Renal calculi (kidney stones) are one of the most common diseases in the world, often causing acute pain and worsening of kidney function. It also occasionally develops urinary tract infections and sepsis, which can be fatal. Kidney stones are the most expensive urological problem in health care in the United States, costing more than $10 billion annually [1]. Its lifetime prevalence is approximately 10% in men and 6% in women and is increasing in many developed countries [2] , [3] . Furthermore, its recurrence rate is 30-40% within 5 years and 50% within 10 years [4]. Most kidney stones are calcium oxalate (CaOx) stones [3] , [4] . Although kidney stones can be removed in a minimally invasive manner through endoscopic surgery, there are still no effective preventive or therapeutic drugs for CaOx stones [5] , [6] , [7] , and a better understanding of the molecular mechanisms of this disease is needed.
Sodium-glucose cotransporter 2 (SGLT2, encoded by SLC5A2) inhibitors are a new class of antidiabetic drugs that induce urinary glucose excretion by inhibiting SGLT2 in the S1 and S2 segments of the proximal tubule [8] , [9] . In addition to glycemic control, SGLT2 inhibitors have multiple functions, including reducing blood pressure [10], improving cardiovascular outcomes [11], protecting the kidneys [12], [13], [14], improving blood lipids and reducing body weight, visceral fat and serum uric acid levels [15], [16]. Furthermore, SGLT2 inhibitors cause reverse cardiac remodeling by improving myocardial energetics [17] , [18] , [19] and reducing aortic stiffness [20] while also improving quality of life [21] . Multiple mechanisms underlie the function of SGLT2 inhibitors, including diuretic and anti-inflammatory effects [12], [22].
Inflammatory factors, including osteopontin (OPN), are key to kidney stones [23], [24], [25], [26], [27], [28]. Opn is a pleiotropic glycoprotein secreted by renal tubule cells and is one of the most important nucleoproteins in the calcium stone matrix [23, 24, 25]. SGLT2 inhibitors inhibit OPN production in proximal tubules [29] , which may prevent CaOx nephrolithiasis. However, the therapeutic potential of SGLT2 inhibitors against kidney stones is controversial in humans [30] , [31] and needs to be investigated in more detail. Therefore, we hypothesized that SGLT2 inhibitors could be a potential treatment for kidney stones. To test this hypothesis, we conducted a large-scale epidemiological study to assess the relationship between kidney stone prevalence and SGLT2 inhibitor prescription status. We also studied the effects of SGLT2 inhibition on CaOx nephrolithiasis in animal models and human cell culture experiments.
highlight
• Based on a Japanese database, SGLT2 inhibitors reduce kidney stones in male diabetic patients.
•Phlorizin improves renal CaOx stone formation and subsequent damage in rats.
•SGLT2 deficiency suppresses renal CaOx stones, inflammation, and injury in mice.
•SGLT2 silencing inhibits OPN upregulation and CaOx crystal cell adhesion in vitro.
•SGLT2 inhibitors may be a promising treatment for kidney stones.
Background and purpose: Kidney stones are a common kidney disease, and there is currently no specific drug treatment. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are antidiabetic drugs that have diuretic and anti-inflammatory effects and may prevent kidney stones. Here, we investigated the potential of SGLT2 to inhibit kidney stones using large-scale epidemiological data, animal models, and cell culture experiments.
Methods: This study included data on diabetes patients (n = 1,538,198) available in a Japanese administrative database and divided them according to SGLT2 inhibitor prescription status. In animal experiments, ethylene glycol induced renal calcium oxalate stones in Sprague-Dawley rats, which were treated with the SGLT1/2 inhibitor phlorizin . The effects of SGLT2-specific inhibition on renal stone formation were evaluated in SGLT2-deficient mice and the human proximal tubular cell line HK-2.
Results: The prevalence of kidney stones was significantly lower in men with diabetes in the SGLT2 inhibitor prescription group compared with the non-SGLT2 inhibitor prescription group. Phlorizin attenuated kidney stone formation and downregulated the expression of kidney injury molecule 1 (Kim1) and osteopontin (Opn) in rats, with unchanged water consumption and urine output.It suppressed the expression of inflammatory and macrophage markers, suggesting a role for SGLT2 inhibitors in reducing inflammation. SGLT2-deficient mice are resistant to -glyoxylate--induced calcium oxalate stone formation, accompanied by reduced Opn expression and renal damage. High glucose-induced OPN and CD44 upregulation and reduced cell surface adhesion of calcium oxalate after SGLT2 silencing in HK-2 cells.
Conclusion: Overall, our findings suggest that inhibition of SGLT2 prevents kidney stone formation and may be a promising treatment for kidney stones.
