Protective Effect of Alkaline Mineral Water on Calcium Oxalate-Induced Kidney Injury in Mice

Background Kidney stone disease induces chronic renal insufficiency by crystal-induced renal tubular epithelial cell injury. It has been reported that the prevalence of kidney stone disease is increasing, accompanied by the high recurrence rate. Alkaline mineral water has been reported to possess beneficial effects to attenuate inflammation. Here, we explored the potential protective effects and underlying mechanisms of alkaline mineral water against calcium oxalate-induced kidney injury. Methods We performed the mice kidney stone model by administering glyoxylate at 100 mg/kg once daily for 7 days. To assess the effects of alkaline mineral water on oxalate-induced kidney injury, mice drank different water (distilled water, natural mineral water at pH = 8.0, as well as natural mineral water at pH = 9.3) for 7 days, respectively, followed by glyoxylate exposure. After collection, crystal formation, kidney injury and cell apoptosis, fibrosis, oxidative stress, as well as inflammation were measured. Results Our results showed that glyoxylate treatment led to kidney crystal formation and fibrosis, which can be attenuated by drinking alkaline mineral water. Furthermore, alkaline mineral water also reduced kidney injury and cell apoptosis, oxidative stress, and inflammation. Conclusion Alkaline mineral water supplement prevents progression of glyoxylate-induced kidney stones through alleviating oxidative stress and inflammation.


Introduction
Nephrolithiasis (kidney stones) is one of the most prevalent urologic diseases worldwide, with a lifetime occurrence over 10% in the United States [1] and 6.4% in China [2] according to the recent data.Research has found that the formation of kidney stone is due to the imbalance of inhibitors and promoters of crystallization [3].Te characteristic symptoms of kidney stones are cramping and intermittent abdominal and fank pain, as well as hematuria, nausea or vomiting, and malaise, which impart substantial long-term disease burden.Tere are growing data for an increasing incidence and recurrence of kidney stones in all age, sex, and racial and/or ethnic subgroups, posing a serious threat to human health.
Understanding of pathophysiology of kidney stones is important to develop efcient strategies of prevention and treatment.Globally, approximately 80-90% of all kidney stones are composed of calcium oxalate (CaOx) mixed with calcium phosphate or uric acid [4,5].CaOx forms, grows, aggregates, and fnally retains within the kidneys due to urinary supersaturation [6].CaOx crystals induce intrarenal infammation and kidney tubular cell injury, which is strongly associated with oxidative stress injury and reactive oxygen species (ROS) [7,8].Inhibition of renal infammation and ROS production has been identifed to alleviate oxidative stress damage and reduce intrarenal crystal deposition [9].
Te risk factors contributing to kidney stones are various, including renal anatomic abnormalities, family history, older age, metabolic syndrome, climate changes, lifestyle, microbiomes, and so on [10][11][12][13][14].In practice, lifestyle interventions are shown efective and economic from all available treatment options.People have been trying to fnd the commonly espoused nonprescription agents or dietary recommendations to prevent stone formation [15].Recently, compelling links are beginning to emerge between high and appropriate fuid intake and reduced stone recurrence [16].Te quality of drinking water, including the hydrogen bond network [17], high pH [18], disturbed ratio of Ca and Mg [19], high salt [20], increased intake [21], and even specifc beverage types, is believed to be conducive to the formation of kidney stones, ofering drinking behavior along with dietary modifcation designed to restore normal renal biochemistry.
Despite these advances, the inescapable elephant in the room remains about treatment and prevention of kidney stones.Here, we focused on the pH of drinking water and explored the preventive and protective roles of alkaline drinking water (pH � 9.3) on the development of kidney stones.We found that a high pH drinking water reduced calcium stones' deposition in renal tubular epithelial cells, which further uncovering the underlying mechanism involved in kidney stones' formation.

