Beta thalassemias are a group of inherited hematological disorders characterized by reduced synthesis of beta globin chains of the hemoglobin molecule. Beta thalassemia major is characterized by severely decreased production of beta-globin chains resulting in increased hemolysis, ineffective erythropoiesis, and severe anemia. These patients require chronic blood transfusions starting in infancy for survival. Beta thalassemia intermedia is characterized by a milder reduction in beta-globin production resulting in a less severe phenotype. Patients may need transfusion support later in life to suppress complications of ineffective erythropoiesis and worsening anemia. A key feature of transfusion-dependent thalassemia (TDT) is the development of iron overload from repeated red cell transfusions, which is a significant cause of morbidity and mortality secondary to liver cirrhosis, cardiac failure, and endocrinopathies such as hypogonadism, diabetes, osteoporosis, and hypothyroidism. Survival of patients with TDT has significantly increased in the last several decades due to advances in monitoring and treatment of iron load [9]. As patients are living into their sixties and beyond, chronic conditions such as nephrolithiasis, are emerging as a new challenge to their health and the quality of life [7] [8]. In the past decade, reviews have focused on risk factors related to renal dysfunction and proposed biomarkers for renal disease [10] [11] [12] [13] [14], yet there remains a paucity of evidence-based information for clinicians in monitoring nephrolithiasis, an increasing problem for patients with TDT.

Currently, the prevalence of kidney stones in the general population is 9%, which represents a 70% increase since the 1990s [15]. Historically, kidney stones were most common among middle-aged white men, but the incidence of stones has increased substantially among women, children, and African-Americans [16]. Though the cause of these shifts is unclear, hypotheses include increased use of diagnostic imaging, and increasing incidence of dietary, metabolic, and environmental impacts including global warming [17] (Table 1). There is also a growing awareness that nephrolithiasis can be associated with other chronic diseases, as exemplified by thalassemia, where prevalence estimates are as high as 18% to 59% [3] [18].

Kidney stones are a consequence of pathophysiological biomineralization at the interface of the renal papilla or collecting duct with the urine, or seeded within the urine itself. Stone formation may be triggered by changes in saturation, solubility, hydrodynamics, or the balance between stone promoting and inhibiting constituents [19]. Most stones are calcium-based, and for this review, we will focus on risk factors for the most common, calcium oxalate stones. There is at least one report that patients with thalassemia may have a different profile of stone components, that is, both struvite and calcium oxalate type stones are equally observed [18], but this has not been well studied.

Adapted from information found in: Scales CD et al. 2016 [1].

Several pathological conditions have been associated with an increased risk of developing kidney stones, including diabetes, hypertension, hyperlipidemia, and chronic kidney disease [21] [22]. Nephrolithiasis, therefore, may be just one manifestation of a more complex syndrome. In thalassemia, disease-specific factors are some of the most unique aspects of additional risk factors for nephrolithiasis, including splenectomy, increased erythropoiesis, iron overload and chelation therapy.

1) Splenectomy and Excess Erythroblasts. Splenectomy has been identified as a major risk factor for stone formation in thalassemia [6]. In one study, 91% of patients with kidney stones were splenectomized [6]. Higher circulating erythroblasts due to decreased clearance post-splenectomy may lead to increased urinary uric acid. Hyperuricemia and hyperuricosuria in patients with thalassemia may also be due to the increased cell turnover and higher number of erythroblasts as a result of the increased ineffective erythropoiesis [23]. Up to 40% of

TDT patients have increased uric acid excretion. Patients with thalassemia intermedia have a high prevalence of hyperuricemia needing treatment as well as nephrolithiasis [6]. Further research is needed to evaluate the role of hyperuricemia and hyperuricosuria in the pathogenesis of nephrolithiasis in thalassemia.