In this study, we describe the effects of SGLT2 inhibition on kidney stones. We determined that SGLT2 inhibitors are effective in treating nephrolithiasis in male diabetic patients using a Japanese acute DPC hospital database. Furthermore, in rats with EG-induced renal CaOx stone formation, phlorizin treatment was effective against renal stone formation and subsequent renal injury without affecting water intake, urine output, and oxalate excretion. This beneficial effect of SGLT2 inhibition was replicated in SGLT2-deficient mice. Furthermore, SGLT2 silencing inhibited OPN and CD44 mRNA upregulation and CaOx crystal cell adhesion caused by high glucose in the human HK-2 proximal tubular cell line.
Although SGLT2 inhibitors have multiple functions [10] , [11] , [12] , [13] , [14] , [15] , [16] , reports on their effects on kidney stones are inconsistent [30] , [31] ]. A meta-analysis of randomized clinical trials using SGLT2 inhibitors showed that SGLT2 inhibitors did not affect the risk of kidney stones [30]. However, the sample size of this study was limited because kidney stones were not a prespecified endpoint in the included trials and the number of kidney stone cases was small. In contrast, a nationwide cohort study using the Danish Health Registry showed that SGLT2 inhibitor users had a lower risk of kidney stones than dipeptidyl peptidase 4 (DDP4) inhibitor users [31]. The study speculates that increased urine output by SGLT2 inhibitors may be a reason for their protective effect against kidney stones. In addition, a clinical trial has been initiated to verify the effect of the SGLT2 inhibitor empagliflozin on the prevention of kidney stone events [63]. Some pathophysiological functions of SGLT2 inhibitors, such as increased urine pH, increased urine output, and anti-inflammatory effects [12], [22], [39], [40], [41], [42], suggest a possible protective effect against kidney stones; however, the detailed mechanisms have not been studied.
The rat model of renal CaOx stone formation with EG supplemented in ad libitum drinking water has been well-established and studied as a clinical kidney stone formation model rather than a simple experimental model because this model does not show severe renal injury [34], [35]. Administration of EG at appropriate concentrations leads to the accumulation of CaOx on the surface of renal tubular cells, while other metabolic intermediates are not toxic to renal cells [35] , [64] . However, due to the diuretic effect of SGLT2 inhibitors, especially in the acute phase, which may be complementary to conventional diuretics [12] , [22] , [39] , [40] , [41] , [42] , [43] , [44] , [45] , we hypothesized the possibility of excessive kidney stone formation and organ toxicity accompanying EG overdose. Therefore, we changed the administration procedure of EG from mixing with water to body weight-referenced subcutaneous injection. Kidney stone formation in rats by subcutaneous injection of EG as well as administration through drinking water. Furthermore, water consumption differs between strains of rats [65] and mice [66]. Therefore, precise and accurate dosing by subcutaneous injection may lead to stabilization of the stone formation model and appropriate assessment of medical therapy, including diuretics.
SGLT2 inhibition suppressed proximal tubule inflammation and prevented CaOx stone formation and subsequent damage and fibrosis . However, although the diuretic/natriuretic effects of SGLT2 inhibitors are additive to those of conventional diuretics, no significant effects were observed on urine output and water intake in rats with EG-induced renal CaOx stone formation (EG vs. EG+Ph) in the acute phase, with chronic phase relief in humans [43] , [44] , [45] . In contrast to the diuretic effect, inhibition of SGLT2 inhibits the stone core matrix protein OPN and expression of renal injury, inflammation, and macrophage markers. These phenotypes were replicated in a model of glyoxylate-induced renal CaOx stone formation in SGLT2-deficient mice and in vitro studies on human proximal tubular epithelial cells.Based on these results, we propose that the beneficial effects of SGLT2 inhibition on renal CaOx stone formation may be due to its anti-inflammatory effects.
SGLT2 inhibitors also reduce serum uric acid levels and enhance uric acid excretion while altering urate transport activity associated with glycosuria [67], [68], [69]. Although lowering serum uric acid improves quality of life by improving aortic stiffness [20] and reducing inflammasome activity [70] , hyperuricemia is a risk for renal uric acid stone formation [71] . In this study, phlorizin treatment tended to decrease plasma uric acid and increase urinary uric acid levels. Although the relationship between SGLT2 inhibitors and renal uric acid stones has not been reported [63] , [71] , it cannot be excluded that SGLT2 inhibition increases the risk of renal uric acid stone formation.
crystal deposition is associated with renal cell damage, loss in cells, inflammation and fibrosis [72] , [73] . Renal crystals including CaOx induce reactive oxygen species -mediated inflammation in renal cells. Therefore, targeting inflammation may be effective in treating kidney stones [26]. Infiltrating renal macrophages promote fibrosis in CaOx nephrocalcinosis [28]. We found CD68+ macrophage infiltration surrounding interstitial fibrosis, which was attenuated by SGLT2 inhibition. Furthermore, OPN is a proinflammatory cytokine associated with macrophage responses [59] and OPN levels are elevated in proximal tubules before tubular injury inhibits SGLT2.
