Progress in Understanding the Genetics of Calcium-Containing Nephrolithiasis

Abstract

Renal stone disease is a frequent condition, causing a huge burden on health care systems globally. Calcium-based calculi account for around 75% of renal stone disease and the incidence of these calculi is increasing, suggesting environmental and dietary factors are acting upon a preexisting genetic background. The familial nature and significant heritability of stone disease is known, and recent genetic studies have successfully identified genes that may be involved in renal stone formation. The detection of monogenic causes of renal stone disease has been made more feasible by the use of high-throughput sequencing technologies and has also facilitated the discovery of novel monogenic causes of stone disease. However, the majority of calcium stone formers remain of undetermined genotype. Genome-wide association studies and candidate gene studies implicate a series of genes involved in renal tubular handling of lithogenic substrates, such as calcium, oxalate, and phosphate, and of inhibitors of crystallization, such as citrate and magnesium. Additionally, expression profiling of renal tissues from stone formers provides a novel way to explore disease pathways. New animal models to explore these recently-identified mechanisms and therapeutic interventions are being tested, which hopefully will provide translational insights to stop the growing incidence of nephrolithiasis.

Renal stones are common; the overall lifetime risk of stone disease in the United States is 8.8%.¹ Renal stones may be associated with both metabolic syndrome² and ESRD³ and provide a huge burden on health services. Most stones are calcium-containing, accounting for approximately 75% of all stones. Previous studies, in both twins and families with renal stones, have confirmed the heritability of the risk for renal stones.^4,5 It is likely that multiple genetic and environmental factors predispose to a risk of renal stone disease. Interestingly, the incidence of renal stones is increasing globally in both children and adults.^1,6–8 This increase relates to a whole range of environmental effects, including dietary changes such as high animal protein and high sodium intake.^9,10 In addition, the traditional pattern of more stone disease in men compared with women is being narrowed, due to a rise in stones among women.¹¹ Here we will discuss the basic aspects of calcium stone disease and review the current understanding of monogenic and complex disease causes of calcium stone disease within the context of known metabolic risk factors. Understanding these molecular players, together with new animal models of renal stone disease, will help define the underlying disease mechanisms and potential novel therapies.

The Basics of Calcium Stone Disease and its Nongenetic Factors

Calcium-containing kidney stones are the most common type of kidney stone. A collection of systemic causes may predispose toward calcium stone formation and include inflammatory bowel disease, primary hyperparathyroidism, and renal tubular acidosis; however, in most people these are absent. Instead, metabolic factors within the urine, the commonest being hypercalciuria, contribute toward a stone-forming environment. The evidence points toward calcium oxalate stones forming on anchored sites in the renal papillae made of interstitial apatite deposits and known as Randall plaques.^12,13 However, in conditions where there is urinary supersaturation of calcium salts, formation of stones may develop from crystal deposits at the tip of the Bellini duct and within inner medullary collecting ducts.¹⁴ The most obvious environmental risk factor for stone formation is dehydration from low fluid intake and other risk factors include high dietary animal protein and sodium. Monogenic forms of kidney stones are rare but their study offers a chance to understand the underlying pathophysiology and develop novel therapies.

The Investigation of Monogenic Renal Stone Disorders

As discussed above, the etiology of idiopathic calcium nephrolithiasis is likely to be a result of complex interactions involving genes and environmental factors,^15,16 however a growing list of monogenic causes (Table 1), many of which can present in childhood, has provided some important insights. The commonest type of renal stone is calcium oxalate and underlying metabolic factors such as hypercalciuria, hyperoxaluria, and hypocitraturia may point toward a specific cause.

Table 1.