2) Iron Overload and Chelation Therapy. Control of systemic iron burden is the most important determinant of morbidity and overall survival in thalassemia. However, Wong and colleagues found serum ferritin to be significantly lower in thalassemia patients with nephrolithiasis, suggesting a complex relationship with chelation therapy [3]. Urinary iron is elevated in thalassemia compared to the general population, although it is mostly present as iron-chelator complexes and varies widely, based on the type of chelator [24]. In the general population, the iron content in calcium-based kidney stones is highly variable (0.005 - 5.6 mg/g) [25], but comparable studies have not been published for iron-overload conditions such as thalassemia. Like calcium, iron excreted in the urine can bind to oxalate and phosphate anions, which might interfere with calcium mineralization and slow the rate of stone formation. However, iron binding to citrate and other important endogenous inhibitors of calcium oxalate nucleation, aggregation, and crystal growth can indirectly promote stone formation [26]. Interestingly, lower urinary iron excretion was correlated with poor shockwave lithotripsy success rates for calcium oxalate stones [27]. The balance of these chemical interactions of iron on lithogenic potential has not been defined for patients with or without iron overload.

Currently, the three options for iron chelation are deferoxamine, deferasirox and deferiprone. Each chelator has its own profile of efficacy, toxicity and acceptability, and some patients are prescribed a combination of two drugs to overcome these limitations. Of the three chelators, deferasirox has the greatest effect on renal function, causing a reversible mild elevation of creatinine and proximal tubular dysfunction; patients most affected appear to be those with low total body iron burden [10] [28] [29]. A few studies have suggested that deferasirox might increase the incidence of nephrolithiasis [8] [30]. However, baseline abnormalities in renal function are present in thalassemia irrespective of the chelation regimen [14]. Mean urinary calcium excretion is 3-fold higher compared with non-thalassemia controls in younger patients (4 - 23 years), with hypercalciuria observed in 36% [31]. Urine calcium excretion is significantly associated with proteinuria, but not with ferritin, serum creatinine, Cystatin C, beta2-microglobulin or creatinine clearance. Urine calcium excretion is greater with deferasirox compared with deferoxamine and is correlated with the dose of deferasirox. The effect of deferasirox on the incidence of nephrolithiasis was suggested by one retrospective study [30], but not by another [32]. Thus, baseline hypercalciuria is present in many patients with TDT from an early age, which may be exacerbated by deferasirox. The mechanism by which deferasirox increases tubular damage may be related to mitochondrial swelling from the excessive removal of iron from cells [28]. Prospective studies to evaluate the risk of nephrolithiasis using different chelators are lacking.

3) Zinc. Both insufficient and excess dietary zinc have been associated with a higher risk of nephrolithiasis in the general population [33] [34]. Urinary zinc loss is nearly 4 times higher in patients with thalassemia compared with controls [35], which is further increased in the presence of diabetes [36] and the use of iron chelators [37]. Zinc deficiency is observed in 18% of patients using deferiprone for chelation [37], and up to 30% of non-diabetic TDT patients [38]. However, the impact of zinc deficiency on the risk of nephrolithiasis in thalassemia has not been evaluated.

4) Diabetes. In the general population, diabetes is an independent risk factor for the development of kidney stones, increasing the risk by 29% - 60% [39]. The mechanism may be related to the effect of insulin resistance on ammoniagenesis and the resultant changes in urine composition, a lower urine pH, and hypercalciuria. Diabetes is a common complication of pancreatic iron overload in TDT occurring in approximately 20% [40] of adult patients, and thus could contribute to the risk of nephrolithiasis in this population.

In the general population, vegetarians have reduced stone incidence due in part to low animal protein intake and higher intakes of citrate and phytate, which act as inhibitors of stone formation in the urine by competing for the binding of calcium and other minerals [19]. Elevated consumption of animal protein increases stone risk, presumably by increasing calciuria and uricosuria, reducing citrate excretion, and increasing oxaluria [41]. Elevated consumption of fructose increases urinary excretion of calcium, oxalate, and uric acid, and has therefore been associated with an increased risk of incident kidney stones among adults [42]. High dietary sodium chloride intake also increases stone risk by increasing calciuria and reducing urinary citrate excretion [43]. Diets containing average amounts of calcium, moderate protein, and low sodium content reduce the risk of kidney stone recurrence [44]. Although dietary risk factors have not been specifically studied in thalassemia, diets tend to contain less meat (purine-rich foods), sodium (processed food), and foods with high iron and zinc content (meats and seafood) due to nutrition and disease management recommendations [45].

