Effects of a Chinese herbal extract on the intestinal tract and aquaporin in Adriamycin-induced nephropathy.

Wuling Decoction is a traditional Chinese medicine that has been used to open knots, benefit water, transform Qi, return fluid, and has a significant effect on strengthening the spleen and removing dampness. To explore the effects of Wuling Decoction on the intestinal tract and aquaporin in Adriamycin-induced nephropathy, 45 specific pathogen free (SPF) Wistar rats were randomly divided into a blank control group (5 rats), Dosing control group (10 rats), Adriamycin nephropathy model group (10 rats), diarrhea group (10 rats), and an Adriamycin nephropathy diarrhea model group (10 rats). The tissue localization of aquaporin (AQP) was determined by immunohistochemistry. The expression of AQP mRNA and protein was measured by RT-PCR and western blot analysis, respectively. The results indicated that Wuling Decoction causes excretion of AQP2 through the urine, regulates AQP2 levels, and exerts diuretic and anti-diarrheal effects. It also regulates the levels of antidiuretic hormone (ADH) and arginine vasopressin (AVP), affects water absorption rate, and reduces the level of cyclic adenosine monophosphate (cAMP) in each tissue, thus reducing the absorption of AQP2 to water. Wuling Decoction promoted AQP2 expression in the nephropathy model group and inhibited AQP2 expression in the diarrhea group. Wuling Decoction increased the expression of aquaporin in the intestinal tract, reduced the water content of stool by promoting the absorption of water in the intestinal tract, inhibited the expression of aquaporin and its regulatory factors in nephridia tissue, and reduced the reabsorption of water to increase urine volume, to decrease the occurrence of diarrhea.

Immunohistochemical Expression of AQP2 and HSP70 in Broiler Kidney Tissue Treated with Salix tetrasperma Roxb. Extract under Heat Exposure.

The administration of plant extracts to broilers may be a way to mitigate the effects of heat stress. The importance of AQP2 and HSP70 compounds in maintaining the homeostasis of the chicken body when it is subjected to heat stress is well established. This study aims to determine the effect of giving the ethanolic extract of the leaves of Salix tetrasperma Roxb. on the immunohistochemical expression of AQP2 and HSP70 in exposed and unexposed broiler kidney tissue. This study used 36 samples of 28-day-old chicken kidneys. Chickens were kept in individual cages, provided with feed and drinking water ad libitum. The design used was a completely randomized design with 6 treatments and 6 replications: (a) chickens were reared in conditions exposed to heat (HS + 0); (b) chickens were reared in conditions exposed to heat and given Salix extract at a dose of 50 mg/L drinking water (HS + 50); (c) chickens were reared under heat-exposed conditions and given Salix extract at a dose of 100 mg/L drinking water (HS + 100); (d) chickens were reared in conditions without exposure to heat (n-HS + 0); (e) chickens were reared in conditions without exposure to heat and given Salix extract at a dose of 50 mg/L drinking water (nHS + 50); and (f) chickens were reared in conditions exposed without exposure to heat and given 100 mg/L drinking water (nHS + 100) of Salix extract. Salix extract was given for 24 hours and was renewed every 6 hours. The results showed that giving Salix extract 100 mg/L in drinking water to chickens exposed to heat (HS + 100) reduced the value of the H/L ratio. Giving Salix extract 50-100 mg/L in drinking water caused an upregulated AQP2 expression; on the other hand, it downregulated HSP-70 expression, in chicken kidney tubules both exposed to heat stress and nonexposed to heat stress. In conclusion, exposure to heat stress in broiler chickens and giving Salix extract can increase the formation of aquaporin 2 compounds and suppress the formation of HSP70.

Organ Specific Differences in Alteration of Aquaporin Expression in Rats Treated with Sennoside A, Senna Anthraquinones and Rhubarb Anthraquinones.

