Potential low toxic alternative for Na-Cl cotransporter inhibition: A diuretic effect and mechanism study of Pyrrosia petiolosa.

BACKGROUND: Hydrochlorothiazide, a diuretic commonly used for the treatment of hypertension, is often associated with serious metabolic side effects. Pyrrosia petiolosa (Christ) Ching is a traditional Chinese medicine that possesses diuretic properties, without any obvious side effects. AIM: To evaluate the diuretic effect of P. petiolosa (Christ) Ching and to elucidate its underlying mechanism of action. METHODS: Extracts obtained from different polar components of P. petiolosa (Christ) Ching were analyzed for toxicity in a Kunming mouse model. The diuretic effects of the extracts were compared to that of hydrochlorothiazide in rats. In addition, compound isolation procedures, cell assays of Na-Cl cotransporter inhibition and rat diuretic test of monomeric compounds were conducted to identify the active ingredients in the extract. Subsequently, homology modeling and molecular docking were performed to explain the reason behind the diuretic activity observed. Finally, LC-MS analysis was used to elucidate the underlying mechanism of action of P. petiolosa (Christ) Ching. RESULTS: No toxicity was observed in mice administered P. petiolosa (Christ) Ching extracts. The ethyl acetate fraction showed the most significant diuretic effect. Similar results were obtained during the analysis for Na+ content in rat urine. Further separation of P. petiolosa (Christ) Ching components led to the isolation of methyl chlorogenate, 2',3'-dihydroxy propyl pentadecanoate, and ß-carotene. Results from cell assays showed that the Na-Cl cotransporter inhibitory activity of methyl chlorogenate was greater than that of hydrochlorothiazide. This result was again confirmed by the diuresis tests of monomeric compounds in rats. The molecular simulations explain the stronger interactions between the methyl chlorogenate and Na-Cl cotransporter. Of the compounds determined using LC-MS analysis, 185 were identified to be mostly organic acids. CONCLUSIONS: P. petiolosa possesses significant diuretic activities without any obvious toxicity, with least two possible mechanisms of action. Further study on this herb is warranted.

Human renal organic anion transporter 4 operates as an asymmetric urate transporter.

Human organic anion transporter 4 (hOAT4) is located at the apical membrane of proximal tubule cells and involved in renal secretion and reabsorption of endogenous substances as well as many drugs and xenobiotics. This study reevaluated the physiologic role, transport mode, and driving forces of hOAT4. 6-Carboxyfluorescein (6-CF) uptake into HEK293 cells that stably expressed hOAT4 was saturable, resulting in a K(m) of 108 muM. 6-CF as well as [(3)H]estrone sulfate ([(3)H]ES) accumulation by HEK293-hOAT4 cells were abolished by ES, dehydroepiandrosterone sulfate, sulfinpyrazone, benzbromarone, and probenecid, whereas several OA, including p-aminohippurate (PAH), lactate, pyrazinoate, nicotinate, glutarate, and the diuretic hydrochlorothiazide (HCTZ) exhibited a slight or a NS inhibitory effect. PAH and glutarate are not taken up by HEK293-hOAT4 cells, but they trans-stimulated 6-CF and [(3)H]ES uptake, indicating an asymmetric interaction of hOAT4 with these substrates. In chloride-free medium, HEK293-hOAT4-mediated [(3)H]PAH efflux was almost abolished, whereas addition of ES restored it comparable to Ringer solution, consistent with a physiologic ES/PAH or PAH/Cl(-) exchange mode of hOAT4. Moreover, an acidification of the uptake medium increased 6-CF as well as [(3)H]ES uptake, which was reduced by nigericin, suggesting that hOAT4 also can operate as an OA/OH(-) exchanger. hOAT4 facilitates substantial uptake of [(14)C]urate, which was elevated 2.6-fold by intracellular HCTZ. Thus, hOAT4 is the long-postulated, low-affinity apical urate anion exchanger that facilitates HCTZ-associated hyperuricemia.

Renal effects of 26-week administration of olmesartan medoxomil/hydrochlorothiazide in rats.

The combination of an angiotensin II type 1 receptor blocker (ARB) and a diuretic is effective clinically in treatment of hypertension. As a non-clinical safety evaluation of a combination of the ARB olmesartan medoxomil (OM) and the diuretic hydrochlorothiazide (HCTZ), male and female normotensive rats were administered OM/HCTZ (fixed ratio of 8 : 5) orally by gavage for 26 weeks at dose levels of 0, 4.88, 16.25, 48.75, 162.5, 487.5, or 1625 mg/kg/day. Additional groups were given 1000 mg/ kg/day OM or 625 mg/kg/day HCTZ. Statistically significant and marked decreases in urinary protein excretion were observed in males and females given doses of 16.25 mg/kg/day or higher compared to vehicle-control groups. Increases in blood urinary nitrogen (BUN) were observed in males and females given doses of 16.25 and 162.5 mg/kg/day or higher, respectively. Increased incidence of chronic progressive nephropathy (CPN), a rat-specific spontaneous renal lesion, was observed in males and females given doses of 48.75 mg/kg/day or higher. An additional mechanistic study, consisting of male and female rats given 0, or 162.5 mg/kg/day OM/HCTZ, was conducted to clarify the toxicological significance of the increases in BUN and the increased incidence of CPN described above. This additional study clearly demonstrated that saline-supplementation through free access to saline in the drinking water ameliorated the elevation in BUN and also ameliorated the incidence of CPN. Consequently, the effects on BUN and CPN observed in the first study can be explained by the hemodynamic disturbances caused by the large doses and an exaggerated pharmacological action in volume-depleted normotensive animals. Importantly, the marked decreases in urinary protein were not affected by the saline-supplementation, and indicated that OM/HCTZ elicited a renoprotective effect, probably by an effect on the glomeruli. An additional toxicokinetic study revealed no drug interactions between OM and HCTZ. In conclusion, OM/HCTZ induced a renoprotective effect as well as changes probably attributed to the exaggerated pharmacological action of the ARB with diuretic in normotensive rats. These results suggest that OM/HCTZ may have renoprotective effects in clinical treatment of hypertensive patients.

Toxicology and carcinogenicity studies of diuretics in F344 rats and B6C3F1 mice. 1. Hydrochlorothiazide.

Toxicology and carcinogenesis studies of hydrochlorothiazide, a benzothiadiazide diuretic, were conducted by administering diets containing the drug to both sexes of F344 rats and B6C3F1 mice in 15-day, 13-week and 2-year studies. No rats died during the 15-day or 13-week studies at dietary concentrations of up to 50,000 ppm. Deaths of male mice in the top dose group in the 13-week study were likely to be related to chemical administration. In the prechronic studies, increased nephrosis and mineralization at the kidney corticomedullary junction were the primary toxic effects of hydrochlorothiazide observed in rats. In mice, chemical-related effects included nephrosis and calculi, inflammation and epithelial hyperplasia in the urinary bladder. In 2-year studies using dietary concentrations of 0, 250, 500 and 2000 ppm in rats and 0, 2500 and 5000 ppm in mice, survival of dosed and control groups of rats and mice was similar, as were body weights of mice. Dosed groups of male and female rats were uniformly lighter than controls (up to 25%) throughout the studies. Severe chronic renal disease with secondary parathyroid hyperplasia and fibrous osteodystrophy of the bone were attributed to chemical administration in rats. No neoplasms in rats or female mice or non-neoplastic lesions in mice were associated with hydrochlorothiazide. In high-dose male mice, liver neoplasms were increased but were not considered to be related to hydrochlorothiazide administration because of an unusually low incidence in the control group relative to historical controls.