There are two conflicting hypotheses regarding the role of OPN in urinary stone formation: as an inducer [23], [24], [25], [26], [27], [28] and an inhibitor [74], [75]. The N-terminal OPN fragment is the main active form of OPN and can trigger pathological changes, including pro-inflammatory effects [53], [54]. In addition to being a pro-inflammatory cytokine, it is also one of the most important core proteins in the formation of CaOx crystals in the kidney [23], [24], [25]. Complete knockout or calcium-binding site mutation of OPN ameliorates glyoxylate-induced CaOx crystal deposition in mouse kidneys [24], [25]. The deposition of CaOx crystals in renal tubular cell lines is reduced by expression of OPN antisense [76] or OPN neutralizing antibodies [77] . Strong expression of OPN in renal tubular cells was detected during the initial process of renal calcium crystallization in vitro and in vivo [78]. Cyclosporine A blocks the opening of the mitochondrial permeability transition pore by inactivating cyclophilin D, thereby inhibiting OPN expression and renal crystallization [78]. Taken together, our results indicate that OPN is an inducer of kidney stone formation. Furthermore, the SGLT2 inhibitor inhibits OPN production by inhibiting glucose uptake in damaged proximal tubular epithelial cells, thereby shifting the metabolic pathway to the glycolytic system [29]. However, the precise intracellular mechanisms linking glucose consumption and OPN transcriptional activity remain unclear. Alterations in mitochondrial function by oxalate and calcium lead to changes in energy production, regulation of oxidative stress and intracellular calcium homeostasis, and inflammatory cascades [79]. Furthermore, reprogramming of energy metabolism in proximal tubular cells from fatty acid oxidation to glycolysis in injured proximal tubules may contribute to the transcriptional activation of OPN due to several intracellular pathways such as mammalian target of rapamycin (mTOR), AMP-activated protein kinase (AMPK), and silent message regulator 1 (SIRT1). are involved in this reprogramming [80]. Further studies are needed to discern the transcriptional regulation of OPN by SGLT2 inhibitors. OPN is also required for TGFB1-mediated fibrotic signaling and myofibroblast differentiation in cultured cardiac or dermal fibroblasts [60]. Therefore, inhibition of OPN by inhibiting SGLT2 may be effective against renal CaOx stone formation.
We also found that SGLT2 expression is reduced in injured proximal tubules after CaOx stone formation. Tubular injury is associated with loss of transporters, including SGLT2 [58] . Therefore, the effects of SGLT2 inhibition may be limited to damaged proximal tubules. However, SGLT2 inhibitors also have renoprotective effects in chronic kidney disease patients [14] and in mice initiated after ischemia-reperfusion renal injury [57]. During acute kidney injury , glucose metabolism is altered in the proximal tubule [81], and SGLT2 inhibition significantly reduces glucose uptake [57]. Furthermore, phlorizin and SGLT2 inhibitors increase urinary pH, -H+ exchange activity and bicarbonate reabsorption in the proximal tubule due to inhibition of Na+ [82], [83], [84].In this study, EG administration decreased urinary pH and phlorizin treatment increased urinary pH. This may affect the adhesion of CaOx crystals to tubular surfaces, as the highest CaOx crystal cell adhesion has been shown to occur at the most acidic pH values [37]. Collectively, these reports and our data suggest the potential of SGLT2 inhibition for use after kidney stone development; however, further investigation is needed to explore this aspect.
This study has some limitations. First, detailed information on diabetes severity and duration of SGLT2 inhibitor administration is lacking in the Japanese DPC database. Therefore, future studies of the effects of SGLT2 inhibitors on kidney stones in diverse ethnic and non-diabetic patients are needed. Second, in animal experiments, we only evaluated the formation of renal CaOx stones, which account for approximately 75% of human kidney stones [85]. The effect of SGLT2 inhibition on the remaining 25% of stones composed of calcium hydroxyphosphate, magnesium ammonium phosphate, urate, or cystine remains to be studied. Third, we initiated phloridzin treatment and EG administration simultaneously in our rat experiments. The treatment period of SGLT2 inhibitors is important to evaluate their therapeutic potential via diuresis or anti-inflammatory after the development of kidney stones.
5 . Conclusion
SGLT2 inhibition prevents kidney stone formation and subsequent renal damage. This effect may be due to reduced OPN gene expression and inflammation inhibiting glucose uptake in the proximal tubule via SGLT2 (Supplementary Figure 13). Therefore, the use of SGLT2 inhibitors may be a promising treatment for kidney stones.
Anan G, Hirose T, Kikuchi D, Takahashi C, Endo A, Ito H, Sato S, Nakayama S, Hashimoto H, Ishiyama K, Kimura T, Takahashi K, Sato M, Mori T. Inhibition of sodium-glucose cotransporter 2 suppresses renal stone formation. Pharmacol Res. 2022 Dec;186:106524. doi: 10.1016/j.phrs.2022.106524. Epub 2022 Oct 28. PMID: 36349594.
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