Cofilin1 is involved in hypertension-induced renal damage via the regulation of NF-?B in renal tubular epithelial cells

Animals

Twenty-week-old male spontaneous hypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs)
were obtained from the Vital River Laboratory Animal Co. Ltd (Beijing, China). The
animals were housed under standard conditions and 12 h light/dark cycles. They were
given free access to tap water and standard rat chow. Following 1 week of acclimation,
the rats were randomly assigned to the following four groups (n = 10 per group): SHR-C
(a SHR control group administered 1 mL 0.9 % sodium chloride orally), WKY-C (a WKY
control group administered 1 mL 0.9 % sodium chloride orally), SHR-H (SHRs administered
GSPE at a dose of 250 mg kg
^?1
 day
^?1
), SHR-L (SHRs administered GSPE at a dose of 100 mg kg
^?1
 day
^?1
). The animals were subjected to drug treatments as indicated above via oral gavage
for 22 weeks. GSPE was provided by Jianfeng Inc. (Tianjin, China) and contained ?95.0 %
proanthocyanidins. The components of GSPE were analysed via high-performance liquid
chromatography (HPLC)-UV. All animals were cared for in accordance with the National
Institute of Health Guide for the Care and Use of Laboratory Animals, with the approval
of the Scientific Investigation Ethics Committee of Qilu Hospital, Shandong University.

Systolic blood pressure measurements

Tail systolic blood pressures (SBP) were measured weekly until the end of the experiment
(22 times in total) in conscious rats using a Softron tail-cuff BP-2006A non-invasive
sphygmomanometer (Softron Incorporated, Beijing, China).

Urine and serum measurement

The rats were housed in individual metabolic cages for the collection of urine at
2-week intervals. The urine was centrifuged and stored at ?80 °C. Treatment was continued
for 22 weeks, at which time the rats were weighed and anaesthetized using chloral
hydrate. Blood was collected from the abdominal aorta and centrifuged to obtain serum,
which was stored at ?80 °C. Twenty-four-hour urinary protein, serum creatinine and
urea nitrogen were determined using an automatic biochemical analyser and served as
indices of renal injury.

Histology

Immediately following anaesthesia, the kidneys were removed. The left kidney was processed
for histology and immunostaining, whereas the right kidney was used for Western blotting.
The kidneys from ten rats per group were fixed in 4 % neutral buffered formalin and
subsequently embedded in paraffin. The 4 µm sections of paraffin embedded tissues
were subsequently stained with haematoxylin and eosin (HE) and analysed under a light
microscope by two investigators blinded to the treatments. To assess the degree of
tubulointerstitial inflammation, sections from each animal were evaluated. In each
section, 10 randomly selected observation fields were examined. The number of inflammatory
cells in the tubulointerstitium was counted under high-power fields (40×) using a
0.0625-mm
²
graticule fitted to the eyepiece of the microscope as previously described 13].

Immunohistochemical analysis

Following deparaffinization, hydration, blockage of endogenous peroxidase and antigen
retrieval routinely, the renal sections were incubated with primary antibodies for
MCP1, IL-1? (1:200, Abcam, Shanghai, China) or NF-?B(1:300, CST, USA) at 4 °C overnight.
The slides were subsequently incubated with goat anti-rabbit IgG (ZSGB-BIO, Beijing,
China) at 37 °C for 30 min, followed by staining with diaminobenzidine. A negative
control was prepared by omitting the primary antibody (data were not shown). The stained
tissue slides were observed under an Olympus microscope (model IX81, Japanese). The
expression levels of MCP-1, IL-1? and NF-?B p65 in the cortical tubulointerstitium
(cross section of the renal cortex) were determined using quantitative Image-Pro plus
software as described previously 14].

ELISA

The protein levels of IL-1? and MCP1 in the renal tissues were measured using an ELISA
kit (EBioscience, USA) according to the manufacturer’s instructions. Briefly, protein
samples from four groups (n = 7) were homogenized with ice cold extraction buffer
(1 mol L
^?1
HCl and neutralized with 1.2 mol L
^?1
NaOH/0.5 mol L
^?1
HEPES). The homogenate was centrifuged at 12,000g for 15 min at 4 °C. The supernatant was used to assay the amounts of IL1? and MCP1.
Absorbance was determined at 450 nm using an ELISA plate reader (INIFINITE M200, TECAN,
Switzerland).

Cell culture and treatment

Human renal proximal tubular cells (HK-2) were purchased from the American Type Culture
Collection (Manassas, VA, USA) and maintained in DMEM/F12 (Gibco, Carlsbad, USA) medium
supplemented with 10 % foetal bovine serum (FBS, Gibco, Carlsbad, USA), 100 U mL
^?1
penicillin and 100 µg mL
^?1
streptomycin (Solarbio, Beijing, China). These cells were routinely cultured at 37 °C
in a humidified atmosphere of 95 % air-5 % CO
₂
and nourished at intervals of 2–3 days. Subconfluent HK2 cells were preincubated in
either the presence or the absence of GSPE (50 µg mL
^?1
) for 12 h before being stimulated either with or without AngII (10
^?6
mol L
^?1
, Sigma, Shanghai, China) for 12 h. GSPE was dissolved in DMSO and diluted so that
the final concentration of DMSO was 0.1 %.