Fluid intake has an inverse relationship with stone formation in the general population likely by increasing the dilution of minerals and other components in the urine [46]. Tea beverage consumption is encouraged in thalassemia since the bioactive components in tea including epicatechins decrease absorption of dietary non-heme iron [45] [47]. While tea consumption was associated with 10% fewer stones in the general population, in part due to higher urine output, its association with stone risk has not been specifically studied in thalassemia [43].

1) Calcium. In the general population, increasing dietary calcium progressively from one to five servings daily has been associated with a decreased risk of incident nephrolithiasis [43]. This seemingly paradoxical effect has been attributed to dietary calcium-binding oxalate in the gut, which reduces intestinal oxalate absorption and urinary excretion. There is, however, a difference between dietary and supplemental calcium, with supplemental calcium increasing the risk of stone formation [48]. Many thalassemia patients avoid dairy foods, the optimal dietary source of calcium, due to either self-reported lactose intolerance, personal choice, or cultural beliefs [45]. The prevalence of hypolactasia can reach nearly 100% in East-Asian populations [49]. Given the majority of patients with thalassemia in North America are Asian, dairy avoidance leading to low dietary calcium intake is a concern. Calcium supplementation is used to fill the gap between inadequate dietary intake and the requirement for osteoporosis prevention. In one small case-control study evaluating renal calculi in patients with thalassemia on iron chelation with deferasirox or deferoxamine, vitamin D and calcium supplementation did not appear to increase the risk for stone formation [32].

2) Vitamin D. Vitamin D deficiency is common in patients with thalassemia due to limited sun exposure and avoidance of dairy foods that are usually fortified with vitamin D. High-dose, infrequent supplementation of vitamin D (50,000 IU every 3 weeks) has become an effective tool to improve vitamin D status. It is uncertain if daily or intermittent supplementation with vitamin D increases the risk of kidney stones. In a 2016 meta-analysis, vitamin D supplementation was not associated with a higher risk of nephrolithiasis [50]. However, an increased incidence of hypercalciuria in thalassemia patients with serum 25OH vitamin D concentration > 30 ng/mL has been observed [14], but the impact on stone formation or recurrence was not studied. Since patients have baseline hypercalciuria, it is possible that vitamin D deficiency normalized the urinary calcium levels, which is reversed by supplementation. Clearly, further evaluation of the role of vitamin D supplementation in the development of nephrolithiasis in thalassemia is warranted.

3) Vitamin C. The use of high-dose vitamin C (≥1000 mg/d) has been associated with nephrolithiasis in healthy men, likely resulting from the metabolism of excess vitamin C to oxalate [51]. Therefore, patients at risk for renal stones are warned against taking high-dose vitamin C. In thalassemia, iron overload and the concomitant increase in non-transferrin bound iron leads to an exhaustion of circulating antioxidants, most notably vitamin C [52]. The majority of patients with TDT have low serum ascorbate levels, which further decrease with increasing iron overload [52]. At very high liver iron concentrations (>25 mg Fe/g liver tissue), nearly 100% of patients have serum ascorbate levels characteristic of vitamin C deficiency. Moderate doses of vitamin C are recommended to improve the efficacy of iron chelation. However, given the relationship between high-dose ascorbate supplementation and oxalate metabolism, it is prudent to avoid large doses of vitamin C [51].

4) Obesity. Obesity is an important risk factor for nephrolithiasis among adults in the general population. The association of obesity with incidence and prevalence of nephrolithiasis is stronger in women than in men, possibly because women have greater adiposity than men at a given body mass index [53]. While patients with thalassemia typically have a body mass index (BMI) within the normal range, their fat content may be unusually high for body weight [54] [55]. This is due to a combination of lower lean mass for height and low bone mass. Since body fat is a predictor of stone formation, the disproportionate body fat in thalassemia may be a risk factor for kidney stones. Patients with a history of nephrolithiasis have been observed to have a higher BMI than those without kidney stones [3] [18].

