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.

Effects of SGLT2 inhibitor ipragliflozin alone and combined with pioglitazone on fluid retention in type 2 diabetic mice with NASH.

Several antidiabetic agents, including thiazolidinediones and sodium-glucose cotransporter (SGLT) 2 inhibitors, attenuate the symptoms of nonalcoholic steatohepatitis (NASH). However, thiazolidinediones have serious side effects such as fluid retention and increased risk of congestive heart failure. We examined the effects of SGLT2 inhibitor ipragliflozin, pioglitazone, and ipragliflozin + pioglitazone on fluid retention in type 2 diabetic mice with NASH. Four-week repeated administration of pioglitazone caused significant increases in heart weight (31% increase in 30 mg/kg pioglitazone-treated group compared to vehicle-treated group) concomitant with fluid retention, as estimated by a decrease in plasma osmolality and increase in water intake/urine volume ratio. In addition, pioglitazone significantly increased (by 1.5 to 2-fold) mRNA expression of α, ß, and γ subtypes of ENaC and AQP2 and 3 subtypes in the renal medulla. Thus, pioglitazone-induced fluid retention may arise from enhanced reabsorption of sodium and water associated with increased expression of these channels in the kidney. In contrast, ipragliflozin alone did not induce these symptoms and did not affect ENaC or AQP expression. Combination treatment with ipragliflozin + pioglitazone attenuated these symptoms by ipragliflozin-induced osmotic diuresis. These findings demonstrate that treatment with ipragliflozin monotherapy or coadministered with pioglitazone may be a potential therapeutic option for the treatment of type 2 diabetes with NASH without fluid retention as a side effect.

Altered Iris Aquaporin Expression and Aqueous Humor Osmolality in Glaucoma.

Purpose: Aquaporins (AQPs) facilitate transmembrane osmotic water transport and may play a role in iris fluid conductivity, which is implicated in the pathophysiology of glaucoma. In this study, we compared the iris expression of AQPs and aqueous osmolality between primary angle closure glaucoma (PACG), primary open-angle glaucoma (POAG), and nonglaucoma eyes. Methods: AQP1-5 transcripts from a cohort of 36 PACG, 34 POAG and 26 nonglaucoma irises were measured by quantitative real-time PCR. Osmolality of aqueous humor from another cohort of 49 PACG, 50 POAG, and 50 nonglaucoma eyes were measured using an osmometer. The localization of AQP1 in both glaucoma and nonglaucoma irises was determined by immunofluorescent analysis. Results: Of the five AQP genes evaluated, AQP1 and AQP2 transcripts were significantly upregulated in both PACG (3.48- and 8.07-fold, respectively) and POAG (3.12- and 11.58-fold, respectively) irises relative to nonglaucoma counterparts. The aqueous osmolalities of PACG (303.68 mmol/kg) and POAG (300.79 mmol/kg) eyes were significantly lower compared to nonglaucoma eyes (312.6 mmol/kg). There was no significant difference in expression of AQP transcripts or aqueous osmolality between PACG and POAG eyes. Conclusions: PACG and POAG eyes featured significant increase in AQP1 and AQP2 expression in the iris and reduced aqueous osmolality compared to nonglaucoma eyes. These findings suggest that the iris may be involved in altered aqueous humor dynamics in glaucoma pathophysiology. Because PACG did not differ from POAG in both properties studied, it is likely that they are common to glaucoma disease in general.

Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in Pichia pastoris for Solid-State NMR.

Solid-state nuclear magnetic resonance (SSNMR) is a powerful biophysical technique for studies of membrane proteins; it requires the incorporation of isotopic labels into the sample. This is usually accomplished through over-expression of the protein of interest in a prokaryotic or eukaryotic host in minimal media, wherein all (or some) carbon and nitrogen sources are isotopically labeled. In order to obtain multi-dimensional NMR spectra with adequate signal-to-noise ratios suitable for in-depth analysis, one requires high yields of homogeneously structured protein. Some membrane proteins, such as human aquaporin 2 (hAQP2), exhibit poor expression, which can make producing a sample for SSNMR in an economic fashion extremely difficult, as growth in minimal media adds additional strain on expression hosts. We have developed an optimized growth protocol for eukaryotic membrane proteins in the methylotrophic yeast Pichia pastoris. Our new growth protocol uses the combination of sorbitol supplementation, higher cell density, and low temperature induction (LT-SEVIN), which increases the yield of full-length, isotopically labeled hAQP2 ten-fold. Combining mass spectrometry and SSNMR, we were able to determine the nature and the extent of post-translational modifications of the protein. The resultant protein can be functionally reconstituted into lipids and yields excellent resolution and spectral coverage when analyzed by two-dimensional SSNMR spectroscopy.

