**2.2. Renal calcium handling**

Of all the calcium filtered by the kidney, 98% is reabsorbed by the tubules, with the proximal tubule reabsorbing about 65%, the TAL of the loop of Henle accounting for approximately 20–25%, and 8–10% being reabsorbed in the distal tubule [15]. Hypercalciuria is an important, identifiable, and reversible nephrocalcinosis risk factor. It is a complex trait, caused by both environmental and genetic factors. It is not a disease per se, but represents the upper end of a continuum, rather like height, weight, and blood pressure, and—like these polygenic traits urinary calcium excretion should be considered a graded risk factor [16]. **Table 2** shows a summary of the clinically and experimentally identified monogenic causes of hypercalciuria, pointing to the genetic causes of renal calcium leak.

It is worth remembering that the crystallization of calcium salts is a physiological event linked to biomineralization, i.e., the capacity of calcium, like other inorganic crystalline or non‐crystal‐ line minerals, to interact with and deposit around biomolecules, becoming an integral part of organic tissues to provide hardness and strength. Biomineralization is often arbitrarily distin‐ guished as physiological or pathological. It would be more appropriate to say that pathological calcium crystallization is a physiological process occurring in the wrong place and at the wrong time [17]. Nephrocalcinosis might fit this definition.


Conditions predisposing to medullary nephrocalcinosis may be either those that raise the urinary concentration of inductors of calcium crystal deposition or those that lower the con‐ centration of the inhibitors of this process. The former category includes hypercalciuria, hyp‐ eroxaluria, the latter hypocitraturia and hypomagnesuria. Renal tubular acidosis (RTA), on the other hand, is responsible for changes in urinary pH, which has a fundamental role in favoring crystallization. In some cases, there may also be specific anatomical abnormalities that predispose to the onset of nephrocalcinosis, as in medullary sponge kidney (MSK).

6 Updates and Advances in Nephrolithiasis - Pathophysiology, Genetics, and Treatment Modalities

Several genetic disorders have been found associated with conditions that predispose individu‐ als to the development and progression of nephrocalcinosis. Most of them are tubular disorders associated with epithelial cellular and paracellular ion transport disruptions that result in the urinary excretion of higher levels of calcium, phosphate or oxalate and lower levels of citrate and magnesium. **Table 1** shows the genetic basis for the link between some inherited disorders and medullary nephrocalcinosis [7]. **Figure 1** shows the list of intrarenal transport defects that prompt a dysfunctional renal handling of the two most important divalent cations Ca2+ and Mg2+ [14]. As can be noted, not all cation‐handling disorders are associated with nephrocalcinosis. The kidney handles calcium, phosphate, and oxalate in the proximal tubules, in the thick ascending limb (TAL) of the loop of Henle, and in the distal convolute tubule (DCT), shown in **Figure 2** [6]. Knowing the site where the tubular exchanger and transporter proteins involved in regulating urinary calcium, phosphate, and oxalate work help us better understand the

Bearing in mind that 98% of interstitial crystal deposition occurs in the medulla around each

Of all the calcium filtered by the kidney, 98% is reabsorbed by the tubules, with the proximal tubule reabsorbing about 65%, the TAL of the loop of Henle accounting for approximately 20–25%, and 8–10% being reabsorbed in the distal tubule [15]. Hypercalciuria is an important, identifiable, and reversible nephrocalcinosis risk factor. It is a complex trait, caused by both environmental and genetic factors. It is not a disease per se, but represents the upper end of a continuum, rather like height, weight, and blood pressure, and—like these polygenic traits urinary calcium excretion should be considered a graded risk factor [16]. **Table 2** shows a summary of the clinically and experimentally identified monogenic causes of hypercalciuria,

It is worth remembering that the crystallization of calcium salts is a physiological event linked to biomineralization, i.e., the capacity of calcium, like other inorganic crystalline or non‐crystal‐ line minerals, to interact with and deposit around biomolecules, becoming an integral part of organic tissues to provide hardness and strength. Biomineralization is often arbitrarily distin‐ guished as physiological or pathological. It would be more appropriate to say that pathological calcium crystallization is a physiological process occurring in the wrong place and at the wrong

We focus our attention on the genetic defects that alter the kidney's homeostatic capacity.

pyramid, Sayer et al. proposed the model of nephrocalcinosis shown in **Figure 3**.

mechanisms underlying nephrocalcinosis.

pointing to the genetic causes of renal calcium leak.

time [17]. Nephrocalcinosis might fit this definition.