5) Physical activity. Physical activity protects against stone formation in the general population, especially among postmenopausal women [56]. Typically, levels of daily physical activity are lower in thalassemia and patients are more sedentary due to, fatigue, pain and other factors [41]. While feasible, the association of obesity, metabolic syndrome, or limited physical activity with nephrolithiasis in thalassemia has not been evaluated.

Chronic exposure to the extremes in temperature due to season, climate and occupational conditions increases stone risk. The risk of kidney stone formation increases up to 68% within 2 days of high (>28˚C) daily temperatures [57]. Occupations that promote exposure to high temperatures are associated with higher stone risk [58]. The increase due to high ambient temperatures is probably mediated by low urine volume and increased concentration of lithogenic minerals (e.g. calcium, oxalate), leading to stone growth in susceptible patients. An inverse dose-response relationship between fluid intake and risk of stones has been observed with the greatest reduction (RR 0.52) occurring with daily intake > 2.5 L [43]. A urine output volume of 2.5 L is thus recommended for decreasing kidney stone recurrence [59]. The majority of patients with thalassemia reside in climates where the average annual temperature is above 26˚C, although the environmental risk factors for kidney stones have not been specifically evaluated in thalassemia.

Metabolic risk factors for stone formation are hypercalciuria, hypocitraturia, hyperuricosuria, hyperoxaluria, low urine volume, and alkaline urine pH. Hypercalciuria is most commonly due to increased intestinal calcium absorption, increased bone resorption, or increased postprandial renal fractional excretion of calcium. It is exacerbated by high salt and protein diets. Hypocitraturia is usually related to metabolic acidosis, as in chronic kidney disease, chronic diarrheal states, or a high protein diet. Hyperuricosuria, typically due to dietary purine excess or genetic predisposition, is a risk factor for calcium oxalate stones [60]. Hyperoxaluria may be due to a genetic mutation (primary hyperoxalurias), intestinal fat malabsorption, high dose vitamin C supplements, high dietary oxalate, or low dietary calcium.

1) Hypercalciuria. Although symptomatic kidney stones have only been reported in 18% of thalassemia patients [3], the prevalence of hypercalciuria is much higher. In a study of 216 thalassemia patients (51% female, average age 23 yrs), nearly 30% had hypercalciuria as defined by 24-hour urinary calcium to creatinine ratio (UCa: UCr) ≥ 0.21 mg/mg [14]. Hypercalciuria was more common (32.2%) in the 180 subjects who were regularly transfused, compared to those not regularly transfused (11.1%). Most patients were on deferoxamine (89%) for iron chelation, while a small number were receiving deferasirox (10%), or deferiprone (1%). A 25-OH vitamin D level > 30 ng/ml was associated with a higher prevalence of hypercalciuria compared levels < 11 ng/ml (OR 4.1, CI 1.3 - 13.1) [14]. The possible association of iron chelators with stone risk is important to explore. In a prospective study of 30 young thalassemia patients, 24-hour urine collections were checked at baseline and 6 months after starting deferasirox (20 mg/kg/d) [61]. Only 2 patients had hypercalciuria (urine calcium > 4 mg/kg/d) at baseline, while 8 had hypercalciuria after deferasirox (p = 0.037). In a more recent study of 152 subjects with transfusion-dependent hemoglobinopathies (86% thalassemia, 58% female, and average age of 34 yrs), hypercalciuria was reported in 92% of the patients on deferasirox and 83% of the patients on deferoxamine [62]. In this study, hypercalciuria was defined as UCa:UCr ≥ 0.4 mol/mol (equivalent to UCa:UCr of 0.14 mg/mg), a lower threshold that may help explain the higher prevalence. Severe hypercalciuria (UCa:UCr > 1.5 mol/ mol), however, was restricted to those receiving deferasirox. These two studies raise the possibility of the association of deferasirox with urinary calcium excretion. The true prevalence of hypercalciuria in thalassemia remains unknown since 24-hour urine collections are rarely performed. In addition, the definition of hypercalciuria in published studies is variable, which makes comparisons difficult. In thalassemia, the etiology of hypercalciuria is likely to be multifactorial, including renal dysfunction, hypoparathyroidism, iron chelation, diabetes, and high bone turnover. However, it is plausible that hypercalciuria is more common in patients on regular transfusions and exacerbated by deferasirox [62].

