Introduction
The renal collecting duct system contributes to the regulation of water reabsorption and final urine
concentration (Mamuya et al. 2016). Aquaporin-2 (AQP-2), a channel
protein located in the collecting duct, works in regulating urine concentration
and maintaining electrolyte homeostasis in the body. It is regulated by
arginine vasopressin, greatly improving water reabsorption in collecting tube
(Jung and Kwon 2016). AQP-2 improves water permeability when
transported to the apical membrane of the cell. The evidence showed that it
can promote the reabsorption of water and the concentration of urine, reducing
plasma osmotic pressure finally (Huang et al. 2018).
Alisma orientale (Sam.) Juzep (Alisma), a
Chinese materia medica, has been used for thousands of years in China and other
Asian countries. It has been found to hold water
infiltration, heat release, lipid reduction and other bio-activities, and has
been used for the prevention and treatment of adverse urination, full swelling
of water, little diarrhea and urine, dizziness caused by phlegm, hot dripping
and acerbity pain, hyperlipidemia. Modern phytochemical studies show that terpenoids are their main active ingredients, including
triterpenes, sesquiterpenes and diterpenes (Jeong et al. 2016; Zhang et
al. 2017a). In recent years, the accumulating evidence showed that alisma extract can significantly increase urine
volume and increase the levels of Na+, K+ and Cl-
in rats (Zhang et al. 2017b). Studies have suggested that this
regulatory activity may be closely related to AQP-2 (Mose et al. 2019). However, the effects of AQP-2 on the regulation of alisma on water-liquid metabolism remain unclear.
The research is
aimed to study the roles of terpenoids from alisma in regulating urine concentration and
maintaining electrolyte homeostasis and also
explore the role of AQP-2 in this regulation and its mechanism.
Materials and Methods
Drugs and reagents
Furosemide injection and TRIzol were respectively purchased from
Wellhope Pharmaceutical Co., Ltd. (Shanghai, China) and Invitrogen (USA).
cDNA first strand synthesis kit was purchased from ThermoFisher (USA). SYBR
Premix Ex Taq II was purchased from TaKaRa
Japan Company. Agarose I was purchased from Amresco (USA). All protein
extraction kit, Braford protein content detection kit, SDS-PAGE gel preparation
kit, pre-stained protein molecular weight, ECL detection kit, developing and
determining reagents were purchased from Beyotime Biotechnology Co., Ltd.
(Nantong, China). PVDF membrane was purchased from Merckmillipore Company
(Germany). Rabbit beta-actin and AQP2 antibodies were purchased from BOSTER
Biological Engineering Co., Ltd. (Wuhan, China). Secondary antibody (sheep
anti-rabbit IgG, sheep anti-rat IgG) was purchased from Abcam Company.
Immunohistochemical kit was purchased from Hongshan Jinqiao Biotechnology Co.
(Beijing, China). DMEM hyperglycemic-f12 1:1 incomplete medium, Dimethyl
sulfoxide (DMSO) and FBS were respectively purchased from Corning (USA),
Amresco and Gibco (USA). Tetramethylazoles salt (MTT) was purchased from Sigma
Company (USA). Other reagents are commercial and analytical grade.
Chromatographic conditions
Spusil C18 chromatographic
column (150×4.6 mm, 5 micron) was used. Mobile phase: (0.1% formic acid -
acetonitrile (95:5)) - (0.1% formic acid - acetonitrile (15:85)); gradient
elution (0–10 min, 100–40% A; 10–25 min, 40–0% A; 25–35 min, 0% A; 35–36 min, 0–100%
A; 36–48 min, 100% A). The flow rate was 1.0 mL/min and column temperature was
30°C.
Test products preparation
The
extract (1 mL) was taken and diluted with pure water to 5 mL for test.
Dry powder (0.5 g) was
taken and put into a conical bottle with plug, 25 mL of 50% acetonitrile was
added into the conical bottle accurately and treated with ultrasonic for 4 h.
Then the extraction was filtered and the filtrate was taken for further
experiments.
