**3.1. Tubular nephrocalcinosis**

It is commonly assumed that crystals of CaOx or CaP form in the tubular fluid because of supersaturation and are presumably a renal mechanism for excreting excess waste [65–69]. In physiological conditions, this process is well controlled and lowers the risk of supersatura‐ tion [70–72]. When these control mechanisms fail, however, or changing conditions alter the solubility of the urinary calcium salts, there is a consequent crystal retention and renal cal‐ cium deposition. This may involve epithelial crystal adhesion when the crystals are smaller than the diameter of the tubular lumen or lead to crystals obstructing the tubules when crys‐ tal formation and/or aggregation becomes excessive (**Figure 5A**).

#### *3.1.1. Adhesion of crystals to the tubular epithelial cells: the fixed particle theory*

cystic disease and familial juvenile hyperuricemic nephropathy (Uromodulin‐related dis‐ eases). Mutations lead to a defective intracellular trafficking of THP, and to a reduced THP excretion and secretion. No renal stone disease has been described in patients with any of

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

Medullary nephrocalcinosis is a frequent finding in medullary sponge kidney (MSK), a renal malformation associated with renal stones, urinary acidification and concentration defects, cystic anomalies in the precalyceal ducts, a risk of urinary infections, and renal failure. In a large series of 375 patients with macroscopic nephrocalcinosis, it was found that the clinical diagnoses most frequently associated with MSK were hyperparathyroidism and dRTA [9]. The prevalence of MSK in the general population is not known because no systematic autopsy searches have been performed. In a large series of subjects undergoing iv urography for vari‐ ous reasons, pictures ranging from clearly evident MSK to faint radiological signs of the disease were seen in 0.5–1% of cases [59]. MSK is relatively well represented in renal stone patients, however, and has been found in up to 20% of recurrent renal calcium stone formers [60, 61].

Why this malformative condition may predispose to medullary nephrocalcinosis remains to be established. MSK is considered a rare and sporadic disorder, but a recent study showed that 50% of MSK stone formers had relatives with milder forms of MSK, suggesting that it is relatively common for MSK to be familial, and it may be inherited as an autosomal dominant trait [62]. It has also been reported that 12% of unrelated MSK patients carried in heterozygos‐ ity two very rare variants of the glial cell line‐derived neurotrophic factor (GDNF) gene, and these variants were inherited and co‐segregated with the MSK phenotype in some families [63]. Mezzabotta et al. [64] had the chance to conduct an *in vitro* analysis on the behavior of papil‐ lary renal cells coming from the healthy portion of a kidney resected due to renal cancer in a MSK patient with medullary nephrocalcinosis, who harbored one of these rare GDNF gene variants. They found an unexpected and previously never reported phenomenon involving

They demonstrated that silencing the GDNF gene in a human renal cell line and cultivating

demonstrate the functional role of GDNF gene mutation in determining the medullary neph‐ rocalcinosis associated with the MSK phenotype. They also provide the first experimental evidence of human renal tubular cells having a pivotal role in driving a calcification process.

It is commonly assumed that crystals of CaOx or CaP form in the tubular fluid because of supersaturation and are presumably a renal mechanism for excreting excess waste [65–69]. In physiological conditions, this process is well controlled and lowers the risk of supersatura‐ tion [70–72]. When these control mechanisms fail, however, or changing conditions alter the

The role of renal cells in nephrocalcinosis is discussed in the subsequent paragraphs.

nodules very similar to those of calcifying vascular cells.

PO4

. These results

these mutations to date, however [13].

**2.10. Medullary sponge kidney**

the spontaneous formation of Ca2

**3.1. Tubular nephrocalcinosis**

PO4

**3. Proposed mechanisms of nephrocalcinosis**

the silenced cells in osteogenic conditions triggered the deposition of Ca2

The first step in crystal formation is nucleation, i.e., the process by which free ions in solu‐ tion become associated forming microscopic particles. Crystallization can occur in solution micro‐environments, such as those potentially existing in certain parts of the nephron [73], as well as on surfaces (like those of cells), and in the extracellular matrix [74]. Nucleation is followed by an aggregation of the crystals forming in the free solution, giving rise to larger particles. Finlayson and Reid [75] postulated that crystals cannot grow large enough dur‐ ing the short time it takes them to transit through the tubules to be retained in the tubules because of their size ("free particle" mechanism). This led to the hypothesis that crystals can only remain in the kidney if they adhere to the tubular epithelium (the 'fixed particle' theory) [74–76]. As a general mechanism for the etiology of tubular nephrocalcinosis, it was therefore suggested that crystallization starts at particular sites on the epithelial surface, not

**Figure 5.** Proposed mechanisms of nephrocalcinosis. (A) Processes of tubular and interstitial calcium crystal deposition. (B) Possible mechanisms of interstitial crystal formation.

freely in the tubular fluid. A nascent crystal then becomes aggregated with other crystals, forming a mass large enough to occlude the nephron, leading to an obstructive tubulopathy (**Figure 5A**).

