Heterogeneous nuclear ribonucleoprotein f suppresses angiotensinogen gene expression and attenuates hypertension and kidney injury in diabetic mice.
ABSTRACT We investigated the impact of heterogeneous nuclear ribonucleoprotein F (hnRNP F) overexpression on angiotensinogen (Agt) gene expression, hypertension, and renal proximal tubular cell (RPTC) injury in high-glucose milieu both in vivo and in vitro. Diabetic Akita transgenic (Tg) mice specifically overexpressing hnRNP F in their RPTCs were created, and the effects on systemic hypertension, Agt gene expression, renal hypertrophy, and interstitial fibrosis were studied. We also examined immortalized rat RPTCs stably transfected with control plasmid or plasmid containing hnRNP F cDNA in vitro. The results showed that hnRNP F overexpression attenuated systemic hypertension, suppressed Agt and transforming growth factor-β1 (TGF-β1) gene expression, and reduced urinary Agt and angiotensin II levels, renal hypertrophy, and glomerulotubular fibrosis in Akita hnRNP F-Tg mice. In vitro, hnRNP F overexpression prevented the high-glucose stimulation of Agt and TGF-β1 mRNA expression and cellular hypertrophy in RPTCs. These data suggest that hnRNP F plays a modulatory role and can ameliorate hypertension, renal hypertrophy, and interstitial fibrosis in diabetes. The underlying mechanism is mediated, at least in part, via the suppression of intrarenal Agt gene expression in vivo. hnRNP F may be a potential target in the treatment of hypertension and kidney injury in diabetes.
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ABSTRACT: AIMS/HYPOTHESIS: We investigated whether heterogeneous nuclear ribonucleoproteins F and K (hnRNP F, hnRNP K) mediate insulin inhibition of renal Agt expression and prevention of hypertension and kidney injury in an Akita mouse model of type 1 diabetes. METHODS: Adult male Akita mice (12 weeks old) were treated with insulin implants and killed at week 16. Untreated non-Akita littermates served as controls. The effects of insulin on blood glucose, systolic BP (SBP), renal proximal tubular cell (RPTC) gene expression and interstitial fibrosis were studied. We also examined immortalised rat RPTCs stably transfected with control plasmid or with plasmid containing rat Agt promoter in vitro. RESULTS: Insulin treatment normalised blood glucose levels and SBP, inhibited renal AGT expression but enhanced hnRNP F, hnRNP K and angiotensin-converting enzyme-2 expression, attenuated renal hypertrophy and glomerular hyperfiltration and decreased urinary albumin/creatinine ratio, as well as AGT and angiotensin II levels, in Akita mice. In vitro, insulin inhibited Agt but stimulated Hnrnpf and Hnrnpk expression in high-glucose media via p44/42 mitogen-activated protein kinase signalling in RPTCs. Transfection with Hnrnpf or Hnrnpk small interfering RNAs prevented insulin inhibition of Agt expression in RPTCs. CONCLUSIONS/INTERPRETATION: These data indicate that insulin prevents hypertension and attenuates kidney injury, at least in part, through suppressing renal Agt transcription via upregulation of hnRNP F and hnRNP K expression in diabetic Akita mice. HnRNP F and hnRNP K may be potential targets in the treatment of hypertension and kidney injury in diabetes.Diabetologia 04/2013; · 6.49 Impact Factor
Heterogeneous Nuclear Ribonucleoprotein F Suppresses
Angiotensinogen Gene Expression and Attenuates
Hypertension and Kidney Injury in Diabetic Mice
Chao-Sheng Lo,1Shiao-Ying Chang,1Isabelle Chenier,1Janos G. Filep,2Julie R. Ingelfinger,3
Shao Ling Zhang,1and John S.D. Chan1
We investigated the impact of heterogeneous nuclear ribonucleo-
protein F (hnRNP F) overexpression on angiotensinogen (Agt)
gene expression, hypertension, and renal proximal tubular cell
(RPTC) injury in high-glucose milieu both in vivo and in vitro.