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.

Morphometric changes and AQP2 expression in kidneys of young male rats exposed to chronic stress and a high-sucrose diet.

OBJECTIVE: Consumption of a cafeteria-like diet and chronic stress have a negative impact on kidney function and morphology in adult rats. However, the interaction between chronic restraint stress and high-sucrose diet on renal morphology in young rats is unknown. A high-sucrose diet does not modify serum glucose levels but reduces serum corticosterone levels in stressed young rats, in this way it is confusing a possible potentiate or protector effect of this diet on kidney damage induced by stress. METHODS: Wistar male rats at 4 weeks of age were randomly assigned into 4 groups: control (C), stressed (St), high-sucrose diet (S30), and chronic restraint stress plus a 30% sucrose diet (St + S30). Rats were fed with a standard chow and tap water (C group) or 30% sucrose diluted in water (S30 group). Chronic restraint stress consisted of 1-h daily placement into a plastic cylinder, 5 days per week, and for 4 weeks. RESULTS: Stressed rats exhibited a low number of corpuscles, glomeruli, high number of mesangial cells, major deposition of mesangial matrix and aquaporin-2 protein (AQP-2) expression, and low creatinine levels. Meanwhile, high-sucrose diet ameliorated AQP-2 expression and avoided the reduction of creatinine levels induced by chronic stress. The combination of stress and high-sucrose diet maintained similar effects on the kidney as stress alone, although it induced a greater reduction in the area of proximal tubules. CONCLUSIONS: Our results show that both chronic stress and a high-sucrose diet induce histological changes, but chronic stress may generate an accelerated glomerular hypertrophy associated with functional changes before puberty.

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.

Steviol slows renal cyst growth by reducing AQP2 expression and promoting AQP2 degradation.

Overexpression of aquaporin 2 (AQP2) was observed and suggested to be involved in fluid secretion leading to cyst enlargement in polycystic kidney disease (PKD). The cyst expansion deteriorates the renal function and, therefore, therapies targeting cyst enlargement are of clinical interest. Of note, inhibition of vasopressin function using vasopressin 2 receptor (V2R) antagonist which decreased cAMP production along with AQP2 production and function can slow cyst growth in ADPKD. This finding supports the role of AQP2 in cyst enlargement. Steviol, a major metabolite of the sweetening compound stevioside, was reported to retard MDCK cyst growth and enlargement by inhibiting CFTR activity. Interestingly, its efficacy was found to be higher than that of CFTRinh-172. Since steviol was also found to produce diuresis in rodent, it is likely that steviol might have an additional effect in retarding cyst progression, such as inhibition of AQP2 expression and function. Here, we investigated the effect of steviol on AQP2 function and on cyst growth using an in vitro cyst model (MDCK and Pkd1-/- cells). We found that steviol could markedly inhibit cyst growth by reducing AQP2 expression in both Pkd1-/- and MDCK cells. Real-time PCR also revealed that steviol decreased AQP2 mRNA expression level as well. Moreover, a proteasome inhibitor, MG-132, and the lysosomotropic agent, hydroxychloroquine (HCQ) were found to abolish the inhibitory effect of steviol in Pkd1-/- cells. Increased lysosomal enzyme marker (LAMP2) expression following steviol treatment clearly confirmed the involvement of lysosomes in steviol action. In conclusion, our finding showed for the first time that steviol slowed cyst growth, in part, by reducing AQP2 transcription, promoted proteasome, and lysosome-mediated AQP2 degradation. Due to its multiple actions, steviol is a promising compound for further development in the treatment of PKD.

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.

Sirtuin1 (SIRT1) Regulates Tumor Necrosis Factor-alpha (TNF-α-Induced) Aquaporin-2 (AQP2) Expression in Renal Medullary Collecting Duct Cells Through Inhibiting the NF-κB Pathway.

