Reduction of Na/K-ATPase affects cardiac remodeling and increases c-kit cell abundance in partial nephrectomized mice

Abstract

The current study examined the role of Na/K-ATPase α1-subunit in animals subjected to 5/6th partial nephrectomy (PNx) using Na/K-ATPase α1-heterozygous (α1(+/-)) mice and their wild-type (WT) littermates. After PNx, both WT and α1(+/-) animals displayed diastolic dimension increases, increased blood pressure, and increased cardiac hypertrophy. However, in the α1(+/-) animals we detected significant increases in cardiac cell death in PNx animals. Given that reduction of α1 elicited increased cardiac cell death with PNx, while at the same time these animals developed cardiac hypertrophy, an examination of cardiac cell number, and proliferative capabilities of those cells was carried out. Cardiac tissues were probed for the progenitor cell marker c-kit and the proliferation marker ki-67. The results revealed that α1(+/-) mice had significantly higher numbers of c-kit-positive and ki-67-positive cells, especially in the PNx group. We also found that α1(+/-) mice express higher levels of stem cell factor, a c-kit ligand, in their heart tissue and had higher circulating levels of stem cell factor than WT animals. In addition, PNx induced significant enlargement of cardiac myocytes in WT mice but has much less effect in α1(+/-) mice. However, the total cell number determined by nuclear counting is higher in α1(+/-) mice with PNx compared with WT mice. We conclude that PNx induces hypertrophic growth and high blood pressure regardless of Na/K-ATPase content change. However, total cardiac cell number as well as c-kit-positive cell number is increased in α1(+/-) mice with PNx.

Keywords: ATPases; apoptosis; cardiac hypertrophy; cardiac progenitor cells; cardiotonic steroids; caspase; cell proliferation; stem cells; uremic cardiomyopathy.

Fig. 1.

Partial nephrectomy (PNx) induces blood pressure (BP) and marinobufagenin (MBG) concentration increases in both wild-type (WT) and Na/K-ATPase α₁ heterozygous knockout (α₁^+/−) mice. BP was measured by tail cuff as described in materials and methods. Both systolic BP (A and B) and mean BP (C and D) showed significant increases after PNx vs. sham, but there were no significant differences between the WT and α₁^+/− animals. MBG concentration increases significantly with PNx in both WT and α₁^+/− animals. E: means ± SE of plasma [MBG] determined by immunoassay. *P < 0.05, significantly different than sham.

Fig. 2.

PNx induces cardiac hypertrophy and fibrosis in WT and α₁^+/− mice. Heart weight (HW) and left ventricle weight (LVW) (A and C) were measured after mice were euthanized, and the heart weight/body weight ratio (HW/BW, C) and LVW/BW ratio (D) were calculated based on the final body weight before euthanasia. Both HW/BW and LVW/BW data indicate a significant increase in cardiac hypertrophy. E: Masson's trichrome staining in left ventricle tissue from experimental mice: representative images from each group taken by an Olympus FSX100 microscope with a ×20 lens (left) and quantification data (n = 8 in each group; right). *P < 0.05, significantly different than sham of same genotype; **P < 0.01, significantly different than sham of same genotype; !!P < 0.01, significantly different than PNx of WT.

Fig. 3.

PNx promotes cardiomyocyte hypertrophy in WT animals and hyperplasia in α₁^+/− mice. A: cardiomyocyte cross-sectional area was determined by wheat germ agglutinin staining as illustrated in materials and methods: representative images from each group (left) and quantification data analyzed by ImageJ from 5 animals in each group (right). Scale bar = 25 μm. B and C: representative frequency distribution curves illustrating the frequency of cells of a given size as a percentage of the total measured cells in the WT (B) and α₁^+/− (C). ***P < 0.001, significantly different than sham of shared genotype; !P < 0.001, significantly different than WT receiving PNx.

Fig. 4.

PNx induces increased nuclei numbers in α₁^+/− mice. Left ventricle sections from each experimental group were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue) and cardiac troponin I (cTnI, red) as described in materials and methods. A: representative images of DAPI and cTnI staining. Scale bar = 25 μm. B: quantification data of nuclei number by counting the DAPI-stained particles that overlaid with cTnI staining. C: quantification data for nuclei that are outside of the cTnI staining area. *P < 0.05, PNx vs. sham in α₁^+/− animals.

Fig. 5.

PNx induces apoptosis in α₁^+/− mice but not in WT mice. Active caspase-3 levels in mouse heart tissue were measured using immunohistochemical methods as described in materials and methods. A, left: representative ×20 photomicrographs of active caspase 3 in sham- or PNx-operated animals. A, right: quantification data from 5 to 6 animals in each group. B, left, representative terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) images from sham- and PNx-operated animals. B, right: quantification of TUNEL-positive nuclei. Red arrows indicate TUNEL-positive nuclei. WT sham, n = 6; WT PNx, n = 5; α₁^+/− sham, n = 6; PNx+/−, n = 6. *P < 0.05, significantly different than sham; !significantly different than WT PNx. Scale bar is 60 μm.

Fig. 6.

PNx activates the mammalian target of rapamycin pathway in WT mice but not in α₁^+/− mice. Heart tissue homogenates from experimental mice were analyzed for Na/K-ATPase α₁ (NKA)-content, phosphorylation of Akt (pAkt) at threonine-308, pAkt at serine-473, and phosphorylation of S6 ribosomal protein (pS6) using Western blot analysis. Top, right: Western blots. tAkt, total Akt. A–D: respective quantification data of Na/K-ATPase α₁ (A), pS6 (B), pAkt Thr308 (C), and pAkt Ser473 (D) taken from 8 animals in each group. *P < 0.05, significant difference vs. WT control; !P < 0.05, PNx α₁^+/− significantly different than sham α₁^+/−; #P < 0.01, significant difference between PNx WT and PNx α₁^+/−.

Fig. 7.

Heart tissues exhibit more c-kit-positive and ki-67-positive cells in α₁^+/− mice vs. WT mice. Left ventricle tissues fixed in 4% formaldehyde solution were used for immunofluorescent staining with anti-c-kit antibody and anti-ki-67 antibody as described in materials and methods. Top: representative images of c-kit (red) and ki-67 (green) staining. DIC, differential interference contrast. Bottom: quantification data from 5–7 animals from each group. *P < 0.05 and **P < 0.01 vs. WT sham. Scale bar is 25 μm.

Fig. 8.

Coimmunostaining of CD45 and c-kit in heart tissue. Left ventricle tissues fixed in 4% formaldehyde solution were used for immunofluorescent staining with anti-c-kit antibody and anti-CD45 antibody as described in materials and methods. Data show that over 90% of c-kit-positive cells are CD45 negative, indicating that most of the c-kit cells are not of hematopoietic origin. Scale bar is 25 μm.

Fig. 9.

Stem cell factor (SCF) in heart tissue and in plasma. For SCF expression in heart tissue, left ventricle homogenates from experimental mice were analyzed using Western blot analysis, whereas the SCF level in plasma was measured using an ELISA kit from Abcam. A: representative Western blots of heart tissue SCF (top) and the quantification data (bottom). B: concentration of SCF in plasma collected after euthanasia from mice subjected to sham or PNx surgeries. **P < 0.01, PNx vs. sham; !P < 0.05, PNx α₁^+/− vs. PNx WT.