Diabetic Akita transgenic (Tg) mice specifically overexpressing
hnRNP F in their RPTCs were created, and the effects on systemic
hypertension, Agt gene expression, renal hypertrophy, and inter-
stitial fibrosis were studied. We also examined immortalized rat
RPTCs stably transfected with control plasmid or plasmid contain-
ing hnRNP F cDNA in vitro. The results showed that hnRNP F
overexpression attenuated systemic hypertension, suppressed
Agt and transforming growth factor-b1 (TGF-b1) gene expres-
sion, and reduced urinary Agt and angiotensin II levels, renal
hypertrophy, and glomerulotubular fibrosis in Akita hnRNP F-Tg
mice. In vitro, hnRNP F overexpression prevented the high-glucose
stimulation of Agt and TGF-b1 mRNA expression and cellular
hypertrophy in RPTCs. These data suggest that hnRNP F plays
a modulatory role and can ameliorate hypertension, renal hyper-
trophy, and interstitial fibrosis in diabetes. The underlying mech-
anism is mediated, at least in part, via the suppression of
intrarenal Agt gene expression in vivo. hnRNP F may be a poten-
tial target in the treatment of hypertension and kidney injury in
diabetes. Diabetes 61:2597–2608, 2012
tensinogen (Agt; the sole precursor of all angiotensins) gene
expression and induces renal proximal tubular cell (RPTC)
hypertrophy and profibrotic gene expression in vitro (2–5).
RAS blockers and transfer of antisense rat Agt cDNA prevent
high-glucose stimulation of transforming growth factor-b1
(TGF-b1) and RPTC hypertrophy in RPTCs (6,7). RAS
blockers also attenuate hypertension, proteinuria, and renal
injury in diabetic transgenic (Tg) mice specifically over-
expressing rat Agt in their RPTCs (8,9). Taken together,
these data support a crucial role for intrarenal Agt gene
expression in hypertension and kidney injury in diabetes.
n addition to the systemic renin-angiotensin system
(RAS), the existence of a local intrarenal RAS is well-
established (1). We previously demonstrated that high
glucose (25 mmol/L D-glucose) stimulates rat angio-
We have established that insulin inhibits high-glucose
stimulation of Agt gene expression and RPTC hypertrophy
(10,11) via a putative insulin-responsive element (IRE) in
the rat Agt gene promoter that binds two nuclear proteins,
heterogeneous nuclear ribonucleoprotein F (hnRNP F) and
hnRNP K (12–14). Overexpression of hnRNP F and/or
hnRNP K inhibits Agt gene transcription in RPTCs in vitro
(13,14). The physiological roles of hnRNP F and hnRNP K in
RPTCs in vivo, however, remain undefined.
In the present studies, we investigated the effect of
hnRNP F overexpression on Agt gene expression, hyper-
tension, and RPTC injury in high-glucose milieu both in vivo
and in vitro. Our results demonstrate that hnRNP F over-
expression indeed attenuated hypertension, suppressed
Agt and TGF-b1 gene expression, and ameliorated RPTC
hypertrophy and glomerulotubular fibrosis in diabetes.
RESEARCH DESIGN AND METHODS
Chemicals and constructs. D(+)-Glucose, D-mannitol, and monoclonal anti-
bodies against b-actin were purchased from Sigma-Aldrich Canada (Oakville,
Ontario, Canada). Rabbit polyclonal antibodies specific for hnRNP F (13) and
rat Agt (15) were generated in our laboratory. Anti-p27Kip1and anti-green
fluorescence protein (GFP) antibodies were obtained from BD Biosciences
(Mississauga, Ontario, Canada) and Zymed (South San Francisco, CA), re-
spectively. Polyclonal anti–TGF-b1 antibody was procured from R&D Systems
(Minneapolis, MN). Anti-angiotensin II (Ang II) antibody (immunohistochemis-
try grade) was bought from Phoenix Pharmaceuticals (Burlingame, CA). The
plasmid pKAP2 containing the kidney-specific androgen-regulated protein
(KAP) promoter that is responsive to androgen was a gift from Dr. Curt D.