Human genes implicated in monogenic and polygenic forms of calcium stone disease

Gene Symbol	Gene Name	Expression and Function	Phenotype and Disease Mechanisms
ADCY10	Adenylate cyclase 10 (soluble)	Multiple tissues including kidney and jejunum. Acts as a bicarbonate sensor	Risk allele: Variants in this gene are risk alleles and may cause absorptive hypercalciuria and stone formation
AGXT	Alanine-glyoxylate aminotransferase	Peroxisome enzyme in liver	Monogenic: Inactivating mutations lead to deficiency of alanine:glyoxylate aminotransferase leading to oxalosis and progressive kidney failure (PH type 1). Phenotype includes recurrent stones and nephrocalcinosis
ALPL	Tissue-nonspecific Alkaline phosphatase	Membrane-bound glycoprotein that hydrolyses pyrophosphate and provides inorganic phosphate. Expressed in proximal tubule	Monogenic: Inactivating variants lead to hypophosphatasia with hypercalcaemia, hypercalciuria, nephrocalcinosis, and rickets. Activating variants lead to autosomal dominant hypocalcemia with low PTH and hypercalciuria
AQP1	Aquaporin-1	Water channel protein expressed in proximal tubule segments	Risk allele: Variant 14.2 kb upstream of AQP1. May be important in urinary concentration by regulating AQP1
ATP6V0A4	Vacuolar H(+)-ATPase A4 subunit	Apical membrane of collecting duct, mediating distal nephron acid secretion and inner ear	Monogenic: Inactivation mutations lead to distal RTA with sensorineural deafness in young adults
ATP6V1B1	Vacuolar H(+)-ATPase B1 subunit	Apical membrane of collecting duct, mediating distal nephron acid secretion and inner ear	Monogenic: Inactivation mutations lead to distal RTA with sensorineural deafness. Renal phenotype includes nephrolithiasis, nephrocalcinosis, hypokalemia, hypercalciuria, hypocitraturia
CA2	Carbonic anhydrase 2	Expressed in osteoclasts during bone resorption and expressed in proximal and distal nephron segments	Monogenic: Inactivating variants cause carbonic anhydrase 2 deficiency resulting in osteopetrosis and RTA. Defect causes a mixed proximal-distal RTA with bicarbonate wasting and decreased ammonia excretion
CASR	Calcium-sensing receptor	Expressed in parathyroid-producing chief cells and renal tubule (proximal, TALH, DCT, and CD). Acts as a cell surface receptor to regulate set point of PTH secretion	Monogenic: Inactivating variants lead to familial hypocalciuria hypercalcaemia with raised PTH
			Risk allele: Polymorphisms are associated with kidney stone disease
CLCN5	Chloride channel, voltage-sensitive 5	Chloride ion transporter, expressed in proximal tubule	Monogenic: Inactivating mutations in males lead to Dent disease and hypophosphatemia rickets. Urinary phenotypes include LMWP, aminoaciduria, phosphaturia, hypercalciuria, nephrolithiasis, and nephrocalcinosis. Female carriers may have hypercalciuria and renal stones
CLCNKB	Chloride channel, voltage-sensitive Kb	Expressed in the basolateral membrane of the TALH and distal nephron	Monogenic: Bartter syndrome type 3 with hypercalciuria and occasionally Gitelman syndrome phenotype (with hypocalciuria). Nephrocalcinosis is not usually seen
CLDN14	Claudin 14	Tight junction protein in TALH and inner ear	Risk allele: Variants associated with calcium stones
CLDN16	Claudin 16	Tight junction protein in TALH	Monogenic: Renal hypomagnesaemia, hypercalciuria, and renal stones
CLDN19	Claudin 19	Tight junction protein distal tubules	Monogenic: Renal hypomagnesaemia, hypercalciuria, and renal stones
CYP24A1	Vitamin D 24-hydroxylase	Mitochondrial enzyme responsible for inactivating vitamin D metabolites	Monogenic: Inactivation mutations lead to excess active 1,25 dihydroxy-vitamin D leading to infantile hypercalcaemia type 1. Adult phenotypes include hypercalcaemia, suppressed PTH, hypercalciuria nephrocalcinosis, and renal stones
GRHPR	Glyoxylate reductase/hydroxypyruvate reductase	Widespread tissue expression, converts glyoxylate to glycolate	Monogenic: Inactivation mutations lead to oxalate overproduction and hyperoxaluria (PH type 2).
HOGA1	4-hydroxy-2-oxogluarate aldolase 1	Mitochondrial enzyme, expressed in liver and kidney	Monogenic: Inactivation mutations lead to oxalate over-production and hyperoxaluria (PH type 3). Phenotype includes recurrent renal stone formation in early life
KCNJ1	Potassium inwardly-rectifying channel, subfamily J, member 1	Expressed in the apical membrane of the TALH allowing potassium recycling back to tubular lumen	Monogenic: Bartter syndrome type 2 with salt wasting, hypercalciuria, nephrocalcinosis, and renal stones
OCRL	Phosphatidylinositol 4,5-bisphosphate-5-phosphatase	Protein is localized to the trans-Golgi network that is involved in actin polymerization	Monogenic: Inactivating mutations cause Oculocerebrorenal syndrome of Lowe and Dent disease type 2, with LMWP and hypercalciuria and renal stones
SLC12A1	Sodium-potassium-chloride transporter 2	Expressed in the apical membrane of the TALH	Monogenic: Bartter syndrome type 1 with salt wasting, hypercalciuria, nephrocalcinosis, and renal stones
SLC26A1	Sulfate anion transporter	Plasma membrane chloride/bicarbonate/oxalate/sulfate exchanger. Highly-expressed in kidney (basolateral membrane of proximal tubule and collecting duct cells) and also basolateral membrane of ileum and colon	Monogenic: Inactivation mutations lead to loss of transporter function and calcium oxalate stone formation
SLC26A6	Solute carrier family 26, member 6	Anion exchanger expressed in renal tubules (apical membrane) and intestine (apical membrane) facilitating oxalate secretion	Risk allele: May limit net intestinal absorption of oxalate (by allowing secretion of absorbed oxalate. Missense variants are associated with increased oxalate absorption and calcium oxalate stone formation
SLC34A1	Sodium-dependent phosphate cotransporter type 2	Expressed in the proximal tubule, allows proximal tubular reabsorption of phosphate	Monogenic: Inactivating variants lead to renal phosphate loss and hypophosphatemic nephrolithiasis/osteoporosis. Inactivating mutations may also present with infantile hypercalcemia
SLC34A3	Sodium-dependent phosphate transporter 2c	Expressed in the proximal tubule, allows proximal tubular reabsorption of phosphate	Monogenic: Biallelic inactivating variants cause hereditary hypophosphatemic rickets with hypercalciuria. Heterozygous carriers have a biochemical phenotype that mimics idiopathic hypercalciuria
SLC4A1	Solute carrier family 4, anion exchanger, member 1	Expressed in erythrocyte plasma membrane and basolateral membrane of α-intercalated cell	Monogenic: Distal RTA, spherocystosis, renal stones, and nephrocalcinosis
SLC9A3R1	Solute carrier family 9, subfamily A	Cytoplasmic adaptor protein present in kidney small intestine and liver epithelia	Monogenic: Hypophosphatemic nephrolithiasis/osteoporosis type 2
TRPV5	Transient receptor potential cation channel V5- calcium permeable channel	Expressed at apical epithelial surface of small intestine and DCT of kidney	Risk allele: variants lead to a risk of renal stones
TRPV6	Transient receptor potential cation channel V6 – calcium-permeable channel	Widely expressed, including at apical epithelial surface of small intestine and DCT of kidney	Risk allele: Activating variants lead to increased calcium transport (absorptive hypercalciuria) and a risk of renal stones
UMOD	Uromodulin	GPI-anchored glycoprotein and most abundant protein in urine. Expressed in TALH and DCT	Protective allele: Intronic SNP may be protective for renal stones
VDR	Vitamin D receptor	Encodes a nuclear receptor for 1,25-dihydroxy vitamin D. Expressed in small and large bowel and renal tubules	Risk allele: VDR polymorphisms associated with urinary stone formation in certain populations