**2.2. Renal calcium handling**


**Table 1.** Inherited disorders associated to medullary nephrocalcinosis (NC).

#### **2.3. Renal phosphate handling**

The kidney's control over systemic phosphate homeostasis is crucial. About 80% of filtered phosphate is reabsorbed from the urine by transporters located in the proximal tubule and mostly in the juxtamedullary nephrons (**Figure 2**). At least three transporters are responsible for renal phosphate reabsorption, and they are precisely regulated by various cellular mecha‐ nisms and factors [18]. They are members of the Type II NA+ ‐dependent phosphate cotrans‐ porter family encoded by the SLC34A1, SLC34A3, and SLC20A2 genes. Though it is not a

**Figure 1.** Schematic representation of the nephron listing the predominant origins of intrarenal transport defects causing dysfunctional renal handling of divalent cations. This figure was originally published in Ref. [14].

Understanding the Pathophysiology of Nephrocalcinosis http://dx.doi.org/10.5772/intechopen.69895 9

**Figure 2.** Tubular transport of calcium, phosphate, and oxalate. A nephron map of exchanger and transporter proteins involved in the regulation of urinary calcium, phosphate and oxalate is shown. In the proximal tubule (PT) apical Na/ Pi cotransporters (NaPi‐2a) mediate phosphate reabsorption, whereas several anion exchange proteins (including those of the SLC26 family) mediate transcellular oxalate (Ox) secretion and recycling. There is no evidence for calcium or phosphate transport in the thin descending limb (DLH) or the thin ascending limb (tALH)of the loop of Henle. The TAL of Henle allows paracellular calcium (and magnesium) reabsorption via paracellin channels (PCLN‐1). The distal convoluted tubule (DCT) allows for regulated transcellular reabsorption of calcium via apical TRPV5 and the basolateral NCX, together with the basolateral PMCA. A calcium entry pathway exists within the IMCD, the molecular identity of which remains uncertain. It is likely that Na/Pi isoforms are also present in this nephron segment and their physiological function may be linked to the PPi transporter protein ANKH. Basolateral calcium exporters NCX and PMCA are also present in the collecting duct. This figure was originally published in Ref. [6].

transporter protein, the Na+/H+ exchanger regulatory factor (NHERF1) plays a crucial part in renal phosphate transport by binding to SLC34A1 in the proximal tubule. Alterations in the genes encoding these transporters result in phosphate wasting, and consequent hyper‐ phosphaturia (**Table 3**). For the sake of completeness, **Table 3** also includes renal phosphate handling impairments due to extrarenal inherited defects.

#### **2.4. Renal oxalate handling**

**Figure 1.** Schematic representation of the nephron listing the predominant origins of intrarenal transport defects causing

The kidney's control over systemic phosphate homeostasis is crucial. About 80% of filtered phosphate is reabsorbed from the urine by transporters located in the proximal tubule and mostly in the juxtamedullary nephrons (**Figure 2**). At least three transporters are responsible for renal phosphate reabsorption, and they are precisely regulated by various cellular mecha‐

**Gene Protein Disorder Mode of inheritance**

AD, autosomal dominant; AR, autosomal recessive; XLR, X‐linked recessive; XLD, X‐linked dominant.

(MSK)

Medullary sponge kidney

Familial idiopathic hypercalciuria

Cystinuria AD

Liddle syndrome AD

Wilson syndrome AR

AD

AD

porter family encoded by the SLC34A1, SLC34A3, and SLC20A2 genes. Though it is not a

‐dependent phosphate cotrans‐

nisms and factors [18]. They are members of the Type II NA+

dysfunctional renal handling of divalent cations. This figure was originally published in Ref. [14].

**2.3. Renal phosphate handling**

GDNF Glial cell line‐derived

SLC7A9 B(0,+)‐type amino acid

ADCY10 Adenylate Cyclase 10,

ATP7B Copper‐transporting

SCNN1G/B Renal epithelium channel

Soluble

ATP‐ase

**Table 1.** Inherited disorders associated to medullary nephrocalcinosis (NC).

neurotrophic factor

8 Updates and Advances in Nephrolithiasis - Pathophysiology, Genetics, and Treatment Modalities

Transporter 1 (BAT1)

(βENaC and αENaC)