Sigmund (University of Iowa, Iowa City, IA) (16). Full-length rat hnRNP F cDNA
[cloned in J.S.D.C.’s laboratory (13)] fused with HA-tag (which encodes amino
acid residues 98–106 of human influenza virus hemagglutinin at the COOH-
terminal with NotI site at both 59 and 39 termini) was inserted into pKAP2
plasmid at the NotI site or enhanced green fluorescent protein (pEGFP) C1
plasmid (Invitrogen, Burlington, Ontario, Canada). Oligonucleotides were syn-
thesized by Invitrogen. Restriction and modifying enzymes were obtained from
commercial sources. Mice heterozygous for the Akita spontaneous mutation of
insulin 2 (Ins2) gene (C57BL/6-Ins2Akita/J) (viable and fertile) were purchased
from The Jackson Laboratory (Bar Harbor, ME; http://jaxmice.jax.org).
Generation of Akita hnRNP F-Tg mice. Tg mice specifically overexpressing
hnRNP F in their RPTCs were generated as described previously for Tg mice
specifically overexpressing rat Agt or catalase in their RPTCs (8,17).
Akita hnRNP F-Tg mice were created by cross-breeding of homozygous
hnRNP F-Tg mice with heterozygous Akita mice (homozygous Akita mice are
infertile). Primers used forPCR genotypingof Akita hnRNP F-Tgmice are listed
in Table 1.
Physiological studies. Male wild-type (WT) adult control littermates, hnRNP
F-Tg, Akita, and Akita hnRNP F-Tg mice (six mice per group) were used. All
animals received standard mouse chow and water ad libitum. Animal care and
procedures were approved by the Centre de Recherche du Centre Hospitalier
de l’Université de Montréal’s Animal Committee.
Systolic blood pressure (SBP) was monitored with a BP-2000 tail-cuff pres-
sure machine (Visitech Systems, Apex, NC) as described previously (8,9,18–20).
Twenty-four hours prior to euthanasia (at 20 weeks of age), they were housed
individually in metabolic cages. Body weight was recorded. Urine was collected
and assayed for albumin and creatinine (ELISA; Albuwell and Creatinine
Companion; Exocell, Philadelphia, PA) (8,9,18–20). Mouse serum and urinary
Montréal, Université de Montréal, Hôtel-Dieu Hospital, Montreal, Quebec,
Canada; the2Research Centre, Maisonneuve-Rosemont Hospital, Montreal,
Quebec, Canada; and the
General Hospital, Boston, Massachusetts.
Corresponding author: John S.D. Chan, firstname.lastname@example.org.
Received 26 September 2011 and accepted 6 April 2012.
This article contains Supplementary Data online at http://diabetes
? 2012 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
1Centre de Recherche du Centre Hospitalier de l’Université de
3Pediatric Nephrology Unit, Massachusetts
diabetes.diabetesjournals.org DIABETES, VOL. 61, OCTOBER 20122597
Agt were assayed by ELISA (Immuno-Biological Laboratories, IBL America,
Minneapolis, MN). The kidneys were removed, decapsulated, and weighed. The
left kidneys were processed for histology and immunostaining study, and the
right kidneys were used for isolation of renal proximal tubules (RPTs) by Percoll
gradient (8,9,18–20). Aliquots of freshly isolated RPTs from individual animals
were used immediately for total RNA isolation and Western blotting (WB).
Histology. Kidneys were collected in Tissue-Tek cassettes (VWR Canlab,
Montreal, Quebec, Canada), dipped immediately in ice-cold 4% parafor-
maldehyde, fixed for 24 h at 4°C, and then processed by the Centre Hospitalier
de l’Université de Montréal Pathology Department. Tissue sections (four to
five sections per kidney) were counterstained with periodic acid Schiff (PAS)
or Masson’s Trichrome staining (8,9,18–20) and analyzed by light microscopy
by two investigators blinded to treatment.
