Progress in the development of drugs for kidney stone disease is comparatively slow, and no new drugs have been clinically implemented for decades 33. A replacement therapy by alkali citrate is still a widely used medication for prevention of stone recurrence. Gastrointestinal side effects and bad taste are the main drawbacks of potassium citrate treatment that cause poor compliance 11–13. The EAU urolithiasis guideline stated that the ideal drug should halt stone formation, have no side effects, and be easy to administer, and these elements are important to achieve good compliance 9. In this study, we developed HydroZitLa to address the drawbacks of potassium citrate and to meet the ideal drug criteria as much as possible. The HydroZitLa beverage has a delicious sour-and-sweet taste. We believed that the poor palatability of potassium citrate could be solved by HydroZitLa similar to reports by Mechlin et al. who demonstrated that the addition of Splenda to potassium citrate helped improve the compliance of potassium citrate therapy 13.
A low fluid intake is an important risk factor for CaOx stone formation. Patients with a stone disease are recommended to increase their fluid intake to 2.5–3.0 L/day, but it is relatively difficult for them to maintain adherence, especially in the long-term. Current stone medications are made in the form of either powder or tablet. Water intake with these medications is usually inadequate and varied between patients. By contrast, drinking HydroZitLa enabled patients to increase their water intake at least 500 mL with every pouch consumed. With long-term use, HydroZitLa could promote changes in dietary habits toward increasing daily fluid intake. The toxicity assessment data ensured that HydroZitLa was safe for consumption. The HydroZitLa beverage concentrate was officially approved by the Thai Food and Drug Administration (FDA) and locally available in Thailand since 2019. To our knowledge, consumers have not reported toxicity and serious side effects.
HydroZitLa significantly inhibited the aggregation of COM crystals in vitro. Although albumin was reported as an inhibitor of CaOx aggregation 34, we did not observe the significant inhibition of CaOx aggregation by BSA in our testing system. By contrast, citric acid strongly inhibited COM aggregation in our assay. Plausibly, citric acid contained in HydroZitLa was responsible for the blockage of COM aggregation.
The phenolic content in HydroZitLa was derived from banana stem, blue pea flower, and sappan wood. The antioxidant properties of banana stem 35, blue pea flowers 36, and sappan heartwood 37 extracts are well documented. The present study showed that HydroZitLa significantly reduced the intracellular ROS production and protein oxidation in HK-2 cells exposed to H2O2 and COM. Furthermore, HydroZitLa profoundly rescued HK-2 cells from apoptotic cell death induced by H2O2 and COM. These data highlighted that HydroZitLa had an antioxidative function to mitigate oxidative stress in renal tubular cells.
In this study, we demonstrated in a rat model of CaOx nephrolithiasis in which HydroZitLa significantly inhibited CaOx deposition in rats’ kidneys, and its antilithogenic efficacy was equivalent to Uralyt-U drug. Interestingly, HydroZitLa significantly decreased intrarenal 4-HNE expression, but Uralyt-U did not. HydroZitLa contained antioxidants, but Uralyt-U did not. Our data suggested that an alleviation of oxidative stress was an advantage of HydroZitLa over potassium citrate. HydroZitLa and Uralyt-U had comparable efficacy on the reduction of urinary oxalate in nephrolithic rats, although it was more pronounced with HydroZitLa. Significant reduction of urinary iCOCI levels (indicator of the urinary competency of CaOx crystallization 38) was also observed in rats treated with HydroZitLa and Uralyt-U. These indicated that the potential of CaOx crystallization in the urine was decreased after HydroZitLa and Uralyt-U treatments. HydroZitLa appeared to deliver antilithogenic activity primarily through the reduction of urinary oxalate excretion. The finding was corroborated with the reports by Poonguzhali and Chegu who showed that hyperoxaluric rats treated with aqueous banana stem extract significantly reduced urinary oxalate excretion 30. Based on the present findings, HydroZitLa induced CaOx stone inhibitory effects equivalent to the currently used potassium citrate, and it could be clinically useful as a new therapeutic alternative for CaOx nephrolithiasis.
