Mir151 conventional knockout mice
8.2. Characterization of Mir151 conventional knockout mice
8.2.1. Mir151 is not essential to survival
We have collected 361 N1F2 pups and recorded their survival at day 10 after birth.
The distribution of three genotype was Mir151+/+ : Mir151+/- : Mir151-/- = 1 : 1.91 : 0.92
(Table 2), that was nearly identical to Mendel’s first law (1 : 2 : 1). Our result indicates
140
that Mir151 loss has no significant deleterious effects on survival. We also found that
Mir151-/- knockout mice were viable, developed well, and fertile.
8.2.2. A kinetic change of erythropoiesis
We have generated a cohort including 10 MiR151+/+ and 9 MiR151-/- male N1F2
mice, the body weight was measured twice a week, and examined the complete blood
count (CBC) every months. We found that MiR151-/- mice showed a significantly higher
RBC count, hemoglobin concentration, and hematocrit during the age between 4 to 8
months, compared with their MiR151+/+ littermates (Figure 5). This alteration was not
continuously persisted in MiR151-/- mice and disappeared after 9 months. We also
examined the clinical chemistry (age: 6~8 months), however, we did not found any
significant alteration in MiR151-/- mice (Table 3). Overall, we observed that the RBC
count, hemoglobin, and hematocrit kept homeostasis in MiR151+/+ mice, but were
induced and kept in higher value between the age of 4-8 months and accompanied by
gradually decline in MiR151-/- mice until the last time point of the experiment
(1-year-old).
8.2.3. Elevated renal Epo mRNA level in young MiR151-/- mice
Erythropoiesis is regulated by erythropoietin (Epo), a 30.4 kDa glycoprotein
141
hormone known as the master regulator of red blood cell production through promoting
erythroid progenitor cell viability, proliferation, and differentiation (22). Production of
Epo is tightly regulated by development, tissue-specific, and physiological signals. It is
produced by hepatocytes in the embryonic stage, and during the late gestation the site
responsible to Epo production switches form the fetal liver to the kidney (23). Because
the erythropoietin synthesis is dependent on the transcriptional activity of Epo gene (24),
we analyzed the transcript level of renal and hepatic Epo in mice with age
corresponding to 3, 6, and 9 months (Figure 6). In agreement with the CBC result, an
expected up-regulation of renal Epo transcription was detected in MiR151-/- mice at 6
months old but not in MiR151+/+ littermates, whereas the hepatic Epo mRNA remained
at low level.
8.2.4. Induction of renal Epo by chronic hypoxia
Epo is highly sensitive to the balance between oxygen supply and demand. A
previous study using isolated rat kidneys with hypoxic perfusate has demonstrated the
increased renal Epo secretion (25). To enhance Epo expression, we performed chronic
hypoxia using the hypoxia chamber (Fig. 7) and evaluated the induction of Epo
transcription (Fig. 8). Mice were exposed to 10% O2 for 0 and 6 hours. At this age, Epo
transcripts at basal level were already elevated in MiR151+/- and MiR151-/- mice
142
(MiR151+/+, MiR151+/-, and MiR151-/- were 1, 2, and 3.5 fold, respectively). Low
oxygen level significantly enhanced Epo transcription in all genotypes after 6 hours
exposure to hypoxia (by ~20-fold). It is interesting that hypoxia-induced increase of
Epo mRNA in all genotypes demonstrated a relative high and regular manner which was
identical to normoxic condition (0 hour) (MiR151+/+, MiR151+/-, and MiR151-/- were 1, 2,
and 3.5 fold, respectively).
8.2.5. No increase in renal Epo-producing cells in MiR151-/- mice
The understanding of which cells produce Epo in kidneys was not clarified until
the use of genetically modified mice which help to identify that interstitial fibroblasts in
the cortex and outer medulla are renal Epo-producing cells (26). In the progression of
renal fibrosis, a common feature of chronic kidney disease (CKD), interstitial
fibroblasts will differentiate into myofibroblasts which are the major collagen producing
cells. During the initial stage of renal fibrosis, interstitial fibroblasts will proliferate
concordant with increased Epo expression, and then lost the Epo producing ability when
become myofibroblasts, resulting in renal anemia at the end stage of the disease. To
elucidate whether the kinetic change of Epo level in MiR151-/- mice was duo to undergo
renal fibrosis, the histological analysis of kidney section was performed (Figure 9).
However, the proliferation of interstitial fibroblasts at 6 months was not observed and
143
mice were free from renal fibrosis when 9 months
Taken together, our data suggests that MiR151 may have a role in regulating renal
Epo transcription through an unknown mechanism.
8.2.6. Increase of Hif-α target gene expressions in young MiR151-/- mice
The mechanisms controlling the expression of the Epo gene is oxygen-dependent.
Increase of Epo synthesis in response to hypoxic stimuli is also based on enhanced
transcriptional activity of the Epo gene, and hypoxia-inducible factors (HIFs) are
responsible for the linkage between changes in tissue oxygenation and altered Epo
transcription (23). Data from animal studies (27, 28) and clinical investigations (29, 30)
showed that hypoxia-inducible factor-2α (HIF-2α) is the critical regulator of Epo under
physiologic and stress conditions in adults. We speculated that MiR151-/- mice may
undergo mild hypoxia because of the up-regulated Epo in kidney but not in liver. To
address whether this alteration in MiR151-/- mice was mediated by Hif-α, we determined
the expression of Hif-α target genes including Epo, Phd3, Pgk, and Vegf A, in kidney of
6-month-old mice (Figure 8). In comparison with MiR151+/+ littermates, Epo and Phd3
expression were significantly increased in MiR151-/- mice, while no difference was
shown in Pgk and Vegf A gene. We also observed that the upregulated Epo mRNA level
was most obvious and reversely correlated with the MiR151 gene dosage. However, it is
144
unexpected that MiR151+/- has physiological higher Pgk and Vegf A mRNA levels.
8.2.7. Induction of Epo expression by CoCl2 treatment
Under normoxic conditions, HIF-α subunits are degraded rapidly by the ubiquitin-proteasome pathway (31). To evaluate whether the high Epo transcription in
MiR151-/- mice was associated with Hif-α expression in the kidney, we injected i.p. with
CoCl2, which is a chemical reagent that can stabilize Hif-α. Renal Epo expression was
significantly increased in MiR151-/- mice after CoCl2 treatment, while the induction in
MiR151+/+ was moderate (Figure 11).
Our data indicates that Hif-α may participate in the phenotype of increasing
erythropoiesis observed in MiR151-/- mice.