BACKGROUND Aquaporin-2 (AQP2) plays a major role in water reabsorption in the renal collecting duct, and is involved in a variety of renal disease. Recent studies have indicate that sirtuin1 (SIRT1) exerts renoprotective properties against kidney diseases. This study aimed to determine the potential role of SIRT1 in AQP2 expression induced by tumor necrosis factor-alpha (TNF-α) and to disclose the underlying mechanism in renal inner medullary collecting duct (IMCD) cells. MATERIAL AND METHODS Quantitative real-time PCR and Western blotting were respectively identified mRNA and protein expression. Immunofluorescence staining was used to detect the localization of AQP2. Small-interfering RNA (siRNA) was carried out for mechanism study. RESULTS Results showed that AQP2 was clearly increased in the plasma membrane and decreased in the cytoplasm of IMCD cells treated with AVP. TNF-α treatment in IMCD cells significantly reduced SIRT1 and AQP2 expression, and increased acetylated NF-κBp65 protein level in time- and concentration-dependent manners. Moreover, SIRT1 overexpression or the activator SRT1720 augmented AQP2 expression and reduced the acetylation of NF-κBp65, which was reversed by SIRT1 siRNA or the inhibitors Ex527 and sirtinol in TNF-α-induced IMCD cells. Knockdown of NF-κBp65 or NF-κBp65 inhibition by pyrrolidine dithiocarbamate (PDTC) enhanced AQP2 expression in IMCD cells exposed to TNF-α. Importantly, knockdown of NF-kBp65 augmented the up-regulation of SIRT1 on AQP2 expression in IMCD cells induced by TNF-α. CONCLUSIONS These findings indicate that SIRT1 increases AQP2 expression in TNF-α-induced IMCD cells via the NF-κB-dependent signalling pathway, which might provide novel insight to understanding the renoprotective effects of SIRT1 in kidney diseases.

Tissue Expression of Aquaporin 2 Is Correlated to Urine Output and Allograft Function in Sensitized Kidney Transplant Patients.

BACKGROUND: Salt and water disturbances often occur during acute kidney allograft dysfunction that contribute to graft failure, but this condition has been poorly investigated in the alloreactivity setting. We evaluated the tissue expression of aquaporins (AQP1 and AQP2) and the epithelial sodium channel (ENAC) in kidney biopsy specimens from sensitized kidney transplant recipients. METHODS: Eighty-six biopsy specimens from 33 sensitized patients were divided into 3 groups according to clinical context: time-zero (n = 9), protocol (n = 9), and indication (n = 68). The indication biopsy specimens were further divided into 3 subgroups according to the presence of acute tubular necrosis or rejection. Normal kidney tissue samples (n = 6) served as the control specimens. Immmunohistochemical expression of AQP1, AQP2, and ENAC was determined by using image analyzing software. RESULTS: Significantly lower AQP1 expression was observed in the time-zero and indication biopsy specimens with rejection compared with control specimens (P = .03 and P = .04, respectively). AQP2 expression was significantly lower in patients with an indication biopsy specimen compared with control and protocol biopsy specimens (P = .05 and P = .005). For ENAC, a lower expression was noted in the indication biopsy specimens compared with the control specimens (P = .04). Both AQP1 and AQP2 tissue expressions were significantly correlated to urine output (r = 0.45 and r = 0.32; P = .001 and P = .02), and AQP2 was correlated with the glomerular filtration rate estimated by using the Modification of Diet in Renal Disease Study equation at biopsy (r = 0.23; P = .05). CONCLUSIONS: These findings partially confirm previous experimental data showing downregulation of AQP1 expression after ischemia/reperfusion injury and during rejection. AQP2 downregulation seems to be rejection-independent, occurring during deteriorating or poor kidney graft function.

Regional heterogeneity of expression of renal NPRs, TonEBP, and AQP-2 mRNAs in rats with acute kidney injury.

To understand the pathophysiology of ischemia/reperfusion (I/R) - induced acute kidney injury (AKI), the present study defined changes in renal function, plasma renotropic hormones and its receptors in the kidney 2, 5, or 7 days after 45 min-renal ischemia in rats. Blood urea nitrogen, plasma creatinine, and osmolarity increased 2 days after I/R injury and tended to return to control level 7 days after I/R injury. Decreased renal function tended to return to control level 5 days after I/R injury. However, plasma concentrations of atrial natriuretic peptide and renin did not change. In control kidney, natriuretic peptide receptor (NPR)-A, -B and -C mRNAs were highly expressed in medulla (ME), inner cortex (IC), and outer cortex (OC), respectively, and tonicity-responsive enhancer binding protein (TonEBP), auqaporin-2 (AQP-2) and eNOS mRNAs were highly expressed in ME. NPR-A and -B mRNA expressions were markedly decreased 2 days after I/R injury. On 5 days after I/R injury, NPR-A mRNA expression increased in OC and recovered to control level in IC but not in ME. NPR-B mRNA expression was increased in OC, and recovered to control level in IC and ME. NPR-C mRNA expression was markedly decreased in OC 2 and 5 days after I/R injury. TonEBP, APQ-2 and eNOS mRNA expressions were markedly decreased 2 days after I/R injury and did not recover in ME 7 days after I/R injury. Therefore, we suggest that there is a regional heterogeneity of regulation of renal NPRs, TonEBP, and APQ-2 mRNA in AKI.