Histological Analysis.
Te kidney tissues were fxed in 4% formalin and parafn-embedded, followed by sectioned at 4 μm.After being deparafnized and rehydrated, the prepared slices were used to conduct pathological staining according to the established standard procedure.
For hematoxylin and eosin (HE) staining, slices were immersed in hematoxylin for 10 min and eosin for 2 min.
Masson trichrome staining, for determining the levels of collagen deposition, was performed by the manufacturer's instruction.After the slices were dyed by Weigert's iron hematoxylin solution and washed by distilled water for 3 times, the slices were sequentially stained by 0.7% Masson-Ponceau-acid fuchsin staining solution for 10 min, diferentiated in phosphomolybdic acid for 4 min, and then stained by 2% aniline blue dye solution.Finally, the collagen deposition was observed under a light microscope.
Periodic acid-Schif (PAS) staining was performed as follows.Te periodate oxidation solution was frstly added on the kidney sections for 5 min, and the Schif reagent was stopped until the color of the tissue changed to red-purple.Te results were observed after hematoxylin staining.
Sirius red staining was conducted according to the manufacturer's instruction with commercial kits.Te positive staining was shown after the kidney sections were stained with Sirius red dye for 1 h.
Von Kossa staining commercial kit was applied to detect the calcium salt in the kidneys.Briefy, silver nitrate and hematoxylin and eosin sequentially immersed the tissues, and the staining was fnished.

Tunel Assay.
In situ cell death detection kit was used to perform the Tunel assay to determine the apoptotic cells in kidney tissue.Briefy, the slices were placed in a humidifed/ dark chamber and incubated with the Tunel reaction mixture for 1 h at 37 °C following deparafnized and permeabilized by 0.1 M sodium citrate, pH 6.0 at 65 °C for 30 min.After that, we observed the positive nuclear staining under the fuorescence microscope.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR).
Te detailed procedure was conducted as our previous description [22].Total RNAs were extracted from mouse kidney samples by the Trizol reagent.Ten, we 2 Evidence-Based Complementary and Alternative Medicine measured RNA concentrations using a spectrophotometer (One Drop, OD-1000+).Next, extracted RNAs were reverse transcribed with the Omniscript RT-PCR kit (Qiagen, Germany), and amplifed products by specifc primers (Sangon, Shanghai) were quantifed following the manufacturer's protocol.Primer sequences are provided in Table 1.Te PCR products were analyzed by electrophoresis using 2% agarose gels, and the density of the bands was used to quantify the mRNA using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA as an internal control.

Alkaline Mineral Water Mitigated Glyoxylate-Induced
Renal Crystal Formation and Fibrosis.Mice were divided into 4 groups to determine the efect of alkaline mineral water supplement on the progression of kidney stones as described in materials' section.Te analysis of HE and Von Kossa staining revealed a high degree of renal damage with dilated and ruptured tubules in the model group, indicating profound crystal deposition staining (Figures 1(a) and 1(b)).
Von Kossa staining showed that CaOx crystals formed and deposited in the renal tubules between cortex and medulla in the model mice.High pH mineral water rather than low pH mineral water signifcantly reduced the number of crystals.Next, we assessed the protein expression levels of the osteopontin (OPN) and CD44 since they are crystalrelated gene and crystal adhesion-related gene, respectively [23].Te results of IHC showed that a drastic increase of OPN and CD44 expression in the model group when compared with the control group (Figure 1(c)).Similarly, high pH mineral water signifcantly downregulated their expression upon CaOx stimulation (Figure 1(c)), while low pH mineral water supplement failed to change their expression levels (Figure 1(c)).Furthermore, we also found high pH mineral water efectively decreased the uric acid level compared with the model group, although urea nitrogen and creatinine failed to be reduced (Figure S1).Tubulointerstitial fbrosis has been reported as a key pathophysiological process in oxalate nephropathy with renal failure [24].To evaluate the alterations of fbrosis levels in glyoxylate-damaged renal tissue in four groups, we performed Masson staining and Sirius red staining.Masson staining of renal tissues showed overt fbrosis in the model group compared to the control group, which was signifcantly reversed after high pH mineral water supplement (Figure 2(a)).Reduced fbrosis was also revealed by Sirius red and α-SMA staining, which are both markers for deposition of extracellular matrix (ECM) (Figures 2(b) and 2(c)).Similar invalid efects were also found in the low pH mineral water supplement group (Figures 2(a)-2(c)).Taken together, these results indicated that high pH mineral water intake prevents glyoxylate-induced renal crystal formation and fbrosis.