A significant increase in urinary citrate levels in nephrolithic rats after HydroZitLa and Uralyt-U treatments was not detected. However, a trend of increased urinary citrate excretion after both treatments was observed. This might be explained by the small sample size (n = 6) or the inadequate dose of citrate (2 mEq per day, equal to 0.4 g/kg per day on average). Yasui et al. used potassium citrate (Uralyt, Nippon Chemiphar, Japan) at 0.5 g/kg (low dose) and 20 g/kg (high dose) per day in their experiments to find a significant increase in urinary citrate levels in experimental rats 39. Ghaeni et al. treated EG rats with potassium citrate (Sepidaj, Iran) at 2.5 g/kg per day to observe a significant elevation of urinary citrate excretion after treatment 40. Thus, a low dose of citrate was likely a reason for not finding a significant elevation of urinary citrate in our rat model following HydroZitLa and Uralyt-U treatments. However, HydroZitLa and Uralyt-U treatments could restore the urinary citrate to the levels observed in normal control rats. This urine citrate normalization could contribute, at least in part, to the inhibition of CaOx formation.
Based on our present findings, HydroZitLa exerted the antilithogenic function through at least two pathways. First, it reduced urinary oxalate excretion, decreased urinary CaOx crystallization capacity, and supplied citrate. Second, it contained herbal polyphenols that were capable of attenuating oxidative stress and inflammation, hence inhibiting the CaOx lithogenesis. Fundamentally, oxalate is endogenously synthesized in the liver using glyoxylate as a prominent precursor 41. Glyoxylate is synthesized from glycolate by the glycolate oxidase (GO) enzyme, and it is further converted to oxalate by lactate dehydrogenase (LDH). We found that HydroZitLa significantly reduced urinary oxalate excretion in EG-treated rats. We speculated that HydroZitLa inhibited oxalate synthetic pathway in the liver through downregulation of hepatic enzymes involved in the glyoxylate metabolism, particularly LDH and GO. The previous study by Kailash and Varalakshmi demonstrated that banana stem extract reduced the hepatic GO and LDH levels in sodium glycolate-induced hyperoxaluric rats 27. Poonguzhali and Chegu also reported that crude extract of banana stem reduced urinary oxalate, glycolic acid, and glyoxylic acid in the glycolate-induced hyperoxaluric rats 30. Recently, Patankar et al. demonstrated that their herbal formulation, called Herbmed Plus (containing banana stem), reduced the level of LDH activity in the liver and kidney tissues 42. To verify our speculation, expression of hepatic and kidney enzymes involving in oxalate synthesis in EG rats treated with HydroZitLa should be further explored.
Several lines of evidence strongly support that oxidative stress, inflammation, and renal tubular injury are critically involved in the CaOx lithogenesis 3,4,15,43−45. Oxidative stress induced by oxalate and/or CaOx crystals causes renal tubular injury that creates the sites for crystal nucleation, adhesion, retention, aggregation and finally a nidus formation 43,46,47. Furthermore, oxidative stress activates the inflammatory response that in turn accelerates the lithogenic process 4,47,48. Thereby, phytomedicine that has antioxidant and anti-inflammatory properties is a promising approach for prevention of kidney stone formation 24. Aggarwal et al. demonstrated in the EG-induced nephrolithic rat model that bergenin (bioactive phenolic compound isolated from Bergenia ligulata) had a potent antilithogenic effect to mitigate oxidative stress, ameliorate mitochondrial dysfunction and decrease tubular injury and inflammation 49. Rutin and curcumin (the well-known indigenous phytochemicals with antioxidant and anti-inflammatory properties) were shown to inhibit tubulointerstitial damage and crystal deposits in the EG-induced nephrolithic rats 50. Polyphenols from grape seeds was also shown to inhibit intrarenal CaOx crystal deposits in the EG-induced lithiasis rats, plausibly through their antioxidant activity 51. An Ayurveda herbal formulation Herbmed was demonstrated to reduce inflammatory injury and crystal deposits in the kidneys of EG-induced nephrolithic rats 42. In this study, we showed that HydroZitLa decreased intrarenal oxidative stress, inflammation and CaOx crystal deposits, suggested that HydroZitLa exerted antioxidant and anti-inflammatory actions to prevent CaOx renal stone formation.
Several plant polyphenols have been experimentally proven to be beneficial for extending lifespan and delaying aging 52. Resveratrol is the most well-characterized life-extending polyphenolic compound. It primarily acts through sirtuin activation and oxidative stress attenuation 53. The longevity effect of resveratrol tested in C. elegans model varies among studies with an increase in median lifespan ranging from 10–65% 54,55. HydroZitLa significantly extended the lifespan of wild-type C. elegans up to 34% with no sign of toxicity, suggesting that the lifespan-extending effect of HydroZitLa was comparable with that of resveratrol. Dietary restriction is a known key mechanism in longevity 56. The pharyngeal pumping rates compared between HydroZitLa worms and control worms were comparable, indicating that the lifespan extension by HydroZitLa was not a result of dietary restriction.