Senna and rhubarb are often used as routine laxatives, but there are differences in mechanism of action and potential side effects. Here, we studied metabolites of senna anthraquinones (SAQ), rhubarb anthraquinones (RAQ) and their chemical marker, sennoside A (SA), in a rat diarrhea model. In in vitro biotransformation experiments, SAQ, RAQ and SA were incubated with rat fecal flora solution and the metabolites produced were analyzed using HPLC. In in vivo studies, the same compounds were investigated for purgation induction, with measurement of histopathology and Aqps gene expression in six organs. The results indicated that SAQ and RAQ had similar principal constituents but could be degraded into different metabolites. A similar profile of Aqps down-regulation for all compounds was seen in the colon, suggesting a similar mechanism of action for purgation. However, in the kidneys and livers of the diarrhea-rats, down-regulation of Aqps was found in the RAQ-rats whereas up-regulation of Aqps was seen in the SAQ-rats. Furthermore, the RAQ-rats showed lower Aqp2 protein expression in the kidneys, whilst the SA-rats and SAQ-rats had higher Aqp2 protein expression in the kidneys. This may have implications for side effects of SAQ or RAQ in patients with chronic kidney or liver diseases.

Er Shen Wan extract alleviates polyuria and regulates AQP 2 and AVPR 2 in a rat model of spleen-kidney Yang deficiency-induced diarrhea.

ETHNOPHARMACOLOGICAL RELEVANCE: Er Shen Wan (ESW), has been empirically used for treating spleen-kidney Yang deficiency (SKYD) syndrome in Traditional Chinese medicine (TCM) for centuries and shows a variety of activities. The medicinal formula is a mixture of two component herbs, Psoraleae Fructus (PF, Bu-Gu-Zhi in Chinese) and Myristicae Semen (MS, Rou-Dou-Kou in Chinese). The current study was designed to evaluate ESWP antidiuretic treatment of polyuria and to explore potential mechanisms of renal water metabolism in the rat model of SKYD-induced diarrhea. MATERIALS AND METHODS: An animal model of 'SKYD-induced diarrhea syndrome' has been established to evaluate the therapeutic effect and action mechanism according to the clinical syndrome and symptoms. The optimal dose (3.5 g/kg) of ESWP was given to rats by gavage for two weeks. Urinary volumes after 24 h were recorded. After the end of the trial, macroscopic morphological and histological examination of the kidney were conducted. Serum levels of Arginine vasopressin (AVP) and aldosterone (ALD) were also measured. Additionally, quantitative real-time RT-PCR (RT-qPCR) and immunohistochemistry (IHC) analyses were performed to clarify the regulation of aquaporin 2 (AQP 2) and arginine vasopressin type 2 receptor (AVPR 2) in the kidney at the gene and tissue expression levels respectively. RESULTS: After the administration of ESWP, urinary output volume after 24 h was found to be significantly decreased in rats. Elevated plasma levels of AVP and ALD were detected. Histological kidney damage appeared to be impeded, and histological disease scores were reduced. In addition, the expression levels of AQP 2 and AVPR 2 were significantly increased. CONCLUSION: This study suggests that ESWP may elicit significant effects on the treatment of polyuria. Potential mechanisms at least partially involve hormone regulation, and alleviating renal pathological damage. Simultaneously, ESWP may alter renal water absorption by increasing AQP 2 and AVPR 2 expression levels. Thus, the in vivo experimental evidence indicates that ESWP has a therapeutic effect on the SKYD syndrome, which is consistent with its traditional usage.

Expression of AQP2, AQP4 and AQP 8 in mouse intestine induced by unprocessed and processed Euphorbia lathyris.

The present research was designed to study expression of AQP2, AQP4 and AQP8 in mouse intestines induced by unprocessed and processed Euphorbia lathyris. KM mice were given by different dose lavage of unprocessed and processed Euphorbia lathyris, Euphorbia factor L1, Euphorbia factor L2, Euphorbia factor L3. Samples of mouse intestine were collected for protein levels of AQP2, AQP 4 and AQP 8 which were assessed by immunohistochemical staining and mRNA expression of AQP2, AQP 4 and AQP 8 which were quantified by Real Time-PCR. Comparing to the normal control group, the protein levels of AQP2, AQP 4 and AQP 8 were significantly decreased (P<0.05)by Semen Euphorbiae group and Semen Euphorbiae Pulveratum group (unprocessed and processed Euphorbia lathyris) induced. Protein expression of AQP2, AQP 4 and AQP 8 in the Euphorbia factor L1, Euphorbia factor L2 and Euphorbia factor L3 group were not significantly lower than normal control group. There had no differences on the levels of AQP2 and AQP 8 mRNA expressions between the high-dose group of semen Euphorbiae group, semen Euphorbiae Pulveratum group and positive control group, while significantly lower than normal control group (P<0.05). Expression of AQP4 mRNA in the Semen Euphorbiae group and Semen Euphorbiae Pulveratum group has not significantly decreased. But levels of AQP2, AQP 4 and AQP 8 mRNA in the Euphorbia factor L1 group had no significant differences in normal control group and positive control group. These findings suggest that semen Euphorbiae could regulate expression of AQP2, AQP 4 and AQP 8 protein and mRNA, which may be the possible one reason of semen Euphorbiae induces diarrhea. The semen Euphorbiae group has more significant effects on the levels of AQP2, AQP 4 and AQP 8 protein and mRNA than semen Euphorbiae Pulveratum group, which may be one of the mechanisms of processing attenuation.