These crystals can be found in contact with the surface of injured/regenerating epithelial cells, apoptotic and/or necrotic cells, and denuded basement membranes [4, 77–84], giving the impression that the composition of the cell surface is crucial in modulating this process. In fact, it has been demonstrated on primary or immortalized tubular epithelial cells exposed to CaOx crystals that the crystal deposits preferentially adhere to injured, apoptotic, depolar‐ ized, immature, migrating, or proliferating tubular epithelial cells, rather than to fully differ‐ entiated, normal epithelia [85–88]. In this context, there is interesting evidence to suggest that proximal tubular cells bind crystals regardless of their differentiation status, whereas distal tubular cells (which are physiologically more likely to encounter crystals) only bind crystals when they are dedifferentiated [70], meaning that the distal tubular epithelium is unable to bind crystals when differentiated.

What we know about crystal adhesion in the proximal and distal tubules stems mainly from having identified the characteristics of the luminal membrane and the molecular composition of the crystal‐binding epithelia, which led to the discovery of several crystal‐binding mol‐ ecules [4, 80, 89–93]. Importantly, these crystal‐binding molecules are upregulated or redis‐ tributed to the apical membrane under certain conditions of cellular dedifferentiation, such as injury or repair, or variations in pathophysiological conditions [78, 87, 88, 94–96], which determine whether or not the crystals are retained in the kidney.

The different categories of crystal‐binding molecules identified *in vitro* to date include: (i) termi‐ nal sialic acid residues [79, 97, 98]; (ii) phospholipids, i.e., phosphatidylserine [78, 84, 99, 100]; (iii) membrane‐bound proteins, i.e., collagen IV [101], OPN [102–106], annexin 2 (ANX2) [107, 108] and nucleolin‐related protein (NRP) [88, 93, 109]; and (iv) glycosaminoglycans, of which hyal‐ uronan (HA) appears to be the most potent crystal‐binding polysaccharide [95, 96, 110]. It has been demonstrated that other proteins, such as matrix Gla protein (MGP), are implicated in this process too [48, 111]. It is intriguing, moreover, that all known crystal‐binding molecules con‐ tribute to inducing a negative cell‐surface charge, a feature that has proved important in crys‐ tal adhesion to renal epithelial cells [85, 97, 112]. This would suggest that an array of aberrant phenotypes could bind crystals if there are appropriate amounts of crystals and appropriately oriented negative charges on the luminal membrane.

On the other hand, crystals and/or concomitant high concentrations of calcium, oxalate, or phosphate have been found to induce injury, proliferation, inflammatory mediator produc‐ tion, and oxidative stress on contact with epithelial cells *in vitro*, suggesting that epithelial dedifferentiation could be a consequence rather than a cause of crystal adhesion [113–120].

#### *3.1.2. Is the crystal‐binding cell phenotype a cause or a consequence of crystal adhesion?*

Crystals do not adhere to normal epithelial cells, so it is highly unlikely that crystal adhesion might be the initial cause of cellular injury and epithelial phenotypic alterations, which are probably triggered instead by forced contact and transient interaction with normal epithelia during the passage of the crystals/oxalate. Either way, it is evident that the tubular epithelium must have a very important direct role in the initiation of intratubular nephrocalcinosis.

freely in the tubular fluid. A nascent crystal then becomes aggregated with other crystals, forming a mass large enough to occlude the nephron, leading to an obstructive tubulopathy

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

These crystals can be found in contact with the surface of injured/regenerating epithelial cells, apoptotic and/or necrotic cells, and denuded basement membranes [4, 77–84], giving the impression that the composition of the cell surface is crucial in modulating this process. In fact, it has been demonstrated on primary or immortalized tubular epithelial cells exposed to CaOx crystals that the crystal deposits preferentially adhere to injured, apoptotic, depolar‐ ized, immature, migrating, or proliferating tubular epithelial cells, rather than to fully differ‐ entiated, normal epithelia [85–88]. In this context, there is interesting evidence to suggest that proximal tubular cells bind crystals regardless of their differentiation status, whereas distal tubular cells (which are physiologically more likely to encounter crystals) only bind crystals when they are dedifferentiated [70], meaning that the distal tubular epithelium is unable to