Alkaline Mineral Water Attenuated Glyoxylate-Induced
Renal Damage and Cell Apoptosis.It is well reported that apoptosis is involved in CaOx crystal formation and renal damage [25].PAS staining revealed signifcant increase of tubular injury in model groups as showed by reduced PAS positively stained cells than the control group (Figure 3(a)).Supplement of high pH mineral water dramatically alleviated tubular injury in model mice (Figure 3(a)).In addition, the Tunel staining results showed that Tunel-positive cells dramatically increased in the model group compared with the control group, whereas impressively fewer apoptotic cells were detected in the high pH group rather than in the low pH group (Figure 3(b)).IHC results of cleaved caspase 3 further clarifed the abovementioned results (Figure 3(c)).Collectively, these results suggested that pretreatment of high pH mineral water had a stronger ability to reduce apoptosis of renal cells than the low pH mineral water, therefore presenting anti-injury against CaOx-induced kidney damage.

Alkaline Mineral Water Alleviated Glyoxylate-Induced
Renal Oxidative Stress.A good many of studies have showed that oxidative stress contributed to CaOx-induced crystal formation [26,27].Terefore, we investigated whether oxidative stress is participated in alkaline mineral waterinduced therapeutic efects.Nrf2/HO-1 is a major antioxidant pathway involved in CaOx-induced crystal deposition treatment [23,28].We found that both Nrf2 and HO-1 were upregulated after CaOx treatment, and the upregulation was further magnifed upon high pH mineral water supplement (Figure 4(a)).Furthermore, SOD downregulation contributed to CaOx-induced oxidative injury, although it was not afected by high pH mineral water supplement (Figure 4(a)).Recently, SIRT1 reduction was reported to participate in CaOx-induced crystal formation [29].Interestingly, high pH mineral water supplement dramatically stimulated the SIRT1 expression level than the model group (Figure 4(a)).Additionally, GSH, MDA, and SOD levels were measured.Although there was no evident change in SOD levels when treated with alkaline mineral water compared to the model group, higher GSH levels were found in the model + high pH group (Figures 4(b) and 4(c)).Meanwhile, MDA levels showed a downward trend (Figure 4(d)).Based on the abovementioned fndings, it was suggested that treatment of alkaline mineral water could ameliorate oxidative damage in glyoxylate-induced renal injury.

Alkaline Mineral Water Attenuated Glyoxylate-Induced
Renal Infammation.Many investigations have indicated that calcium oxalate stone formation is dependent on the infammatory process and secretes many infammatory factors, such as monocyte chemotactic protein 1 (MCP-1) [30].Terefore, we assessed the efects of alkaline mineral water on glyoxylate-induced renal infammation through Table 1: DNA sequences of primers for polymerase chain reaction.

Gene name Primer name
Sequence (5′-3′) Evidence-Based Complementary and Alternative Medicine measuring MCP-1 expression.We found that renal MCP-1 was signifcantly higher in the model group than the control group, but the efect was reversed after high pH mineral water supplement (Figure 5  Evidence-Based Complementary and Alternative Medicine