The mean glomerular and RPTC volumes and tubular luminal areas were
determined as previously described (9,18–20).
peroxidase complex method using four to five sections per kidney and three
mouse kidneys per group (ABC Staining System; Santa Cruz Biotechnology)
(18–20). Staining was analyzed by light microscopy by two independent
investigators blinded to treatment. The collected images were analyzed and
quantified using National Institutes of Health ImageJ software (17–19).
Real-time quantitative PCR. Agt, TGF-b1, TGF-b1 receptor II (TGF-b1 RII),
plasminogen activator inhibitor-1 (PAI-1), collagen type IV Ia, fibronectin, and
b-actin mRNA expression in RPTs was quantified by real-time quantitative
PCR (RT-qPCR) with forward and reverse primers (Table 1) (18–20).
Western blotting. WB was performed as described previously (12–14,18–20).
The relative densities of hnRNP F, Agt, p27Kip1, and b-actin bands were
quantified by computerized laser densitometry (ImageQuant software, version
5.1; Molecular Dynamics, Sunnyvale, CA).
Serum and urinary Ang II measurement. Mouse serum and urine samples
were extracted with a kit and assayed for Ang II by specific ELISA (Bachem
Americas, Torrance, CA) as described previously (20).
Cellular hypertrophy. Immortalized rat RPTCs (21) were stably transfected
with the plasmid pEGFP C1 or pEGFP C1/hnRNP F and cultured in normal
glucose (5 mmol/L D-glucose plus 20 mmol/L D-mannitol) or high glucose (25
mmol/L D-glucose) as described previously (18–20) and observed under an
ECLIPSE TE2000 fluorescence microscope (Nikon, Melville, NY). Cellular
hypertrophy was assessed by several indices of hypertrophy: total cellular
protein content, [3H]leucine incorporation, and cellular p27kip1expression, as
described previously (5–7,11).
Statistical analysis. Statistical significance between the experimental groups
was analyzed initially by Student t test or one-way ANOVA and the Bonferroni
test as appropriate. The data are expressed as means 6 SEM. The P values
,0.05 were considered to be statistically significant.
RPTC-specific expression of the hnRNP F transgene
in Akita and Tg mouse kidneys. KAP2-hnRNP F trans-
genic mice were generated to produce specific and inducible
expression of hnRNP F in RPTs. This was accomplished by
inserting hnRNP F cDNA including the stop codon into
a construct containing the KAP promoter and exons 2–5 of
the human Agt gene, including noncoding DNA at the 39
terminal (Fig. 1A). (Note: human Agt could not be expressed
because exon I of the human Agt gene containing the
starting site of transcription is deleted in the plasmid.)
Southern blot analysis revealed the presence of the trans-
gene in heterozygote and homozygote animals (Fig. 1B).
Testosterone implant induced hnRNP F-HA transgene ex-
pression in the kidney of female and male Tg mice but not in
other tissues, and placebo pellet had no effect (Fig. 1C).
Similarly, hnRNP F-HA transgene was detected in RPTs of
hnRNP F-Tg and Akita hnRNP F-Tg mice but not in WT or
Akita mice (Fig. 1D). Mutated Ins2 gene was detected in
Akita and Akita hnRNP F-Tg mice but not in WT or hnRNP
F-Tg mice (Fig. 1E). These results confirm that the KAP gene
promoter directs hnRNP F-HA transgene expression in
RPTCs of hnRNP F-Tg and Akita hnRNP F-Tg mice.