Rosiglitazone promotes AQP2 plasma membrane expression in renal cells via a Ca-dependent/cAMP-independent mechanism.

BACKGROUND/AIMS: Thiazolidinediones are highly beneficial in the treatment of type II diabetes. However, they are also associated with edema and increased risk of congestive heart failure. Several studies demonstrated that rosiglitazone (RGZ) increases the abundance of aquaporin-2 (AQP2) at the plasma membrane of renal cells. The aim of this study was to investigate whether RGZ might activate a transduction pathway facilitating AQP2 membrane accumulation in renal cells. METHODS: We analyzed the effect of RGZ on renal AQP2 intracellular trafficking in MCD4 renal cells by confocal microscopy and apical surface biotinylation. Cytosolic Ca(2+) dynamics were measured by a video-imaging approach in single cell. Transient Receptor Potential (TRP) channels expression was determined by RT-PCR. RESULTS: We showed that in MCD4 cells, short-term exposure to RGZ dramatically increases the amount of apically expressed AQP2 independently on cAMP production, PKA activation and AQP2 phosphorylation. RGZ elicited a cytosolic Ca(2+) transient due to Ca(2+) influx prevented by ruthenium red, suggesting the involvement of TRP plasma membrane channels. We identified TRPV6 as the possible candidate mediating this effect. CONCLUSIONS: Taken together these results provide a possible molecular mechanism explaining the increased AQP2 membrane expression under RGZ treatment: in renal cells RGZ elicits Ca(2+) transients facilitating AQP2 exposure at the apical plasma membrane, thus increasing collecting duct water permeability. Importantly, this effect suggests an unexplored application of RGZ in the treatment of pathological states characterized by impaired AQP2 trafficking at the plasma membrane.

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.

Integrin-linked kinase regulates tubular aquaporin-2 content and intracellular location: a link between the extracellular matrix and water reabsorption.

One of the clinical alterations observed in chronic renal disease (CRD) is the impaired urine concentration, known as diabetes insipidus (DI). Tubulointerstitial fibrosis of the kidney is also a pathological finding observed in CRD and involves composition of extracellular matrix (ECM). However, an association between these two events has not been elucidated. In this study, we showed that the extracellular-to-intracellular scaffold protein integrin-linked kinase (ILK) regulates expression of tubular water channel aquaporin-2 (AQP2) and its apical membrane presence in the renal tubule. Basally, polyuria and decreased urine osmolality were present in ILK conditional-knockdown (cKD-ILK) adult mice compared with nondepleted ILK littermates. No changes were observed in arginine-vasopressin (AVP) blood levels, renal receptor (V2R), or AQP3 expression. However, tubular AQP2 was decreased in expression and apical membrane presence in cKD-ILK mice, where the canonical V2R/cAMP axis activation is still functional, but independent of the absence of ILK. Thus, cKD-ILK constitutes a nephrogenic diabetes insipidus (NDI) model. AQP2 and ILK colocalize in cultured inner medullary collecting duct (mIMCD3) cells. Specific ILK siRNAs and collagen I (Col) decrease ILK and AQP2 levels and AQP2 presence on the membrane of tubular mIMCD3 cells, which impairs the capacity of the cells to transport water under hypotonic stress. The present work points to ILK as a therapeutic target in NDI.

A human in vitro model that mimics the renal proximal tubule.

Human in vitro-manufactured tissue and organ models can serve as powerful enabling tools for the exploration of fundamental questions regarding cell, matrix, and developmental biology in addition to the study of drug delivery dynamics and kinetics. To date, the development of a human model of the renal proximal tubule (PT) has been hindered by the lack of an appropriate cell source and scaffolds that allow epithelial monolayer formation and maintenance. Using extracellular matrices or matrix proteins, an in vivo-mimicking environment can be created that allows epithelial cells to exhibit their typical phenotype and functionality. Here, we describe an in vitro-engineered PT model. We isolated highly proliferative cells from cadaveric human kidneys (human kidney-derived cells [hKDCs]), which express markers that are associated with renal progenitor cells. Seeded on small intestinal submucosa (SIS), hKDCs formed a confluent monolayer and displayed the typical phenotype of PT epithelial cells. PT markers, including N-cadherin, were detected throughout the hKDC culture on the SIS, whereas markers of later tubule segments were weak (E-cadherin) or not (aquaporin-2) expressed. Basement membrane and microvilli formation demonstrated a strong polarization. We conclude that the combination of hKDCs and SIS is a suitable cell-scaffold composite to mimic the human PT in vitro.