Urinary oxalate is the most important risk factor for CaOx nephrocalcinosis/nephrolithiasis. Oxalate is filtered freely at the glomerulus [6]. Anion exchange proteins in the proximal tubule mediate oxalate excretion and recycling at the brush border membrane (**Figure 2**). These pro‐ teins belong to the SLC26 family, and they allow oxalate loss in exchange for chloride, then uptake oxalate in exchange for sulfate loss, energized by Na‐sulfate transport in the proximal tubules [19]. The main causes of hyperoxaluria relate, however, to genetic defects that alter glyoxylate metabolism in the liver and erythrocytes, leading to endogenous oxalate over‐ production. These hereditary autosomal recessive forms of hyperoxaluria are called primary hyperoxaluria type I, type II, and type III [20]. Defects in the genes responsible for oxalate reab‐ sorption have recently been reported too. Recessive mutations in SLC26A1 gene were identified in two unrelated individuals with calcium oxalate kidney stones. Functional experiments have

**Figure 3.** Renal papillary model of nephrocalcinosis. Collecting ducts (CD) together with descending loops of Henle (DLH) and ascending thin loops of Henle (tALH) and vasa recta (VR) are shown through a cross‐section of a renal papilla. Possible initial sites of nephrocalcinosis are shown as shaded regions. Mechanisms leading to calcification may include: (1) Pi permeability at the papillary thin loops of Henle would allow interstitial loading of phosphate; (2) collecting duct absorption of calcium, possibly via apical calcium channels (e.g., TPC1) and basolateral calcium exit would allow delivery of calcium ions to the interstitium; Na/Pi cotransport at a collecting duct location would also provide additional phosphate ions, which may be derived from PPi delivery into the lumen via ANK; (3) concentration of oxalate from the urinary space into the papillary interstitium allows delivery of oxalate ions; (4) intraluminal crystal formation, both from the loop of Henle (calcium phosphate) and collecting duct (calcium oxalate) may adhere to collecting duct epithelial surfaces, then by endocytosis/transcytosis the crystals are delivered to the papillary interstitium and accumulate. Dissolution by epithelial cells or by interstitial cells (including macrophages) may provide a clearance mechanism. This figure was originally published in Ref. [6].

shown that these mutations resulted in decreased transporter activity [21], thus confirming their role in the disease. In the SLC26A6 gene has also recently been described a single nucleo‐ tide polymorphism associated with increased calcium oxalate kidney stones [22]. **Table 4** sum‐ marizes what we know about the genetics of hyperoxaluria.

#### **2.5. Renal citrate handling**

Citrate is filtrated freely at the glomerulus. In humans, from 65 to 90% of the filtered citrate is reabsorbed, mainly in the proximal tubule [23]. Urinary citrate is an important calcium chelator, consequently reducing the potential of calcium and oxalate to interact. In addition, citrate binds crystals' surface preventing their adhesion to renal epithelial cells [24]. It is intriguing that oxalate transport by SLC26A6 and citrate transport by the sodium dicarboxyl cotransporter SLC13A2 both located in the apical membrane of the proximal tubules and small intestine—have been found to interact. This was demonstrated in SLC26A6 KO mice, which are not only hyperox‐ aluric, but also hypocitraturic [25]. Hypocitraturia is a known risk factor for the development


**Table 2.** Monogenic forms of hypercalciuria: kidney as the primary defect.

shown that these mutations resulted in decreased transporter activity [21], thus confirming their role in the disease. In the SLC26A6 gene has also recently been described a single nucleo‐ tide polymorphism associated with increased calcium oxalate kidney stones [22]. **Table 4** sum‐

**Figure 3.** Renal papillary model of nephrocalcinosis. Collecting ducts (CD) together with descending loops of Henle (DLH) and ascending thin loops of Henle (tALH) and vasa recta (VR) are shown through a cross‐section of a renal papilla. Possible initial sites of nephrocalcinosis are shown as shaded regions. Mechanisms leading to calcification may include: (1) Pi permeability at the papillary thin loops of Henle would allow interstitial loading of phosphate; (2) collecting duct absorption of calcium, possibly via apical calcium channels (e.g., TPC1) and basolateral calcium exit would allow delivery of calcium ions to the interstitium; Na/Pi cotransport at a collecting duct location would also provide additional phosphate ions, which may be derived from PPi delivery into the lumen via ANK; (3) concentration of oxalate from the urinary space into the papillary interstitium allows delivery of oxalate ions; (4) intraluminal crystal formation, both from the loop of Henle (calcium phosphate) and collecting duct (calcium oxalate) may adhere to collecting duct epithelial surfaces, then by endocytosis/transcytosis the crystals are delivered to the papillary interstitium and accumulate. Dissolution by epithelial cells or by interstitial cells (including macrophages) may provide a clearance mechanism. This