HnRNP F overexpression attenuates Agt expression
in Akita hnRNP F-Tg mice. Immunostaining of renal
sections (Fig. 2A), WB of isolated RPTs (Fig. 2D), and RT-
qPCR of hnRNP F-HA transgene (Fig. 2E) revealed that
hnRNP F expression was significantly higher in RPTCs
from hnRNP F-Tg and Akita hnRNP F-Tg mice compared
with WT or Akita mice, respectively.
In contrast, immunostaining (Fig. 2B), WB (Fig. 2F), and
RT-qPCR (Fig. 2G) revealed significantly increased Agt
expression in RPTs of Akita mice as compared with WT
controls and hnRNP F-Tg mice but was reduced in Akita
hnRNP F-Tg mice. The findings of Agt immunostaining in
mouse kidneys (Fig. 2B) were further confirmed by immuno-
staining for Ang II in mouse kidneys (Fig. 2C and Supple-
mentary Fig. 1A and B). These data demonstrated that hnRNP
F overexpression suppresses Agt expression in RPTs of Akita
Overexpression of hnRNP F attenuates hypertension
and kidney hypertrophy in Akita hnRNP F-Tg mice.
Longitudinal SBP measurement showed that SBP was higher
in Akita mice than WT or hnRNP F-Tg mice from week 10 to
20; however, there was no significant difference in SBP be-
tween Akita mice and Akita hnRNP F-Tg mice detected until
week 18 (Fig. 3A). Cross-sectional SBP measurement at
week 20 revealed that SBP was significantly elevated in
Akita mice compared with WT and hnRNP F-Tg mice but
significantly attenuated in Akita hnRNP F-Tg mice (Fig. 3B),
Primers for genotyping and RT-qPCR
(NT_039437.7) S: 59-TGCTGATGCCCTGGCCTGCT-39
(rat) (BC087679)S: 59-CCTCGCTCTCTGGACTTATC-39
(rat and mouse)
(rat and mouse)
(NM_031132) S: 59-TCCTTCAAGCAGACGGATGT-39
(rat and mouse)
(rat and mouse)
Collagen type IV 1a
(rat and mouse)
(NM_009931) S: 59-TGCCGGGTCCACAAGGTTCAC-39
(rat and mouse)
(NM_031144) S: 59-CGTGCGTGACATCAAAGAGAA-39
AS, antisense; S, sense.
hnRNP F AND THE DIABETIC KIDNEY
2598 DIABETES, VOL. 61, OCTOBER 2012diabetes.diabetesjournals.org
demonstrating that hnRNP F overexpression attenuates
Serum Agt and Ang II levels did not differ significantly in
the four different groups of mice studied (Fig. 3C and D,
respectively). In contrast, urinary Agt and Ang II levels
were significantly higher in Akita mice as compared with
WT control littermates or hnRNP F-Tg mice but attenuated
in Akita hnRNP F-Tg mice (Fig. 3E and F, respectively).
Furthermore, Agt (Fig. 3G) and Ang II (Fig. 3H) levels in
whole-kidney extracts were significantly higher in Akita
FIG. 1. Generation of hnRNP F-Tg and Akita hnRNP F-Tg mice. A: Schematic map of the KAP2-rat hnRNP F construct. The isolated 17-kb KAP2-
hnRNP F-HA transgene (digested with SpeI and NdeI) was microinjected into one-cell fertilized mouse embryos obtained from superovulated
C57Bl6 3 C3H mice (performed at the Clinical Research Institutes of Montreal, Montreal, Quebec, Canada). B: Southern blotting of genomic DNA
for founders with radioactive hnRNP F probe. Heterozygous and homozygous F1, F2, and F3were screened by PCR with specific primers (Table 1).
C: RT-PCR product showing tissue expression of hnRNP F-HA mRNA in female and male Tg mice uninduced or induced with testosterone. b-Actin
and hnRNP F-HA fragments are indicated. Transgenic mice (line #937) were induced with placebo pellets or pellets containing 5 mg testosterone
with a 21-day release schedule (catalog number A-121; Innovative Research of America, Sarasota, FL) for 2 weeks prior to RNA isolation.