HydroZitLa significantly decreased an accumulation of the autofluorescent age pigment lipofuscin in C. elegans, suggesting an anti-aging effect. ROS and oxidative stress are well-known inducers of SIPS, and oxidative stress accelerates the shortening of telomeres 57. We recently demonstrated that oxalate, CaOx crystals, and urine from patients with CaOx stones induced premature senescence, p16 upregulation and telomere attrition in renal tubular cells through oxidative stress 58. In this study, we showed that HydroZitLa inhibited telomere attrition, p16 upregulation, and premature senescence in HK-2 cells exposed to H2O2, oxalate, and COM. Although the molecular mechanism for this anti-aging effect was not elaborated, HydroZitLa was suggested to mechanistically prevent premature senescence through inhibition of telomere shortening and downregulation of p16 expression.
Although herbal remedies and phytomedicines progressively become popular and globally accepted, scientific evidence of efficacy and safety must be provided and should not be compromised. Basically, the herb-drug interaction is hardly predicted because it is greatly varied among individuals, depending on several factors such as polymorphisms of cytochrome P450 enzyme and drug transporters, and existing medical conditions 59. Adverse effects of herb and medicine coadministration must be closely monitored to identify the potential herb-drug interactions. The best-known herbs seriously interacted with drugs include garlic, Baizhi, Danggui, ginseng, ginkgo, licorice, St John’s wort, Kava, black and long pepper, Danshen, Huangqin and Milk thistle 59. To our knowledge, there have been no serious side effects or drug interactions reported for banana stem, butterfly blue pea flower and sappan wood. In Thailand, banana stem is consumed as food ingredient. Drinking too much (over-consumption) banana stem juice may cause allergy, abdominal pain, and vomiting, thus moderate consumption is recommended. The blue pea flower is commonly used in food as natural colorant, and it is also used for making tea. Adverse effect of over-consumption of butterfly blue pea flower tea, for instances, upset stomach, nausea, and allergic reactions, is rarely reported. Sappan wood is also frequently used as natural food colorant, and in communities (including communities in Thailand) it is commonly consumed as healthy drink. However, the contraceptive function (anti-fertility in males) of sappan wood extract is demonstrated in rat model, as it interferes the spermatogenesis 60. On the other side, the heartwood of Caesalpinia sappan L. is used as a medicinal treatment for gynecological symptoms, including algomenorrhea and amenorrhea 61. Therefore, safety of the long-term consumption of HydroZitLa in human remains to be ascertained in the clinical trial.
This study has some limitations. The precise active compounds responsible for CaOx stone inhibitory function were not identified and characterized. The exact mechanisms of HydroZitLa to inhibit CaOx stone formation in rats was not extensively investigated. The dose-dependent antilithogenic effect of HydroZitLa was not investigated. The dose of HydroZitLa that could significantly increase urinary citrate level in the nephrolithic rats was not explored. The phenolic profile of HydroZitLa should be further investigated. The mechanism of longevity promotion by HydroZitLa in C. elegans was not intensively explored. Finally, specific compounds in HydroZitLa responsible for longevity and anti-aging were not identified and characterized.
In conclusion, HydroZitLa was developed to simultaneously normalize risk factors of CaOx stone formation by supplying fluid, citrate, and natural antioxidants. Experimentally, HydroZitLa inhibited intrarenal CaOx deposit in nephrolithic rats. The CaOx stone inhibitory efficacy of HydroZitLa was equivalent to the currently used potassium citrate (Uralyt-U). HydroZitLa efficiently attenuated oxidative stress both in vitro and in vivo. Remarkably, HydroZitLa extended the lifespan and delayed the onset of aging in C. elegans. Furthermore, HydroZitLa significantly inhibited telomere shortening, p16 expression, and premature senescence in human kidney cells. These preclinical findings suggested that HydroZitLa was a clinically promising alternative for CaOx nephrolithiasis treatment accompanied by longevity promotion and anti-aging benefits. Clinical trials are necessary to warrant the therapeutic efficacy of HydroZitLa in humans.