a) Renal Dysfunction. Patients with thalassemia are at risk for renal and tubular dysfunction due to anemia, hypoxia, iron overload, and the use of iron chelators [10] [14] [63]. Deferasirox has the most effect due to its role in producing a reversible increase in serum creatinine and proximal tubular dysfunction manifesting as renal tubular acidosis or Fanconi syndrome [61]. The pathogenesis of renal damage with deferasirox is not fully understood, but there is a direct toxic effect on the proximal renal tubule, which is responsible for most calcium reabsorption. Phosphate wasting in Fanconi syndrome causes hypercalciuria by stimulating 1,25-OH vitamin D production and promotes nephrolithiasis and nephrocalcinosis [64]. Whether hypercalciuria due to deferasirox is primarily associated with phosphate depletion has not been investigated.

b) Hypoparathyroidism. Iron overload in thalassemia is associated with several endocrinopathies, including hypoparathyroidism. In a study of 243 patients, subclinical hypoparathyroidism was present in 13.5% [65], although the prevalence of overt hypoparathyroidism is much lower at 1.4% [66]. Low parathyroid hormone (PTH) reduces renal calcium re-absorption. Patients with hypoparathyroidism and hypocalcemia often require calcium supplementation and calcitriol, and since calcium reabsorption is decreased, they can develop hypercalciuria with an increased risk of kidney stones. In these patients, hypercalciuria can be alleviated by targeting lower serum calcium in the range of 8.0 - 8.5 mg/dL.

c)High Bone Turnover. A link between hypercalciuria, kidney stones, osteoporosis and increased risk of fractures has been observed in several studies [3] [4] [5]. Patients had idiopathic hypercalciuria, which is accompanied by either elevated or suppressed PTH, is associated with increased bone turnover. Thiazide diuretics enhance renal tubular calcium re-absorption, and decrease urine calcium, PTH and bone turnover, which is associated with improved bone mineral density [67] [68].

Low bone mineral density with an increased risk of fracture is one of the most prevalent comorbidities in thalassemia. Approximately 50% of patients have low bone mineral density and 30% have had fractures [66]. The etiology for low bone mineral density in thalassemia is multifactorial, due to chronic anemia and bone marrow expansion, vitamin D deficiency, iron overload, iron chelators, hypogonadism and other endocrinopathies, nutritional deficiencies, decreased physical activity, and high bone turnover.

Bisphosphonates are anti-resorptive agents that are routinely employed to treat osteoporosis. In patients without thalassemia, alendronate reduces bone turnover and hypercalciuria [69]. The use of bisphosphonates in thalassemia leads to improvement in bone mineral density and a decrease in bone turnover markers [70]. However, these studies were not powered to determine fractures risk reduction, and the change in urinary calcium excretion was not reported [70]. The mechanism of hypercalciuria and low bone mineral density in thalassemia is multifactorial, but thiazide diuretics and bisphosphonates may have therapeutic potential by correction of hypercalciuria.

In twin studies, approximately 50% of the risk of nephrolithiasis among males was shown to be heritable [20]. While some genetic causes of kidney stones manifest from single-gene mutations (e.g. primary hyperoxaluria, cystinuria, and type 1 renal tubular acidosis), the large majority of heritable stone disease is polygenic [71]. The contribution of single gene polymorphisms to idiopathic calcium stone disease, including polymorphisms of the calcium receptor, vitamin D receptor, and osteopontin has been actively investigated [72] [73]. The role of these polymorphisms in stone formation in thalassemia patients is unknown.