The soluble (Pro) renin receptor does not influence lithium-induced diabetes insipidus but does provoke beiging of white adipose tissue in mice.

Earlier we reported that the recombinant soluble (pro) renin receptor sPRR-His upregulates renal aquoporin-2 (AQP2) expression, and attenuates polyuria associated with nephrogenic diabetes insipidus (NDI) induced by vasopressin type 2 receptor (V2R) antagonism. Patients that receive lithium therapy develop polyuria associated NDI that might be secondary to downregulation of renal AQP2. We hypothesized that sPRR-His attenuates indices of NDI associated with lithium treatment. Eight-week-old male C57/BL6 mice consumed chow supplemented with LiCl (40 mmol/kg diets) for 14 days. For the last 7 days mice received either sPRR-His [30 µg/(kg day), i.v.; sPRR] or vehicle (Veh) via minipump. Control (Con) mice consumed standard chow for 14 days. Compared to Con mice, 14-d LiCl treatment elevated water intake and urine volume, and decreased urine osmolality, regardless of sPRR-His or Veh administration. These data indicate that sPRR-His treatment does not attenuate indices of NDI evoked by lithium. Unexpectedly, epididymal fat mass was lower, adipocyte UCP1 mRNA and protein expression were higher, and multilocular lipid morphology was enhanced, in LiCl-fed mice treated with sPRR-His versus vehicle. The beiging of white adipose tissue is a novel metabolic benefit of manipulating the sPRR in the context of lithium-induced NDI.

How impaired efficacy happened between Gancao and Yuanhua: Compounds, targets and pathways.

As recorded in Traditional Chinese Medicine (TCM) theory, Gancao (Glycyrrhizae Radix et Rhizoma) could weaken the pharmacological effect or increase the toxicity of Yuanhua (Genkwa Flos). However, the theory has been suspected due to lack of evidence. Here, we investigate whether Gancao could weaken Yuanhua's diuretic effect, if so, which chemicals and which targets may be involved. Results showed that Yuanhua exerted diuretic effect through down-regulating renal AQP 2, without electrolyte disturbances such as K+ loss which has been observed as side-effect of most diuretics. Gancao had no diuretic effect, but could impair Yuanhua's diuretic effect through up-regulating renal AQP 2. Glycyrrhetinic acid (GRA) in Gancao could up-regulate AQP 2 and counteract the AQP 2 regulation effect of Yuanhuacine (YHC) and Ginkwanin (GKW) in Yuanhua. Network pharmacology method suggested that YHC, GKW and GRA could bind to MEK1/FGFR1 protein and influence ERK-MAPK pathway, which was verified by Western blotting. This study supports TCM theory and reminds that more attention should be paid to the safety and efficacy problems induced by improper combination between herbs. Moreover, we suggested that promising diuretics with less side effects can be developed from Chinese Medicines such as Yuanhua.

Wiryeongtang regulates hypertonicity-induced expression of aquaporin-2 water channels in mIMCD-3 cells.