PTH, parathyroid hormone; RTA, renal tubular acidosis; TALH, thick ascending loop of Henle; DCT, distal convoluted tubule; CD, collecting duct; LMWP, low molecular weight proteinuria; SNP, single nucleotide polymorphism.

Hyperoxaluria may be due to the rare monogenic disorders of primary hyperoxaluria (PH, type 1, 2, and 3) or more frequently due to enteric hyperoxaluria¹⁷ (Figure 1) or idiopathic hyperoxaluria. The molecular genetics of PH and its associated phenotypes have recently been reviewed and updated.¹⁸ PH comprises a rare autosomal recessive set of conditions (prevalence 1:58,000) leading to oxalate over-production and results in hyperoxaluria, leading to renal stones and nephrocalcinosis. The subsequent CKD leads to a systemic oxalosis. The three known genetic causes of PH (AGXT, GRHPR, and HOGA1) were recently correlated with phenotypes in a cohort of 335 patients belonging to the Rare Kidney Stone Consortium PH registry. The median age of onset of symptoms was very early at 5.2 years.¹⁸

Figure 1.

Oxalate metabolism and a model of intestinal and renal tubular oxalate transport. (A) Dietary oxalate is between 80 and 130 mg/d, of which 5%–15% is absorbed via intestinal anion exchangers (B). Oxalate within the gut binds to calcium and is eliminated via the stool. Low calcium diets increase gut oxalate absorption. Oxalate-degrading bacteria, such as Oxalobacter formigenes, play a role in protecting against oxalate hyperabsorption. Malabsorption syndromes may lead to a lack of such bacteria and promote hyperabsorption of oxalate. Endogenous production of oxalate occurs within the liver (15–45 mg/d). Autosomal recessive inherited PH secondary to mutations in AGXT, GRHPR, or HOGA1 leads to systemic oxalosis (with endogenous production of >100 mg/d) and hyperoxaluria. Figure modified from Hoppe et al. (B) A proposed model of small intestine epithelial cell transporters highlights the molecular players in oxalate transport/exchange. Apical oxalate transporters include anion exchangers SLC26A6 (PAT1) and SLC26A3 (DRA) that mediate oxalate secretion and uptake, respectively. A sulfate anion transporter (SAT1, SLC26A1) is localized at the basolateral membrane and allows sulfate resorption in exchange for anions (including Cl⁻, HCO3⁻, oxalate, and SO₄²⁻). A murine knockout model of Slc26a6 (Pat1) leads to reduced oxalate secretion and an overall net increase in reabsorption, increasing the filtered oxalate load and predisposing to calcium oxalate precipitation. Slc26a3 (DRA) knockout mice exhibit reduced oxalate absorption and reduced urinary oxalate levels (whereas human SLC26A3 mutations cause congenital chloride diarrhea). Murine knockouts of Slc26a1 (Sat1) cause a reduction in intestinal secretion of oxalate, leading to hyperoxalemia and hyperoxaluria. Human SLC26A1 mutations lead to nephrolithiasis by presumed similar mechanisms. (C) Oxalate is freely filtered by the glomerulus. SLC26A6 is localized on the apical membrane of the proximal tubule and forms a complex with NaDC-1, the sodium-dependent dicarboxylate cotransporter (encoded by SLC13A2). SLC26A6 inhibits NADC-1 so that when it is actively transporting oxalate into the filtrate citrate absorption is reduced. In addition, the apical SLC26A6 exchanges chloride for other anions (including Cl⁻, HCO3⁻, and SO₄²⁻) and therefore allows oxalate and sulfate recycling. SLC26A1 (SAT1) is localized to the basolateral membrane. Slc26a1 knockout mice are hyperoxaluric (even in the absence of dietary oxalate) suggesting a role for Slc26a1 in reducing urinary oxalate secretion.