Dry powder (0.5 g) was
taken and put into a conical bottle with plug, 25 mL of pure water was added
into the conical bottle accurately and treated with ultrasonic for 4 h. Then
the extraction was filtered and the filtrate was taken for further experiments.
Alisma terpenoids
extraction and preparation
Alisma
terpenoids was prepared according to the following operation: alisma decoction
pieces of 15 kg were weighed, crushed, and then extracted by reflux with 10
times volume 80% ethanol (v:v) for 30 min. This extraction was carried out twice
to obtain the filtrates for combination. The combined extraction was
concentrated to about 0.5 g/mL under decompression in a rotary evaporator. AB-8
macroporous resin column was used to purify the concentrated extract until the
macroporous resin completely adsorbs. After removing impurities with 4 BV 50%
ethanol, the column was eluted with 4 BV 70% ethanol at speed of 0.3 BV/h. The
eluent was obtained with 70% ethanol and concentrated to obtain alisma
terpenoids (125.03 g, yield 0.83%).
Animals and drug administration
Male
Sprague Dawley (SD) rats weighing 200 ± 20 g were purchased from Nantong
University, China (license number: SCXK (Su) 2014-0001), in line with SPF grade
experimental animal standards. The SD rats were raised in the animal center of
China Pharmaceutical University with room temperature 22–23°C, humidity 45–50%
and diurnal illumination of 12 h of light/12 h of darkness (L/D) cycles. Rats
received food and water ad libitum. This study was approved
by experimental animal ethics committee, and all experiments were in line with
the guiding principles of animal research of Chinese ethics committee. Qualified model rats were screened by Aston method. These rats were
raised adaptively in the metabolic cage for 3 days and then fasted for 8 h on
fourth day. Sequentially, these rats were treated with 2.5 mL/100g distilled
water, and urine was quickly collected for 2 h after administration. Rats whose
urine volume were more than 40% gavage volume were considered qualified rats
and randomly divided into blank control group (2.5 mL/100g distilled water);
positive control group (furosemide of 20 mg/kg); high-dose alisma terpenoids group (equivalent to decoction pieces 10 g/kg/d); medium-dose alisma terpenoids group (equivalent to decoction pieces 5 g/kg/d); low-dose alisma terpenoids group (equivalent to decoction pieces 2.5 g/kg/d). The rats were
administrated by Chao Li. The
rats were treated with distilled water continuously for 3 d and fasted for 8 h
before the last dose. Then rats in each group were given with 0.9% sodium
chloride solution at the dose of 5 mL/100g to simulate water and sodium
retention.
Urine biochemical
indicators detection
After drug administration, the urine of rats were
collected in a metabolic cage for 24 h and quantified accurately. The contents
of Na+, K+ and Cl- in the urine were detected by
automatic biochemical analyzer (BS-490,
Mindray, China) according to manufacturer's protocols.
Western blotting
The kidney tissues or HK-2 cells were added to the lysis buffer
and ground at low temperature. After lysed, the tissues were centrifuged at
13000 rpm at 4°C for 10 min. Then the supernatant was taken for the further
treatment. After the protein was denaturated at 100°C, the same amount of total
protein was separated by SDS-page electrophoresis and transferred to PVDF. The
concentrated glue was kept at a constant pressure of 85 V for about 30 min, and
the separated glue was kept at 120 V for about 80 min. The film transfer time
was kept at 50 min with a constant current of 200 mA. PVDF membrane was sealed
with 5% skim milk overnight and then added with Diluted primary antibody
(1:1000). After shaking at 4℃ overnight, PVDF membrane was washed
with TBST twice (10 min for each time), and was washed with TBS for 10 min.
Secondary antibody diluent was added into the PVDF membrane
and incubated at room temperature for 1–2 h. After PVDF membrane washed
repeatedly with TBST solution and TBS solution, chemiluminescence was
performed. Gel-pro 32 software was used to analyze the results in grayscale.