The kidneys have a key role in the homeostasis of water excretion and reabsorption. Water channels, particularly aquaporin-2 (AQP2), are important proteins in water homeostasis in the body through the short­term and long-term regulation of water permeability. Wiryeongtang (WRT) is a well-known traditional oriental medicine, which is used for the treatment of chronic edema and dysuresia. The aim of the present study was to evaluate the inhibitory effect of WRT on the hypertonicity-induced expression of AQP2 in the inner medullary collecting duct cell line (IMCD­3). Western blotting, reverse transcription­polymerase chain reaction and immunofluorescence analysis were performed to determine the effect of WRT under hypertonic stress. WRT attenuated the 175 mM NaCl hypertonic stress­induced increases in protein and mRNA levels of AQP2 and apical membrane insertion in a concentration­dependent manner. However, no differences were observed in the levels of AQP1, AQP3 or AQP4 between the hypertonic stress and WRT groups. WRT attenuated the hypertonicity-induced phosphorylation of glucocorticoid-inducible protein kinase 1. In addition, the mRNA expression of tonicity­responsive enhancer binding protein was attenuated by WRT under hypertonic stress. Pretreatment with WRT also decreased the hypertonic stress­induced expression of AQP2, as with KT5720, a protein kinase A inhibitor. These results provided evidence of the beneficial effect of the traditional formula WRT in regulating water balance in hypertonic stress of the renal collecting ducts.

Renal response to L-arginine in diabetic rats. A possible link between nitric oxide system and aquaporin-2.

The aim of this study was to evaluate whether L-Arginine (L-Arg) supplementation modifies nitric oxide (NO) system and consequently aquaporin-2 (AQP2) expression in the renal outer medulla of streptozotocin-diabetic rats at an early time point after induction of diabetes. Male Wistar rats were divided in four groups: Control, Diabetic, Diabetic treated with L-Arginine and Control treated with L-Arginine. Nitric oxide synthase (NOS) activity was estimated by [14C] L-citrulline production in homogenates of the renal outer medulla and by NADPH-diaphorase staining in renal outer medullary tubules. Western blot was used to detect the expression of AQP2 and NOS types I and III; real time PCR was used to quantify AQP2 mRNA. The expression of both NOS isoforms, NOS I and NOS III, was decreased in the renal outer medulla of diabetic rats and L-Arg failed to prevent these decreases. However, L-Arg improved NO production, NADPH-diaphorase activity in collecting ducts and other tubular structures, and NOS activity in renal homogenates from diabetic rats. AQP2 protein and mRNA were decreased in the renal outer medulla of diabetic rats and L-Arg administration prevented these decreases. These results suggest that the decreased NOS activity in collecting ducts of the renal outer medulla may cause, at least in part, the decreased expression of AQP2 in this model of diabetes and constitute additional evidence supporting a role for NO in contributing to renal water reabsorption through the modulation of AQP2 expression in this pathological condition. However, we cannot discard that another pathway different from NOS also exists that links L-Arg to AQP2 expression.

Byakkokaninjinto prevents body water loss by increasing the expression of kidney aquaporin-2 and skin aquaporin-3 in KKAy mice.

Byakkokaninjinto (BKN) is an herbal medicine used for the relief of diuresis, thirst and dermal pruritus that are associated with diabetes. The effects of BKN on the expression of aquaporins (AQPs) in the kidney, salivary gland and skin were investigated in order to clarify the mechanism of drug action. Seven-week-old KKAy mice were given feed containing 4.5% BKN for 4 weeks. Compared with the control group, BKN administration did not affect the blood glucose and insulin concentration. However, water intake and urine volume were significantly reduced. AQP2 protein expression in the kidney inner medullary was significantly increased after BKN administration. AQP3 mRNA and protein expression in skin tissue was significantly increased after BKN administration. However, BKN administration did not affect AQP5 mRNA expression in the salivary gland. These results suggest that BKN treatment relieves diuresis, thirst, and dermal pruritis by increasing kidney AQP2 expression and skin AQP3 expression.

Estrogenic effects of a Kampo formula, Tokishakuyakusan, in parous ovariectomized rats.

Female hormone-dependent cancers and other diseases pose a serious health threat for women, and low-risk medicines against such cancers have not yet been discovered. The present study examines the effects of the traditional Chinese herbal mixture, Tokishakuyakusan (TS) and 17beta-estradiol on the uterus of parous ovariectomized rats. Uterine atrophy that causes a reduction in uterine tissue and the uterine cavity area, was induced by ovariectomy, and slightly recovered by the daily oral administration of TS for two weeks (1000 mg/kg body weight). TS restored the decreased plasma estradiol concentration due to ovariectomy. However the yeast two-hybrid assay showed that TS did not bind estrogen receptors alpha and beta and immunohistochemical staining revealed that 17beta-estradiol stimulated the protein expression of estrogen receptor alpha, progesterone receptor, c-fos and c-jun in the uterus, whereas TS did not. These results suggest that TS might be useful for treating menopausal syndromes among women, as well as for patients when hormone replacement therapy (HRT) with estrogen is contraindicated.