Phenotypes of AGXT (PH1) which accounted for approximately two thirds of this cohort were the most severe, with an earlier age of ESRD. GRHPR (PH2) mutations (9% of the cohort) were the next severe, followed by HOGA1 (PH3) (11% of the cohort). Interestingly, patients with HOGA1 mutations presented the earliest, but had the slowest decline in renal function. In the 38 patients from this group there was less nephrocalcinosis and levels of hyperoxaluria were less pronounced, when compared with PH1 and PH2. The low level of ESRD in this group and the more benign phenotype suggests decreased sensitivity to HOGA levels with aging.¹⁸ Eleven percent of families with a clinical and biochemical diagnosis of PH remained unsolved.¹⁸ Metabolic evaluation confirmed patients with PH1 had both high incidence of nephrocalcinosis and higher urinary oxalate levels. The elevation of urinary glycolate, L-glycerate, and HOGA may not allow sufficient difference to confirm PH subtypes and molecular genetic analysis was emphasized as the most precise way to confirm a diagnosis.¹⁸ Late presentations of PH may occur and must be diagnosed before renal transplantation is considered.¹⁹

Additional information regarding monogenic stone disease has been revealed by the examination of renal stone cohorts. Previously thought to account for just 2% of all renal stones,²⁰ two genetic studies suggest that monogenic forms of renal stone disease may be more common. Next generation sequencing has allowed high throughput analysis of genes implicated in monogenic forms of kidney stones. For example, in a mixed pediatric (106) and adult (166) cohort of renal stone formers, 40 patients (almost 15%) had a monogenic cause.²¹ However, it should be noted this was not a randomly-selected population, rather patients were recruited from specialist centers and clinics, probably allowing for an enrichment in patients with rare stone disease and thus potentially over-estimating the contribution of rare monogenic causes.²² In a pediatric cohort of 143 patients with nephrolithiasis or nephrocalcinosis, again recruited from specialist clinics, a monogenic cause was found in 24 patients (16.8%).²³ Homozygous and compound heterozygous mutations were found in the autosomal recessive genes including ATP6V1B1, ATP6V0A4, CLDN16, SLC3A1, CYP24A1, SLC12A1, and AGXT. Heterozygous variants were described in putatively dominantly-inherited disease genes including ADCY10, SLC4A1, SLC9A3R1, SLC34A1, and VDR. Another caution is that not all of the genes associated with stones in this study have had their mechanisms of action, their associated normal physiology, or the phenotypes associated with mutation described. For example, ADCY10 encodes the bicarbonate-sensitive adenylate cyclase, which has been implicated, but not proven, to have a role in autosomal dominant absorptive hypercalciuria.²⁴

New insights from other rare monogenic stone conditions have also been made. Biallelic mutations in SLC34A3, encoding the sodium-dependent phosphate transporter 2c, cause hereditary hypophosphatemic rickets with hypercalciuria.²⁵ Patients with biallelic SLC34A3 mutations may also display hypercalciuria, nephrocalcinosis, and nephrolithiasis without bone phenotypes. Interestingly, heterozygous SLC34A3 carriers have a biochemical phenotype that mimics idiopathic hypercalciuria and also may not necessarily present with bone disease. Dasgupta et al. studied a cohort of 133 individuals from 27 families with SLC34A3 mutations.²⁶ Renal calcification was seen in 16% of heterozygous carriers, especially those with reduced serum phosphate. This is a reminder that detailed biochemical phenotyping of renal stone formers can point toward an underlying molecular genetic diagnosis, and that familial pattern inheritance of stone formation should never be ignored. Furthermore, the concept of these strictly monogenic, notably recessive, disorders may have to be partially redefined, as heterozygosity or digenicity may as well be leading to a (milder) phenotype with manifestation only upon additional environmental or dietary triggers, such as poor fluid intake or sunlight exposure.