Real-time quantitative
polymerase chain reaction (q-PCR) analysis
Kidney tissues or HK-2 cells were added into
precooled TRIzol. The samples were grinded at low temperatures until split
completely then transferred to PCR tube. Chloroform was added into tube and
shook for 15s. After standing at 37°C for 10 min, PCR tube was centrifuged at
12000 rpm at 4°C for 10 min. The residues were dissolved with 50 μL RNase, and stored at -70°C for
later use. Sequentially, concentration and purity of RNA was determined. The primer
was designed by Nanjing Jinsirui technology Co., Ltd, β-actin (175 bp):
upstream primer 5’-GTGCTGAGTATGTCGTGGAGTC-3’; downstream primers
5’-TTGCTGACAAT CTTGAGGGA-3’; AQP-2(132 bp): upstream primer
5’-CCCTCTCCATTGGTTTCTCTGT TA-3’; downstream primers
5’-AGAAGACCCAGTGATCATCAAACTT-3’. The cDNA was synthesized using cDNA first strand synthesis kit. The PCR
protocol was performed according to the procedure for PCR reaction:
pre-denaturizing for 5 min, denaturizing and annealing for 15 s at 95°C successively
and developing for 60 s at 60°C. The reaction without cDNA was included as a
negative control. The date was analyzed by 2-△△Ct method.
Immunochemistry
The 4% formaldehyde solution was used to fix the rat
kidney tissues for 24 h then the tissues were embedded with paraffin and cut
into sections. The tissue was baked at 60°C for 1 h before dewaxing and
immersed into xylene twice to dewaxing, and dehydrated in different gradients
of alcohol (70, 85, 95 and 100%) for 5 min. Sections were placed in antigen
repair buffer, boiled for 20 min and PBS was used to wash the sections for 3
times. The sections were dripped with 3% H2O2-PBS
solution, then sealed for 15 min at room temperature and PBS was used to wash
sections for 3 times. Primary antibody was added on sections (4°C, overnight)
and washed with PBS for 4 times. Secondary antibody was added on sections and
incubated for 30 min at 37°C, then washed with PBS for 3 times. Control
incubation was included with the secondary antibody alone. Appropriate amount
of DAB solution was added on sections for staining, and
color development was stopped with distilled water. The sections were redyed
with
sappanin dyeing solution for 10 min and immersed and dehydrated in different gradients of alcohol
(70, 85, 95 and 100%) for 5 min. Then the sections were immersed into
xylene twice. After the sections drying, neutral gum was added on the section
for sealing. Negative controls were incubated and
processed identically with omission of the primary antibody. The brown
deposition was observed under a microscope.
Cell culture and components
treatment
Human tubular epithelial
cell line HK-2 was purchased from Nanjing KeyGEN Biological Development Co., Ltd.
Cells were cultured with DMEM/F12 incomplete high-sugar medium
containing 10% FBS in an
atmosphere of 95% air and 5% CO2 at 37°C for 24 h. Alisma triterpenes were diluted by DMEM/F12
incomplete high-sugar medium to 1.0×10-3, 5×10-4, 2.5×10-4,
1.25×10-4, 6.25×10-5, 3.125 ×10-5, 1.5625×10-5,
7.8125×10-6 g/mL concentrations for further experiments.