Long-term aldosterone treatment induces decreased apical but increased basolateral expression of AQP2 in CCD of rat kidney.

The purpose of the present studies was to determine the effects of high-dose aldosterone and dDAVP treatment on renal aquaporin-2 (AQP2) regulation and urinary concentration. Rats were treated for 6 days with either vehicle (CON; n = 8), dDAVP (0.5 ng/h, dDAVP, n = 10), aldosterone (Aldo, 150 microg/day, n = 10) or combined dDAVP and aldosterone treatment (dDAVP+Aldo, n = 10) and had free access to water with a fixed food intake. Aldosterone treatment induced hypokalemia, decreased urine osmolality, and increased the urine volume and water intake in ALDO compared with CON and dDAVP+Aldo compared with dDAVP. Immunohistochemistry and semiquantitative laser confocal microscopy revealed a distinct increase in basolateral domain AQP2 labeling in cortical collecting duct (CCD) principal cells and a reduction in apical domain labeling in Aldo compared with CON rats. Given the presence of hypokalemia in aldosterone-treated rats, we studied dietary-induced hypokalemia in rats, which also reduced apical AQP2 expression in the CCD but did not induce any increase in basolateral AQP2 expression in the CCD as observed with aldosterone treatment. The aldosterone-induced basolateral AQP2 expression in the CCD was thus independent of hypokalemia but was dependent on the presence of sodium and aldosterone. This redistribution was clearly blocked by mineralocorticoid receptor blockade. The increased basolateral expression of AQP2 induced by aldosterone may play a significant role in water metabolism in conditions with increased sodium reabsorption in the CCD.

Candesartan prevents long-term impairment of renal function in response to neonatal partial unilateral ureteral obstruction.

Angiotensin II (ANG II) plays an important role in the development of obstructive nephropathy. Here, we examined the effects of the ANG II receptor type 1 (AT1R) blockade using candesartan on long-term renal molecular and functional changes in response to partial unilateral ureteral obstruction (PUUO). Newborn rats were subjected to severe PUUO or sham operation (Sham) within the first 48 h of life. Candesartan was provided in the drinking water (10 mg.kg(-1).day(-1)) from day 21 of life until 10 wk of age. Renal blood flow (RBF) was evaluated by MRI, glomerular filtration rate (GFR) was measured using the renal clearance of (51)Cr-EDTA, and the renal expression of Na-K-ATPase and the collecting duct water channel aquaporin-2 (AQP2) was examined by immunoblotting and immunocytochemistry. At 10 wk of age, PUUO significantly reduced RBF (0.8 +/- 0.1 vs. 1.6 +/- 0.1 ml.min(-1).100 g body wt(-1); P < 0.05) and GFR (37 +/- 16 vs. 448 +/- 111 microl.min(-1).100 g body wt(-1); P < 0.05) compared with Sham. Candesartan prevented the RBF reduction (PUUO+CAN: 1.6 +/- 0.2 vs. PUUO: 0.8 +/- 0.1 ml.min(-1).100 g body wt(-1); P < 0.05) and attenuated the GFR reduction (PUUO+CAN: 265 +/- 68 vs. PUUO: 37 +/- 16 microl.min(-1).100 g body wt(-1); P < 0.05). PUUO was also associated with a significant downregulation in the expression of Na-K-ATPase (75 +/- 12 vs. 100 +/- 5%, P < 0.05) and AQP2 (52 +/- 15 vs. 100 +/- 4%, P < 0.05), which were also prevented by candesartan (Na-K-ATPase: 103 +/- 8 vs. 100 +/- 5% and AQP2: 74 +/- 13 vs. 100 +/- 4%). These findings were confirmed by immunocytochemistry. Consistent with this, candesartan treatment partly prevented the reduction in solute free water reabsorption and attenuated fractional sodium excretion in rats with PUUO. In conclusion, candesartan prevents or attenuates the reduction in RBF, GFR and dysregulation of AQP2 and Na-K-ATPase in response to congenital PUUO in rats, suggesting that AT1R blockade may protect the neonatally obstructed kidney against development of obstructive nephropathy.