It is not surprising that genes involved in the vitamin D pathway have become implicated in renal stone formation.²⁷ Since the discovery of CYP24A1 mutations as the cause of infantile idiopathic hypercalcaemia²⁸ the phenotypic spectrum of mutations in this gene has widened. Adult patients with calcium stone disease may be a presentation of biallelic CYP24A1 mutations, with an asymptomatic childhood.²⁹ The biochemical features that point to this rare monogenic cause of renal stones are raised or high normal levels of serum calcium and a suppressed parathyroid hormone. Mutations lead to a loss of function in 25-hydroxy-vitamin D-24-hydroxylase, a key enzyme acting to 24-hydroxylate 25-hydroxy-vitamin D (to 24,25-dihydroxy-vitamin D) as well as acting to 24-hydroxylate 1, 25-dihydroxy-vitamin D (to 1,24,25-trihydroxy-vitamin D). Renal stone phenotypes may be revealed by increased vitamin D intake and sunlight exposure.³⁰ Nephrolithiasis phenotypes may be seen with single heterozygous mutations as well as biallelic changes.^31,32 Importantly, therapies directed at the inhibition of CYP450-dependent enzymes may be used to control 1,25-dihydroxyvitamin D levels,³³ although long term treatment with agents such as fluconazole may not be viable.

Together, these results from monogenic case series and kidney stone forming cohorts suggest that monogenic stone disorders (Table 1) must be screened for in a systematic way in order to understand the genetic burden among “idiopathic stone formers.” What remains fascinating is that genes responsible for rare monogenic causes of calcium-containing stones if mutated, such as ATP6V1B1 and SLC34A1, have been identified as carrying risk alleles for stone formation in wider population studies and are discussed below.

The Identification of a Novel Monogenic Calcium Oxalate Stone Disorder

Recently, interest in SLC26A1 as a candidate gene for hyperoxaluria, which encodes a sulfate anion exchanger protein SAT1, has grown. The murine model (Sat1^−/−) reveals phenotypes of both liver toxicity and stone formation in renal tubules and bladder secondary to hyperoxaluria.³⁴ SAT1 is an epithelial transport protein and is expressed in the kidney (at the basolateral membrane of renal proximal tubular epithelial cells and collecting ducts), liver (sinusoidal membranes of hepatocytes), and intestine (Figure 1), where it acts as a sulfate exchanger, allowing oxalate secretion.^34–36 The contribution of variants in SLC26A1 to human calcium oxalate formation has recently been reported. In a pilot study Dawson et al. studied 13 patients with recurrent calcium oxalate stone formation. A rare heterozygous variant p.M132T was identified in one patient with severe nephrocalcinosis, however segregation analysis and studies of other family members was not performed. This tantalizing story set the scene for a larger-scale candidate approach in a cohort of 348 patients with nephrolithiasis or nephrocalcinosis, in whom 30 genetic causes had previously been excluded.³⁷ In one patient from Macedonia with calcium oxalate calculi presenting at 5 years of age, and confirmed hyperoxaluria, compound heterozygous mutations were identified in SLC26A1. In a second patient, from consanguineous parents, a single homozygous variant was identified in SLC26A1, however 24-hour urine for oxalate was normal. Functional studies confirmed defects in anion exchange in transfected HEK293T cells.³⁷ These results point toward SLC26A1 as a novel genetic cause of autosomally-recessive calcium oxalate stone disease with roles in intestinal and tubular oxalate transport (Figure 1). However, it remains unclear what specific metabolic phenotype, other than calcium oxalate stone formation, would help clinicians to identify these patients. Unfortunately, a related gene, SLC26A6, was not included in this candidate gene screen, despite recent evidence of Slc26a6-null mice having hyperoxaluria and calcium oxalate urolithiaisis.³⁸ A screen comparing 225 calcium oxalate stone formers from China with 201 healthy controls has recently implicated a missense variant in SLC26A6 in human renal stone disease.³⁹ Thirteen patients in the disease group and two patients in the control group were found to have heterozygous variant p.G539R. In contrast, variants in SLC26A6 were not found to be an important genetic factor in eight patients with hyperoxaluria where AGXT and GRHPR mutations had been excluded.⁴⁰

Complex Disease and Results from Genome-Wide Genetic Studies and Candidate Gene Analysis

The heritability of idiopathic calcium-containing stones has been discussed in detail previously.²⁰ Familial clustering, case-control studies, and statistical modeling all point strongly to a genetic component (up to half of an individual’s risk) for renal stone disease. Indeed, around 35% of stone formers have an affected family member.^41–43