1.11 Cell activity detection of HK-2 cells by MTT
colorimetry
Table 1: The fragment
information of triterpenes in alisma
|
No. |
Compound |
TR
(min) |
(+) ESI-MS m/z |
UVmax
(nm) |
(+) ESI-MS m/z
(ion fragments) |
Formula |
|
1 |
13.752 |
505.20 |
208, 4 |
505.2, 415.25 |
C30H48O6 |
|
|
2 |
16-oxo-alisol A 24-actetate |
14.231 |
547.30 |
208, 4 |
547.25, 529.20,
358.10 |
C32H50O7 |
|
3 |
16-oxo-alisol A 23-actetate |
14.792 |
547.30 |
208, 4 |
||
|
4 |
Alisol C |
14.975 |
487.20 |
208, 4 |
487.20, 431.15,
358.10 |
C30H46O5 |
|
5 |
Alisol C 23-acetate |
17.810 |
529.20 |
208, 4 |
529.20, 358.10 |
C32H48O6 |
%20373-380_files/image002.png)
Fig. 1: The
chemical structure of Alisma Triterpenes including 16-oxo-alisol A,
16-oxo-alisol A 24-actetate, 16-oxo-alisol A 23-actetate, Alisol C, Alisol C
23-acetate
After incubation, the cells were
incubated with the final concentration of drug solution for 24 h in an
atmosphere of 95% air and 5% CO2 at 37°C. The medium without drug
solution was used as blank control. Then 0.5 mg/mL MTT (100 μL) was added into each well of the
media. The cells were incubated for four h. Then 100 μL of dimethyl sulfoxide (DMSO) was added into each well and
vibrated at 37°C for 10 min to dissolve crystals. The absorbance optical
density value of cells was measured in an automatic microplate reader (Thermo, USA). The surviving fraction of cells
was calculated for each assay as the percentage of cell viability = (optical
density of components group - optical density of blank group) / (optical
density of cell blank group - optical density of blank plate) × 100%.
Immunocytochemistry
After being treated
with drugs for 24 h, 4% formaldehyde solution was used to
fix the cells for 24 h and the cells were placed in antigen repair buffer,
boiled for 20 min. The cells were dripped with 3% H2O2-PBS
solution to block endogenous peroxidase at room temperature for fifteen min. Then the
cells were blocked with 10% primary serum or 5% BSA (4°C, overnight). After being washed
with PBS for 4 times, the sections were incubated with biotin-conjugated secondary antibody at 37°C for 30 min.
Appropriate amount of DAB solution was added on sections for staining and color
development. The sections were redyed with hematoxylin staining solution for 10 min and rinsed with distilled water. After the cells drying, neutral gum
was added on the section for sealing. Negative controls were incubated and
processed identically with omission of the primary antibody. The expression of
visualization was observed and photographed under a microscope.
Statistical analysis
In this study, the obtained
data are shown as means ± standard deviation (SD). S.P.S.S. 16.0 software
(Chicago, I.L., United States) was used to analyze the statistical differences
between two groups or among more than two groups using One-way analysis of
variance (ANOVA) with Bonferroni post hoc test. A value of
P < 0.05 was regarded as
statistically significant.
Results
Analysis and determination
for triterpenes in alisma
In this study,
high-performance liquid chromatography-quadrupole time-of-flight mass
spectrometry (HPLC-Q-TOF-MS) was used to analyze the triterpenes in alisma. The results showed that triterpenes in alisma
contain mainly 16-oxo-alisol A, 16-oxo-alisol A 24-actetate, 16-oxo-alisol A
23-actetate, Alisol C, Alisol C 23-acetate, and so on (Fig. 1). The detailed fragments information is listed in Fig. 2 and
Table 1.
Table 2: Diuretic
activities of Alisma triterpenes on urine volume and urine Na+, K+
and Cl- concentrations in rats (means ± SD, n=6). Note: compared with the blank
control group, ***P < 0.001, **P < 0.01, *P < 0.05
|
Groups |
Dosage (mg/kg/d) |
Urine volume (mL) |
Na+
(mmol/L) |
K+
(mmol/L) |
Cl-
(mmol/L) |
|
Blank control |
- |
4.33 ± 1.78 |
19.55 ± 1.91 |
11.79 ± 1.12 |
20.7 ± 2.83 |
|
Furosemide |
20 |
15.75 ± 1.81 *** |
88.87 ± 2.86 *** |
28.91 ± 15.08 * |
95.8 ± 1.65 *** |
|
High-dose triterpenes |
80 |
11.00 ± 1.04 * |
41.4 ± 5.16 ** |
25.02 ± 2.46 * |
36.93 ± 7.32 * |
|
Medium-dose triterpenes |
40 |
9.63 ± 1.53 * |
33.9 ± 16.12 |
20.66 ± 9.72 |
25.6 ± 7.92 |
|
Low-dose triterpenes |
20 |
7.50 ± 3.81 |
19.9 ± 13.72 |
19.58 ± 3.93 |
22.33 ± 9.52 |
%20373-380_files/image004.jpg)
Fig. 2: MS ion flow diagram (A) and ion spectra (B)
Alisma triterpenes
increased urine volume and urine concentrations of Na+, K+
and Cl- in water load model rats
Compared with the blank control, positive control
Furosemide significantly increased urine volume and the concentrations of Na+,
K+ and Cl- in urine (P
< 0.001 or P < 0.05).