Discussion
CaOx-induced nephrolithiasis is one of the primary causes of chronic renal diseases, leading to heavy expenditure burden and poor life quality [31].Te current development of medical treatment mainly focuses on limiting oxalate intake and oxalic acid production [32].However, dietary and drinking habits are believed to be economic, useful, and of good treatment compliance, with a minimal side efect possibility.In the present study, we focused on the protective efect of alkaline mineral water supplement and its potential mechanisms in the glyoxylate-induced kidney injury.Our results show that alkaline mineral water inhibited oxalate-induced crystal formation and deposition in vivo.Specifcally, we found that alkaline mineral water at high pH had a protective efect on oxalate nephropathy by reducing renal fbrosis, apoptosis, oxidation, and infammation.All data implied that alkaline mineral water may be an efective and preventive treatment for oxalate-induced nephrotoxicity.Kidney crystal formation is a complicated process involved with oxidative stress, mitochondrial membrane potential, apoptosis, infammatory response, etc. [33,34].Excessive oxidative stress can induce infammation and cell damage, indicating that oxidative stress is one of the 6 Evidence-Based Complementary and Alternative Medicine most important links in stone formation.Previous studies indicated that oxidative stress contributes to the pathogenesis of nephrolithiasis.While application of antioxidants exerts a therapeutic efect, long-term clinical trials are limited because some of antioxidantrelated interventions are invalid or harmful [35].Tese failures may come from exogenous antioxidants which incur infammation at the same time [36,37].We here showed that alkaline water attenuated oxidative stress without inducing infammation, which overcame the limitation.
Quality of water gas gained much attention for kidney stone formation.Water hardness [38,39], calcium content [40], and beverage types [41,42] have been recently reported to infuence the incidence of kidney stones.However, controversy remains as to whether the pH of drinking water impacts the occurrence of stone formation [43].Urine pH and related assessment provide essential information about stone formation potential that can guide prevention.Furthermore, several antinephrolithiasis drugs have been confrmed to increase urinary pH, thereby providing the possibility of stone dissolution [44].So far, there is no  Evidence-Based Complementary and Alternative Medicine distinguished evidence linking the pH of drinking water to that of urine.Since kidney stone formation is a multiple process composed of CaOx crystallization, crystal growth, aggregation, adhesion, and retention [45], alkaline mineral water has the potential against stone formation in every step.

Conclusion
In summary, we developed a model of glyoxylate-induced kidney stones by repetitive administration of glyoxylate.By using this model, we have demonstrated that mice fed with alkaline mineral water are partly protected from progressive renal impairment.We thus believe that alkaline water may have the potential and promising value for the treatment of CaOx nephrolithiasis.
(a)).Moreover, IL-1β and TNF-α mRNA expression levels were dramatically increased in kidney tissue from the model group, and their expressions further reduced after high pH mineral water supplement (Figures 5(b) and 5(c)).Based on the abovementioned results, it was suggested that alkaline mineral water could assuage glyoxylate-induced renal infammation.

Figure 3 :
Figure 3: Te efects of alkaline mineral water on glyoxylate-induced apoptosis in mice.(a) PAS staining of mice kidney from diferent groups.(b) Tunel staining was used to assess renal apoptosis (scale bar � 100 μm).Te percentages of tunel-positive cells were calculated and showed right.Data are presented as the mean ± SD. * * P < 0.01 vs. the normal control group, ## P < 0.01 vs. the model group.(c) Te expression of apoptosis-related protein c-c3 (cleaved-caspase-3) was evaluated by immunohistochemistry (scale bar � 50 μm).

Figure 4 :
Figure 4: Alkaline mineral water inhibited glyoxylate-induced oxidative in mice.(a) HO-1, Nrf2, SOD-1, and SIRT1 expression assay using immunohistochemistry staining of parafn embedded kidney sections (scale bar � 50 μm).(b-d) Mice were treated as shown, and then relative GSH (b), SOD (c), MDA, and (d) content of kidney tissues were detected by their corresponding kits.All quantitative data are shown as means ± SD, * P < 0.05 vs. the normal control group, * * P < 0.01 vs. the normal control group, # P < 0.05 vs. the model group.
2.6.Statistical Analysis.All data are presented as the mean ± SD.Statistically signifcant diferences between the control group and treated groups were determined by Students t-test or one-way analysis of variance (ANOVA) carried out with GraphPad Prism 5.0.To analyze the correlation between genes, Pearson's correlation test was applied.P values <0.05 were considered statistically signifcant.