Some progress has been made in our understanding of this genetic predisposition to form calcium-containing renal stones by using modern technologies that allow large-scale genetic studies to be performed. Genes previously associated with calcium nephrolithiasis identified by genome-wide association studies (GWAS) include SLC34A3,⁴⁴ CLDN14,⁴⁵ SLC34A1, AQP1, DGKH,⁴⁶ and UMOD⁴⁷ (Table 1). More recently, Oddsson et al. performed a GWAS using data from whole-genome sequencing of Icelanders,⁴⁸ a population with a high stone prevalence.⁴⁹ This approach, with a sample size of 5419 patients, led to the identification of an intronic sequence variant (rs1256328) associated with kidney stones in ALPL (encoding an alkaline phosphatase isoenzyme) and a suggested association with CASR (encoding the calcium-sensing receptor), a known candidate gene for kidney stone formation.⁵⁰ In addition, missense variants in SLC34A1 (encoding the sodium-dependent phosphate cotransporter type 2) and TRPV5 (encoding the transient receptor potential cation channel V5, alias epithelial calcium channel 1) were also associated with recurrent kidney stones.⁴⁸

ALPL is an interesting candidate gene for stone disease. The intronic risk allele was associated with raised serum ALP levels. In addition, a protective missense allele (rs149344982) which associated with low serum ALP was also detected.⁴⁸ ALPL is expressed in the proximal tubule of the kidney where it acts to hydrolyze pyrophosphate, an inhibitor of stone formation,⁵¹ to free phosphate. Renal stone risk therefore appears to be a balance between tubular pyrophosphate and phosphate production. Biallelic loss of function mutations in ALPL cause hypophosphatasia syndrome, which may present with infantile hypercalcemia, hypercalciuria, and nephrocalcinosis and is associated with low 1,25-dihydroxy Vitamin D levels.⁵² ALPL also has a crucial role in bone formation with membrane-associated ALPL on osteoblasts being essential for bone mineralization. An imbalance of bone resorption and bone mineralization would contribute to the risk of renal calcification.

The associated and predicted to be dysfunctional variant in SLC34A1, encoding a sodium/phosphate cotransporter protein, found by Oddsson⁴⁸ also implicates renal tubule luminal phosphate levels with kidney stone risk. Rare variants in SLC34A1 are seen in patients with hypophosphatemic nephrolithiasis/osteoporosis (Online Mendelian Inheritance in Man: 612286)⁵³ and autosomal recessive mutations in SLC34A1 have been identified in patients with infantile hypercalcemia and renal phosphate wasting. Heterozygous relatives show an increased frequency of nephrocalcinosis and nephrolithiasis.⁵⁴

Complementary to GWAS studies, candidate gene approaches have identified variants in numerous genes that may be associated with calcium nephrolithiasis, some of which have been replicated in different populations. These include CASR,^55–57 VDR,^58,59 OPN,^60,61 MGP,^62,63 and PLAU.^64,65 Surprisingly, a recent analysis of 40 known candidate genes that may influence urinary calcium excretion, including CASR, did not confirm a role for rare variants in known genes in modulating urinary calcium excretion.⁶⁶ Importantly, the variant in CLDN14 identified in an Icelandic GWAS⁴⁵ did not differ between subjects with low and high urinary calcium levels. The combined outputs of these studies confirm the complexity and challenges of renal stone genetics.

Mutations in ATP6V1B1 encoding the vacuolar H(+)-ATPaseB1 subunit are a rare monogenic form of nephrolithiasis and nephrocalcinosis. Biallelic mutations cause autosomal recessive distal renal tubular acidosis with hearing loss in which patients may present with nephrocalcinosis and renal stones.⁶⁷ In a recent genotype-phenotype study 555 stone registry patients were genotyped for an single nucleotide polymorphism (rs114234874) within ATP6V1B1 (p.E161K),⁶⁸ which has been shown to be functionally important in vitro despite in silico predictions of the variant being a benign polymorphism. Thirty-two patients were identified to be heterozygous for this variant, and under certain dietary restrictions and after ammonium chloride loading, were found to have a higher urinary pH, with a tendency toward calcium phosphate stone formation. This single nucleotide polymorphism, even in a heterozygous state, may therefore confer a risk of nephrolithiasis. The frequency of this variant in the ExAC database (http://exac.broadinstitute.org/) is approximately 3%, thus this variant could be a key variable in conferring risk of calcium phosphate stones in the general population.

Investigating Pathogenic Mechanisms and Hypercalciuria as an Inherited Trait

Different pathways are implicated in idiopathic calcium stone formation¹³ and have been recently reviewed.^69,70 These include the hypothesis that urine within the Bellini duct may develop intraluminal crystal aggregates allowing the deposition of calcium-phosphate and that Randall plaques (formed from interstitial hydroxyapatite deposits) may allow urinary calcium oxalate precipitation.^13,71 Both these mechanisms rely on urinary supersaturation with respect to calcium salts and it is the genetics underlying these metabolic risk factors that has proved elusive.