Interestingly, the treatment of medium-dose triterpene group (40 mg/kg/d) and
high-dose triterpene group (80 mg/kg/d) enhanced urine volume remarkably when
compared with the blank group. Similarly, urine Na+, K+
and Cl- concentrations were also significantly increased by the
treatment with triterpene of 80 mg/kg/d (P
< 0.05) (Fig. 3 and Table 2). These results suggested that alisma
triterpenes could increase the urine volume and promote the excretion of
electrolytes in the urine of water - stressed rats, showing a certain diuretic
effect.
Effects of alisma triterpenes on the expression of AQP-2 protein and
mRNA in rat kidney
%20373-380_files/image006.png)
Fig. 3: Effects of Alisma triterpenes on the urine volume and
the excretion of electrolytes in the urine of water - stressed rats (means ± SD, n=6)
A. The
urine volume in the urine of water - stressed rats; B. The excretion of Na+ in the urine of water - stressed
rats; C. The excretion of K+
in the urine of water - stressed rats; D.
The excretion of Cl- in the urine of water - stressed rats. ***P < 0.001, **P < 0.01, *P < 0.05
compared with the blank control group
%20373-380_files/image008.png)
Fig. 4: Effects of Alisma triterpenes on AQP-2 in rats kidney (means ± SD, n=6)
A. The
examination of AQP-2 level in kidney tissue by western blotting; B. The examination of AQP-2 mRNA level
in kidney tissue by RT-PCR; C. The
relative expression of AQP-2 in kidney tissue by immunochemistry. a, b, c, d, e
respectively represent blank control group, furosemide group, high-dose
triterpenes group, medium-dose triterpenes group and low-dose triterpenes
group; ***P < 0.001, **P < 0.01, *P < 0.05, compared with the blank
control group
As
shown in Fig. 4A and 4C, both Western Blot and Immunochemistry results showed
that a significant reduction on AQP-2 expression in rat kidney tissue treated with
alisma triterpenes, especially in high-dose group and medium-dose group (P < 0.01 or P < 0.05). Furthermore, RT-PCR results showed that the
expression of AQP-2 mRNA in renal medullary was significantly reduced in
furosemide, high-dose triterpenes and medium-dose triterpenes groups, when
compared with blank group (Fig. 4B; P <
0.01 or P < 0.05). Therefore, it
could be concluded that alisma triterpenes could reduce the expression level of
renal AQP-2 in rats.
Optimal concentration for alisma triterpenes to survival rate of HK-2
cells
Table 3: Cell viability of
different concentration of ZTCs in HK-2 cells (means ± SD, n=6)
|
Groups |
Dosage (g/mL) |
Relative cell survival
rate (%) |
|
Blank control |
- |
100 |
|
Triterpenes |
7.8125 × 10-6 |
96.13 ± 4.37 |
|
1.5625 × 10-5 |
95.37 ± 2.86 |
|
|
3.125 × 10-5 |
94.64 ± 3.91 |
|
|
6.25 × 10-5 |
92.97 ± 2.41 |
|
|
1.25 × 10-4 |
91.24 ± 3.83 |
|
|
2.5 × 10-4 |
92.75 ± 2.30 |
|
|
5.0 × 10-4 |
90.52 ± 3.11 |
|
|
1.0 × 10-3 |
86.86 ± 1.91 |
%20373-380_files/image010.png)
Fig. 5: Effects of
Alisma triterpenes on AQP-2 in HK-2 cells
(means ± SD, n=6)
A and B. The examination of AQP-2 level
in HK-2 cells by western blotting
C and E. The relative expression of AQP-2
in HK-2 cells by immunochemistry. a, b, c, d, respectively represent blank
control group, high-dose triterpenes group, medium-dose triterpenes group and
low-dose triterpenes group
D. The examination of AQP-2 mRNA level in HK-2 cells by
RT-PCR; *P < 0.05,
compared with blank control group
In order to explore the regulation of alisma triterpenes on AQP-2 in vitro, the suitable concentration, namely, minimum concentration
affecting cell viability. When the cell survival rate is greater than 95%, the
drug is considered to be non-toxic to cells. MTT results showed that the cell survival
rate was (95.37 ± 2.86%), indicating that alisma triterpenes has no obvious cytotoxicity to HK-2 cells at this concentration (Table
3). Therefore, 3.125×10-5 g/mL, 1.5625×10-5 g/mL and 7.8125×10-6 g/mL were used for subsequent experimental studies.