D: Specific PCR analysis of hnRNP F-HA transgene in offspring of hnRNP F-Tg line 937 crossbred with heterozygous Akita mice. Akita hnRNP F-Tg
mice displaying hnRNP F-HA transgene were used in subsequent experiments. E: Genotyping of Ins2 gene mutation in WT control littermates,
hnRNP F-Tg mice, Akita, and Akita hnRNP F-Tg mice with primers of the mouse Ins2 gene (Table 1). The PCR-DNA fragment (280 base pairs) of
mouse Ins2 gene was then digested with the restriction enzyme Fnu4H1 for 1 h at 37°C. If an allele was mutated, two DNA fragments with 280 and
140 base pairs were observed on 3% agarose gel electrophoresis. Br, brain; Hr, heart; Ki, kidney; Li, liver; Lu, lung; Sp, spleen; Ts, testis.
C.-S. LO AND ASSOCIATES
diabetes.diabetesjournals.orgDIABETES, VOL. 61, OCTOBER 20122599
FIG. 2. HnRNP F, Agt, and Ang II expression in Tg mouse kidneys at week 20. Immunohistochemical staining for hnRNP F (A), Agt (B), and Ang II
(C) in mouse kidneys. a: WT control mouse. b: hnRNP F-Tg mouse. c: Akita mouse. d: Akita hnRNP F-Tg mouse. Original magnification 3200. D: WB
analysis of hnRNP F expression in RPTs from kidneys of WT controls, hnRNP F-Tg, Akita, and Akita hnRNP F-Tg mice. E: RT-qPCR of hnRNP F
mRNA expression in RPTs of WT controls, hnRNP F-Tg, Akita, and Akita hnRNP F-Tg mice. F: WB analysis of Agt expression in RPTs from kidneys
of WT controls, hnRNP F-Tg, Akita, and Akita hnRNP F-Tg mice. G: RT-qPCR of Agt mRNA expression in RPTs of WT controls, hnRNP F-Tg, Akita,
and Akita hnRNP F-Tg mice. Values are expressed as means 6 SEM; N = 6. WT and hnRNP F-Tg mice, empty bars; Akita and Akita hnRNP F-Tg mice,
filled bars. *P < 0.05; **P < 0.01; ***P < 0.005. (A high-quality digital representation of this figure is available in the online issue.)
hnRNP F AND THE DIABETIC KIDNEY
2600DIABETES, VOL. 61, OCTOBER 2012diabetes.diabetesjournals.org
FIG. 3. Overexpression of hnRNP F attenuates systemic hypertension in Akita Tg mice. A: Longitudinal changes in mean SBP in male WT control
(□), hnRNP F-Tg (△), Akita (▲), and Akita hnRNP F-Tg mice (▼). The mice were trained in the procedure for at least 15–20 min per day for
5 days before the first SBP measurements. B: Cross-sectional analysis of SBP (measured two to three times per animal per week in the morning
without fasting, week 20). Serum Agt levels (C), serum Ang II levels (D), urinary Agt levels (E), urinary Ang II levels (F), Agt levels in whole-
kidney extracts (G), and Ang II levels in whole-kidney extracts (H) in WT controls, hnRNP F-Tg, Akita, and Akita hnRNP F-Tg mice. Serum and
urinary Agt were assayed by specific enzyme-linked immunosorbent assay (ELISA), and urinary Agt levels were normalized with urinary creatinine
levels. Serum and urinary Ang II were extracted, assayed by specific ELISA, and normalized with urinary creatinine levels. Agt and Ang II levels in
whole-kidney extracts were assayed by respective ELISA for Agt and Ang II. All data are expressed as means 6 SEM; N = 6. WT and hnRNP F-Tg
mice, empty bars; Akita and Akita hnRNP F-Tg mice, filled bars. *P < 0.05; **P < 0.01; ***P < 0.005.
C.-S. LO AND ASSOCIATES
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