Idiopathic hypercalciuria was originally defined by Albright in 1953, where 22 patients with kidney stones were shown to have high urine calcium levels in the context of normal serum calcium levels and low serum phosphate levels.⁷² Such patients seem to hyperabsorb calcium from the intestine as well as having a reduced renal tubular reabsorption of calcium, leading to a metabolic tendency toward calcium stone formation. This tendency toward hypercalciuric stone formation is heritable⁷³ but the underlying molecular genetics have remained obscure.⁷⁴ Indeed, idiopathic hypercalciuria remains the most common metabolic abnormality associated with renal stones.⁷⁵ Some care must be taken in defining hypercalciuria, as urinary calcium excretion rates are continuous variables, and its effect on stone formation is also likely to be continuous. Curhan has suggested that specific cutoffs for abnormal urinary calcium are not always appropriate as the risk of stone formation increases as calcium excretion increases, even within its “normal” ranges.⁷⁶ It is also interesting that associations between metabolic risk factors such as hypercalciuria and the risk of stone formation vary by age.⁷⁶ For example, in younger cohorts females had more pronounced hypercalciuria which did not persist in older cohorts. Higher than “normal” calcium excretion was also frequently noted in control populations. These nuances must therefore be taken into consideration when interpreting genetic and other studies involving idiopathic hypercalciuric stone formers, as the risk factor is a continuous variable and not binary.

Randall plaques themselves may be seen as a risk factor for stone formation, although this remains controversial. The area of Randall plaques at the papillary surface has been shown to correlate with both calcium excretion and stone-forming rate.⁷⁷ A recent genome-wide expression profile analysis of tissue from Randall plaques in human calcium oxalate stone formers revealed altered regulation of multiple genes, with pathway analysis that implicates oxidative stress, proinflammation, renal dysfunction, and tubular ion transport.⁷⁸ Such novel studies herald the beginning of molecular targeted therapies for Randall plaques and renal stones.

Genes versus Environment in Nephrolithiasis

Gastric bypass surgery for the treatment of obesity is a direct example of how a change in environment can influence the incidence of stone formation.⁷⁹ This extreme example of changing gut anatomy and microbiota points to a close relationship between the bowel and the kidney and the maintenance of serum electrolytes. The intestinal microbiome plays a fundamental role in determining the urinary chemistry and future strategies of understanding renal stone disease will need to examine both the host genome and its interaction with the genomes of the gut flora, such as Oxalobacter formigenes.⁸⁰ In addition, dietary changes may regulate gene expression via epigenetic mechanisms.⁸¹ The progressive rise in renal stone incidence that we are seeing globally thus provides an opportunity for certain kidney stone phenotypes to be revealed more readily (and understood) in genetically-susceptible individuals and their relatives.

Progress in Animal Models of Nephrolithiasis

Animal models for renal stone formation have historically relied on toxins, such as ethylene glycol, to induce calcification within the renal tract. Pure genetic models have been more difficult to generate, however there are some good examples of recent progress (Table 2). Drosophila melanogaster has for a long time been an established disease model for xanthinuria and the Drosophila tubule remains a model system in which to explore the genetics of crystallization.⁸² Indeed, manipulation of the xanthine dehydrogenase pathway in Drosophila has recently implicated zinc as a driver for mineralization, which was then confirmed using genetic models of zinc transport knockdown.⁸³ Zinc foci were also noted within Randall plaques in human renal papilla biopsy tissues, which implicates zinc in the initiation of calcification.⁸³ There are several genes associated with renal stone disease that are enriched in Drosophila tubules. Indeed, the Drosophila homolog of SLC26A6 (encoding dPrestin) seems to have conserved chloride and oxalate exchange function and has been used very recently to explore therapeutic inhibitors of calcium oxalate crystallization.⁸⁴ Such basic biologic research may have important future translational effects in human stone disease.

Table 2.

Animal models of calcium crystallization and nephrolithiasis and latest insights

Animal Model	Latest Findings	Reference
Drosophila	Zinc transport within renal tubules implicated in driving mineralization	83
Drosophila	Drug manipulation of SLC26a5/6 to inhibit oxalate transport	84
Zebrafish	Phenotype of trpm7 mutants show defects in skeletal development and produce kidney stones	85
Mice	Sat1−/− model of intraluminal calcium oxalate stone formation	34
Mice	Slc26a6-null model of calcium oxalate stone formation	38
Mice	Cldn10−/− mice exhibit nephrocalcinosis upon tissue-specific Claudin 10 deficiency in thick ascending loop of Henle	91
Mice	Trpv5−/− mice exhibit hypercalciuria and intestinal hyperabsorption of calcium	92
Genetic hypercalciuric rat	Therapeutic interventions and urinary biochemical changes	97

The zebrafish (Danio rerio) is a useful model organism in which to study renal development, however, the development of renal stones would not be expected within the zebrafish pronephros due to a lack of urinary concentrating ability. Remarkably, trpm7 mutants, who exhibited severe growth retardation and skeletal defects, also developed mineralized deposits resembling kidney stones within the lumen of the developing renal tubules as early as 5 days postfertilization,⁸⁵ and this was associated with reduced levels of calcium and magnesium in serum.⁸⁶ The defect points toward a role for zebrafish trpm7 in calcium and magnesium homeostasis during skeletal development. Mammalian TRPM7 encodes a cation channel that is widely-expressed and is essential for magnesium homeostasis⁸⁷ but hasn’t been implicated in human nephrolithiasis.