The regulation of Alisma triterpenes on
AQP-2 in HK-2 cells
As shown in Fig. 5A and 5B, the expression of AQP-2
in cells was significantly decreased in the high-dose and medium-dose
triterpenes groups (3.125 × 10-5g/mL, 1.5625 × 10-5g/mL)
(P < 0.05) when compared with the
blank control. RT-PCR results (Fig. 5D) showed that the relative expression
level of AQP-2 mRNA was also been reduced by the treatment of high-dose
triterpenes (P < 0.05). In
immunocytochemistry experiment, a significant reduction on AQP-2 positive brown
deposit can be seen in triterpenes-treated cells at the concentration of
high-dose and medium-dose (Fig. 5C and 5E). The phenomena in vitro experiments further suggested that the regulation of AQP-2
protein and mRNA contributed to the diuretic effect of alisma terpenoids.
Discussion
Modern pharmacological studies showed that alisma
has a variety of biological activities, such as
diuretics, antisteatotic, antioxidant, antilipoapoptotic, hepatoprotective,
anti-inflammatory, antifibrotic, antiobesity, hypoglycemic and hypolipidemic
activities (Shu et al. 2016; Choi et al. 2019). Many studies were
conducted on the isolation and pharmacological activities of triterpenes and
sesquiterpenes, which are the main and most significant components of alisma
(Ma et al. 2016). Despite ongoing studies, pharmacodynamic
effects and mechanisms of constituents in alisma have still not revealed.
This study focused on looking for the pharmacodynamic effects and mechanisms of
triterpenes in alisma.
Aquaporin is located
in the collecting duct and is regulated by antidiuretic hormone. AVP triggers
the redistribution of AQP-2 from intracellular vesicles (Schrade et al.
2018). AVP is released from the posterior lobe of the neurohypophysis and
promotes the translocation or transport of AQP-2 from intracellular vesicles of
the cells. From vivo animal
experiments and vitro cell
experiments, we found that terpenoids can achieve diuretic effect by
regulating renal aquaporins. It is concluded that terpenoids reduce the expression of aquaporin by reducing the secretion of AVP.
Conclusion
At present, research on pharmacology and toxicology of alisma has made great progress. But there are still
some problems, with modern pharmacology and toxicology research methods and
related scientific research and technology development, alisma pharmacology and
toxicology studies will get further development and perfection, these studies
can provide more safe and reasonable clinical application of alisma guidance
and contribution, also can better promote the development of alisma resources
in clinical application.
Acknowledgements
The author sincerely
acknowledges the support of these funds for the research of this project: National Natural Science Foundation
of P.R. China (No. 81603382), Key research projects on modernization of
traditional Chinese medicine (2018YFC1706900), "333 Project" research
projects of Jiangsu province (BRA5475); and "Double First-Class"
University project of China Pharmaceutical University (CPU2018GF07,
CPU2018PZQ19).
Author Contributions
Important contributions to design and also to
prepare this manuscript: X-BJ
applied for the grant, conceived and designed this study. L.F. prepared the experiment and
designed this study. CL, Z-LL, X-HL
and Q-LL, performed all of the experiments. S-TP analyzed the data and wrote the manuscript. All authors
participated in the preparation of the manuscript and approved the final
version.
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