Knockdown of cofilin-1

Lentiviral-shRNA specific for interfering cofilin-1 expression, recombinant lentiviral
Lent/Cof and a nonspecific lentiviral control were obtained from GeneChem (GeneChem,
Shanghai, China). These lentiviral expression vectors contained the eGFP reporter
gene (enhanced green fluorescent protein). The cells were transfected with lentiviral
suspension using transfection reagent according to the manufacturer’s recommendations.
Following 72–96 h, transfection efficiency was measured by testing the expression
ratio of eGFP via fluorescence microscopy. Moreover, the knockdown of cofilin1 was
evaluated via Western blotting.

Luciferase reporter gene assay

The HK2 cells were seeded in 24-well plates and grown overnight to 80–90 % confluence;
0.8 µg NF-?B of luciferase reporter (pNF-?B-TA-luc) and the internal control plasmid
pGL6-TA (Byotime, Shanghai, China) were transfected into cells via Lipofectamineâ„¢
2000 and placed in fresh medium after 6 h. Following transfection for 30–48 h, the
cells were stimulated with 10
^?6
mol L
^?1
of AngII. Twelve hours later, the cells were harvested to quantify luciferase activity
using a dual luciferase reporter assay kit (Beyotime, Shanghai, China) according to
the manufacture’s protocol. Regarding the experiments investigating the effects of
cofilin1 knockdown on NF-?B activity, the cells were first transfected with either
recombinant lentiviral Lent/Cof or a nonspecific lentiviral control. Following passage,
the cells were again transfected via pNF-?B-TA-luc and analysed as described above.

Immunofluorescence

Cells from different groups were grown on coverslips and washed three times with phosphate-buffered
saline (PBS), fixed in 4 % paraformaldehyde for 20 min and permeabilized with 0.2 %
Triton X-100 for 10 min at room temperature. Following additional washes, the cells
were incubated in blocking solution (1 % bovine serum albumin/PBS) for 30 min at room
temperature in order to remove non-specifically bound antibodies. To localize the
F-actin filaments, the cells were incubated with 5 µg mL
^?1
rhodamine-phalloidin (Sigma, USA) for 30 min at 37 °C in a humid chamber. RelA/p65
was detected using a rabbit monoclonal anti-RelA/p65 antibody (1:100, CST, USA) overnight
at 4 °C. The cells were washed again and incubated in the dark at room temperature
for 1 h with a secondary antibody [1:500, Cy3-labeled goat anti-rabbit IgG (H + L),
Beyotime, Shanghai, China]. Following washing with PBS in the dark, DAPI was used
to counterstain the nucleus for 5 min (in the dark at room temperature). Images were
obtained using an Olympus microscope (model IX-81; Japanese).

Western blotting

The nuclear and cytoplasmic proteins of the HK-2 cells were extracted using a commercially
available assay kit (Byotime, Shanghai, China). The total proteins of the HK2 cells
and renal cortex were extracted as published previously 15], 16]. The protein concentrations were determined using a BCA assay kit (Byotime, Shanghai,
China) as instructed. The proteins (20–80 µg) were separated via SDS-PAGE and transferred
onto PVDF membranes before being blocked in 5 % no-fat milk in TBST (0.1 % Tween20
in Tris-buffered saline) and incubated with dilutions of anti-cofilin1 (1:1000, CST,
USA), anti-phosphate-cofilin1 (1:500, CST, USA), anti-RelA/p65 (1:1000, CST, USA),
anti-I?B-? (1:500, Abcam, Shanghai), anti-MCP1 (1:500, Abcam, Shanghai), anti-IL-1?
(1:1000, Abcam, Shanghai) and anti-GAPDH (1:1000, SAB, USA) overnight at 4 °C. Following
incubation for 2 h at room temperature with secondary antibodies (1:5000, ZSGB-BIO,
Beijing, China), the target bands were visualized using an enhanced chemiluminescence
detection system (Image Quant LAS 4000 mini, GE, USA) and analysed densitometrically
using Quantity one software (Bio-Rad, USA).

Statistical analysis

All experiments were performed in triplicate. The data were analysed using SPSS 13.0
statistical software. All values are presented as means ± standard deviations (SDs).
The one-way ANOVA post hoc multiple comparisons LSD and t test were used for statistical
comparison between the groups and within the groups. P  0.05 (two-tailed) was indicative
of a statistically significant difference.