10 Updates and Advances in Nephrolithiasis - Pathophysiology, Genetics, and Treatment Modalities

Citrate is filtrated freely at the glomerulus. In humans, from 65 to 90% of the filtered citrate is reabsorbed, mainly in the proximal tubule [23]. Urinary citrate is an important calcium chelator, consequently reducing the potential of calcium and oxalate to interact. In addition, citrate binds crystals' surface preventing their adhesion to renal epithelial cells [24]. It is intriguing that oxalate transport by SLC26A6 and citrate transport by the sodium dicarboxyl cotransporter SLC13A2 both located in the apical membrane of the proximal tubules and small intestine—have been found to interact. This was demonstrated in SLC26A6 KO mice, which are not only hyperox‐ aluric, but also hypocitraturic [25]. Hypocitraturia is a known risk factor for the development

marizes what we know about the genetics of hyperoxaluria.

**2.5. Renal citrate handling**

figure was originally published in Ref. [6].


**Table 3.** Genetic basis of altered renal phosphate handling.

of nephrocalcinosis/nephrolithiasis. No monogenic form of hypocitraturia has been reported so far, whereas genetic associations have been demonstrated between polymorphisms in the VDR and SLC13A2 genes and hypocitraturia [26, 27]. Very recently, Rendina et al. [28] provided evidence of an epistatic interaction between VDR and SLC13A2 in the pathogenesis of hypoci‐ traturia. This may come as no surprise because the active form of vitamin D in the nephron uses VDR to modulate citrate metabolism and transport [26]. Finally, Shah et al. [29] have suggested


**Table 4.** Inherited disorders of renal oxalate handling.

other genetic influences on citrate handling too: they propose a codominant inheritance of alleles at a single locus based on their trimodal frequency distribution of citrate excretion.

#### **2.6. Renal magnesium handling**

of nephrocalcinosis/nephrolithiasis. No monogenic form of hypocitraturia has been reported so far, whereas genetic associations have been demonstrated between polymorphisms in the VDR and SLC13A2 genes and hypocitraturia [26, 27]. Very recently, Rendina et al. [28] provided evidence of an epistatic interaction between VDR and SLC13A2 in the pathogenesis of hypoci‐ traturia. This may come as no surprise because the active form of vitamin D in the nephron uses VDR to modulate citrate metabolism and transport [26]. Finally, Shah et al. [29] have suggested

**Disorder Gene Protein Mode of inheritance**

aminotransferase

hydroxypyruvate reductase

aldolase, mitochondria

SLC26A1 Sulfate anion transporter 1 AR

**Gene Protein Disorder Mode of** 

Hypophosphatemic nephrolithiasis/

Hypophosphatemic nephrolithiasis/

Tumoral calcinosis, hyperphosphatemic AR

AR

AR

AR

with hypercalciuria (HHRH)

osteoporosis‐1

osteoporosis‐2

SLC34A1 Sodium‐phosphate transport protein 2A Fanconi renal tubular syndrome 2,

12 Updates and Advances in Nephrolithiasis - Pathophysiology, Genetics, and Treatment Modalities

SLC34A3 Sodium‐phosphate transport protein 2C Hereditary hypophosphatemic rickets

FGF23 Fibroblast growth factor 23 Hypophosphatemic ricket AD KLOTHO Regulator of calcium homeostasis (Klotho) Tumoral calcinosis, hyperphosphatemic AR

PHEX Phosphate‐regulating neutral endopeptidase Hypophosphatemic rickets XLD FAM20C Extracellular serine/threonine protein kinase Raine syndrome AR FGFR1 Fibroblast growth factor receptor 1 Osteoglophonic dysplasia AD DMP1 Dentin matrix acidic phosphoprotein 1 Hypophosphatemic rickets AR

exchange regulatory cofactor

AD, autosomal dominant; AR, autosomal recessive; XLD, X‐linked dominant.

Hyperoxaluria type I AGXT Serine‐pyruvate

Hyperoxaluria type II GRHPR Glyoxylate reductase/

Hyperoxaluria type III HOGA1 4‐hydroxy‐2‐oxoglutarate

acetylgalactosaminyltransferase 3

**Table 3.** Genetic basis of altered renal phosphate handling.

SLC9A3R1 Na+

/H<sup>+</sup>

NHE‐RF1

GALNT3 Polypeptide N‐

Calcium oxalate kidney

PH, primary hyperoxaluria; AR, autosomal recessive.