Murine genetic models of nephrolithiasis are surprisingly limited.⁸⁸ A recent review highlights models of interstitial calcinosis such as mice deficient for Umod, Opn, Npt2a, and Nherf1.⁸⁹ The Sat1^−/− mice provide a novel model of calcium oxalate stone formation, where calcium oxalate crystals were observed in the lumen of kidney cortical tubules as well as the bladder, which could be used to screen novel therapies.³⁴ Given that human mutations have now been identified in the Sat1 homolog SLC26A1, therapies targeting this channel in order to prevent hyperoxaluria need to be explored further and this murine model would facilitate this. In a related genetic model, Slc26a6-null mice developed calcium oxalate stones, secondary to a defect in intestinal oxalate secretion, leading to elevated plasma oxalate levels and hyperoxaluria.³⁸ Another mechanism of calcium stone formation may be due to hypocitraturia, as urinary citrate inhibits stone formation by inhibiting calcium crystallization and precipitation. Interestingly, it was recently shown that SLC26A6 interacts with the neighboring sodium-citrate transporter, NaDC1 (SLC13A2), in the apical membrane of the proximal tubule (Figure 1) and allows a reciprocal regulation.⁹⁰ Disruption of SLC26A6 resulted in increased tubular reabsorption of citrate and subsequent lack of inhibitory substrates for prevention of stone formation under states of relatively high urinary oxalate.⁹⁰

A mouse model exhibiting nephrocalcinosis on the basis of increased tubular absorption of calcium and magnesium was generated by tissue-specific knockout of claudin-10, a tight junction protein, primarily localized to the thick ascending loop of Henle and taking part in regulation of paracellular reabsorption of sodium and H₂O.⁹¹ Together with CLDN16, CLDN19, and CLDN14, there is growing evidence pointing to the importance of an intact paracellular ion uptake for the prevention of calcium stone formation.

TRPV5 encodes a calcium-selective transient receptor potential channel which is expressed in renal epithelial cells. Trpv5^−/− mice exhibit hypercalciuria, and intestinal hyperabsorption of calcium, mediated via Trpv6 upregulation.⁹² Trpv5^−/− mice also have a bone phenotype, with reduced cortical and trabecular bone thickness, with impaired bone resorption implicating TRPV5 in osteoclast function.⁹³ Recently, an autosomal dominant mouse model of hypercalciuria (generated following an N-ethyl-N-nitrosourea mutagenesis program) has been described where a Trpv5 missense allele, p.S682P, was associated with renal hypercalciuria, implicating abnormal calcium handling in the distal convoluted tubule.⁹⁴ However, polymorphisms in human TRPV5 have not been associated with urinary calcium excretion rates⁶⁶ or functional defects in this calcium channel⁹⁵ and no convincingly pathogenic mutations have been described to date.

The genetic hypercalciuric stone-forming rat model, now into its 95th generation of inbreeding, continues to provide informative data regarding calcium stone formation. This is a highly relevant model for considering the underlying complex genetics of hypercalciuria and stone formation as it demonstrates the polygenic nature of the disorder, recapitulates many aspects of the human disorder, and mirrors the human bone mineral disorder. The genetic hypercalciuric stone-forming rats exhibit a dysregulation of calcium transport with intestinal hyperabsorption of calcium, increased bone resorption, and reduced renal tubular calcium reabsorption, mediated by increases in vitamin D receptor expression in these tissues.⁹⁶ The model allows the exploration of dietary and pharmacologic interventions such as exogenous vitamin D supplementation, thiazide diuretics,⁹⁶ and potassium citrate on the disease phenotype.⁹⁷ The polygenic genetic variants underlying this particular model have yet to be fully elucidated but at least one quantitative trait locus (on chromosome 1) has been identified.⁹⁸

Summary

The pathophysiology underlying renal stone formation is complex, but evidence points strongly, in many cases, to a genetic predisposition toward stone formation. Given this heritability, modern genomic techniques have allowed the identification of a growing number of monogenic stone diseases in humans. The contribution of monogenic stone disease to the total population of kidney stone formers, however, is controversial and has to be resolved by more comprehensive clinical genetic studies. GWAS and candidate studies have directed us to the importance of complex inheritance of renal tubular calcium and phosphate handling and crystallization events. Hypercalciuria and hyperoxaluria are key metabolic risk factors underlying stone formation and new genes and model systems in which to explore this and other risk factors will hopefully allow new therapeutic approaches to be made.

Disclosures

None.