flox/+ and homozygous Col1a1(3.6)-Cre; Pkd1flox/flox mice. (B) BrdU incorporation. A gene dose-dependent increase of BrdU incorporation was observed during 6 h of primary osteoblast culture from heterozygous Col1a1(3.6)-Cre; Pkd1flox/+ and homozygous Col1a1(3.6)-Cre; Pkd1flox/flox mice. (C) ALP activity. Osteoblasts from control, heterozygous, and homozygous mice displayed time-dependent increments in ALP activity during 21 days of culture, but ALP activity was gene dose- and time-dependently decreased in heterozygous and homozygous osteoblasts compared to control osteoblasts. (D) Quantification of mineralization. Alizarin Red-S was extracted with 10% cetylpyridinium chloride and quantified as described in Materials and Methods. A time-dependent increment of Alizarin Red-S accumulation was observed in control, heterozygous, and homozygous osteoblasts during 21 days of culture, but the accumulation was gene dose-dependently decreased in heterozygous and homozygous osteoblasts cultures compared to control osteoblasts at day 21 of culture. (E–I) Gene expression profiles by real-time RT-PCR. Osteoblastic markers such as Runx2, Akp2, and FGF23 were gene dose-dependently reduced during 18 days of osteogenic culture from heterozygous and homozygous osteoblasts. In contrast, a marked increase of adipocyte markers such as PPARγ2 and aP2 was observed from heterozygous and homozygous osteoblasts under the same osteogenic media when compared with control osteoblasts. Data are expressed as the mean ± SD from three independent experiments. *Significant difference from control (Pkd1flox/flox); #significant difference from heterozygous Col1a1(3.6)-Cre; Pkd1flox/+ mice at P<0.05, respectively.
Figure 6.
Col1a1(3.6)-Cre-mediated conditional deletion of Pkd1 causes polycystic pancreas and kidney.
(A) Gross appearance of liver, kidney, and pancreas. There was no cyst formation in the liver, kidney, and pancreas in heterozygous Col1a1(3.6)-Cre;Pkd1flox/+ mice, whereas age-matched homozygous Col1a1(3.6)-Cre;Pkd1flox/flox mice developed severe renal and pancreatic cysts at 6 weeks of age. (B–D) Hematoxylin-eosin-stained sections (5X) of liver, pancreas, and kidney from 6-week-old mice. Cysts were not observed in the livers from heterozygous and homozygous mice, and renal and pancreatic cysts were also not found in kidney and pancreas tissues from heterozygous Col1a1(3.6)-Cre;Pkd1flox/+ mice. However, homozygous Col1a1(3.6)-Cre;Pkd1flox/flox mice exhibited massive cyst formation in both the pancreas and kidney. Interestingly, glomeruli formation in the kidney and endocrine islet formation in pancreas appeared to be unaffected. In addition, expansion of pancreatic ducts formed large pancreatic cysts that led to massive acinar cell loss, formation of abnormal tubular structures, and appearance of endocrine cells in ducts.
Figure 7.
Development of cysts in pancreas and kidney caused by Col1a1(3.6)-Cre-mediated conditional deletion of Pkd1.
Hematoxylin-eosin (H&E) staining (5X) for pancreas (A) and kidney (B) between E14.5 and P14. Both pancreatic duct and renal tubule cysts started at E15.5 in homozygous Col1a1(3.6)-Cre;Pkd1flox/flox embryos, and the size of renal cystic lesions developed rapidly between E15.5 and P14.
Figure 8.
Development of fibrosis in kidney caused by Col1a1(3.6)-Cre-mediated conditional deletion of Pkd1.
Masson-trichrome staining for fibrosis in polycystic kidney sections (A)1X, (B) 20X magnification between E16.5 and 8 weeks (8w). Masson-trichrome staining was observed in polycystic kidney tissue at P7 and became more severe at 8 weeks of age, indicating a renal fibrosis formation occurring in Col1a1(3.6)-Cre;Pkd1flox/flox mice.
Figure 9.
Effect of Col1a1(3.6)-Cre-mediated conditional deletion of Pkd1 in kidney on expression of epithelia-mesenchymal-transition (EMT) and fibrosis markers.
(A–C) Expression of EMT markers in polycystic kidneys of 6-week-old mice by real-time RT-PCR. A panel of EMT markers including transforming growth factor β (TGF-β), snail1, and vimentin were significantly up-regulated in polycystic kidney from homozygous Col1a1(3.6)-Cre;Pkd1flox/flox mice compare with age-matched control Pkd1flox/flox mice. (D–F) Expression of fibrosis markers in polycystic kidneys of 6-week-old mice by real-time RT-PCR. A panel of fibrosis markers such as α-smooth muscle actin (α-SMA) and precollagen type I (Col1a1) was markedly increased in the kidney from homozygous Col1a1(3.6)-Cre;Pkd1flox/flox mice compare with age-matched control Pkd1flox/flox mice. 18 S was served as an internal control for gene expressions. There were no differences in EMT and fibrosis markers between heterozygous and control Pkd1flox/flox mice. Data represent the mean ± SD from five to six individual mice. *Significant difference from control (Pkd1flox/flox); #significant difference from Col1a1(3.6)-Cre;Pkd1flox/+ mice at P<0.05, respectively.