**Table 4.** Inherited disorders of renal oxalate handling.

stones

**inheritance**

AD

AR

AD

Hypomagnesuria is the biochemical abnormality found in about 19% of kidney stone patients, alone or in association with other biochemical abnormalities [30]. The kidney has a key role in maintaining a normal magnesium balance. The TAL of the loop of Henle and the DCT are crucially important in regulating serum magnesium levels and body magnesium content (**Figure 2**). Understanding the molecular defects behind rare genetic magnesium loss disor‐ ders has greatly contributed to our understanding of renal magnesium handling. About 80% of all plasma magnesium is filtered through the glomeruli, and 15–20% of it is reabsorbed by the proximal tubules, and 55–70% by the cortical TAL [31]. Magnesium is reabsorbed via a paracellular pathway in this nephron segment. Members of the claudin family of tight junc‐ tion proteins have been attributed a role in controlling magnesium and calcium permeability of the paracellular pathway (**Figure 4**) [31]. Although only 5–10% of the filtered magnesium is reabsorbed in the DCT, this process is finely regulated and plays an important part in deter‐ mining its final urinary excretion [31, 32].

**Figure 4.** Magnesium reabsorption in the cortical thick ascending limb (TAL) of Henle's loop and in the distal convoluted tubule (DCT). The key proteins influencing magnesium reabsorption are indicated. Magnesium reabsorption in the TAL is passive and occurs through the paracellular pathway. The driving force is the lumen‐positive transcellular voltage, which is generated by the transcellular reabsorption of NaCl and the potassium recycling back to the tubular fluid via ROMK. Magnesium transport through DCT cells is active and depends on the negative membrane plasma potential. This mechanism seems to depend on a sodium gradient that results from the coordinate action of NCCT, Na‐K‐ATPase and Kir4.1.


**Table 5.** Inherited disorders of renal magnesium loss.

Hereditary forms of hypomagnesemia include rare, genetically determined disorders that may affect renal magnesium handling either primarily or secondarily. **Table 5** summarizes the spectrum of underlying genetic defects [31].

#### **2.7. Pyrophosphaturia**

Pyrophosphate (PPi) is present in urine and can contribute 50% CaOx monohydrate (COM) crystal growth inhibition in the collecting duct and up to 80% in the urine [33, 34]. It has been postulated that hypopyrophosphaturia is a metabolic risk factor for recurrent stone formers [35]. That the concentration of inorganic PPi is higher in urine than in plasma cannot fully explain the origin of urinary PPi, but does suggest that it is somehow either secreted into the tubule or generated locally [36]. PPi is generated mostly in the mitochondria, and it is a byproduct of about 190 biochemical reactions. The PPi end product must be promptly removed to ensure irreversible, one‐way reactions. PPi may be removed in three ways: by hydrolysis via cytoplasmic phosphatases; by PPi compartmentalization; or by its exporta‐ tion from the cytoplasm via a transporter such as ANKH protein, which is located in the principal cells of the renal collecting duct (**Figure 2**) [6, 37]. Many authors now assume that PPi is removed from the cell by this third means, although the exact physiological function of the ANKH protein has never been clarified [36]. Underexpression or loss of activity of ANK (the mouse homolog of ANKH) is believed to lead to CaP deposition in numerous tis‐ sues, due to loss of PPi's inhibitory effects on CaP formation, and to the ubiquitous nature of CaP mineralization [38]. The majority of known ANKH mutations are assumed to be of the gain‐of‐function type, however, and are responsible for clinical phenotypes character‐ ized by calcium PPi deposition in the joints, i.e., calcium pyrophosphate deposition disease [39]. Loss‐of‐function mutations presumably responsible for the loss of ANKH activity and a lower extracellular PPi were detected in patients with craniometaphyseal dysplasia, which is characterized by overgrowth and sclerosis of the facial bones and abnormal long bone mod‐ eling. No renal calcification was seen in association with this disease, however. Unlike bone, ion content in the tubular environment varies considerably, and the picture is further com‐ plicated by various reabsorption mechanisms, which may in turn be affected by a negative feedback from the tubular ion content [36]. This might explain why ANKH loss of function does not cause nephrocalcinosis or kidney stones.