What we know about crystal adhesion in the proximal and distal tubules stems mainly from having identified the characteristics of the luminal membrane and the molecular composition of the crystal‐binding epithelia, which led to the discovery of several crystal‐binding mol‐ ecules [4, 80, 89–93]. Importantly, these crystal‐binding molecules are upregulated or redis‐ tributed to the apical membrane under certain conditions of cellular dedifferentiation, such as injury or repair, or variations in pathophysiological conditions [78, 87, 88, 94–96], which

The different categories of crystal‐binding molecules identified *in vitro* to date include: (i) termi‐ nal sialic acid residues [79, 97, 98]; (ii) phospholipids, i.e., phosphatidylserine [78, 84, 99, 100]; (iii) membrane‐bound proteins, i.e., collagen IV [101], OPN [102–106], annexin 2 (ANX2) [107, 108] and nucleolin‐related protein (NRP) [88, 93, 109]; and (iv) glycosaminoglycans, of which hyal‐ uronan (HA) appears to be the most potent crystal‐binding polysaccharide [95, 96, 110]. It has been demonstrated that other proteins, such as matrix Gla protein (MGP), are implicated in this process too [48, 111]. It is intriguing, moreover, that all known crystal‐binding molecules con‐ tribute to inducing a negative cell‐surface charge, a feature that has proved important in crys‐ tal adhesion to renal epithelial cells [85, 97, 112]. This would suggest that an array of aberrant phenotypes could bind crystals if there are appropriate amounts of crystals and appropriately

On the other hand, crystals and/or concomitant high concentrations of calcium, oxalate, or phosphate have been found to induce injury, proliferation, inflammatory mediator produc‐ tion, and oxidative stress on contact with epithelial cells *in vitro*, suggesting that epithelial dedifferentiation could be a consequence rather than a cause of crystal adhesion [113–120].

Crystals do not adhere to normal epithelial cells, so it is highly unlikely that crystal adhesion might be the initial cause of cellular injury and epithelial phenotypic alterations, which are probably triggered instead by forced contact and transient interaction with normal epithelia

*3.1.2. Is the crystal‐binding cell phenotype a cause or a consequence of crystal adhesion?*

determine whether or not the crystals are retained in the kidney.

oriented negative charges on the luminal membrane.

(**Figure 5A**).

bind crystals when differentiated.

Some reports have suggested that the renal tubular epithelial cell injury in crystal‐cell inter‐ actions occurs more easily in a setting of prior cell injury [99, 118]. The "incubation" period observed during transient toxic or mechanical crystal‐cell interactions capable of affecting the tubular epithelium is consistent with the need for a shift in the epithelial phenotype prior to crystal adhesion [119–121]. This would mean that crystal adhesion is a consequence, not the initial cause of epithelial injury *in vitro*.

The nucleation of ions from the renal tubule and subsequent growth of a calcium crystal cannot usually occur and, even if it does, such processes do not proceed quickly enough to produce particles of sufficient size to be retained in the kidney, and occlude tubules simply because of their bulk [68, 76, 118, 122, 123]. The crystals are not only the outcome of the physicochemical properties and urinary concentrations of the minerals involved. They are also influenced by crystallization regulators that may promote or inhibit crystallization and by signaling pathways triggered by the crystals, thus leading to different types of renal cell injury [8, 71, 124–127]. Urine or, more properly, tubular fluid probably contains inhibitors of crystal formation that specifically prevent their nucleation, growth, or aggregation. It has been claimed that the inhibitors' role in controlling crystal formation is important in the nor‐ mal defenses against the development of stones, and that abnormalities of these inhibitors may allow for stone formation and growth.

There are different types of such crystallization inhibitors in the urine, including small organic anions such as citrate, small inorganic anions such as PPis, multivalent metallic cations such as magnesium, and macromolecules such as OPN and Tamm‐Horsfall protein, which can take effect on different levels during the crystal formation process (**Table 7**). Citrate lowers the saturation of CaOx by forming complexes with calcium and inhibits the aggregation of preformed crystals and the attachment of crystals to the renal epithelium [97, 128]. PPi is a substance naturally occurring in urine that has been found to inhibit the crystallization of both CaOx and CaP [129]. Magnesium has also been shown to prevent stone formation by inhibiting the growth and aggregation of crystals (and presumably interferes with their nucle‐ ation too) [130]. OPN (known to inhibit the spontaneous nucleation of crystals from solutions) was found to prevent the growth of preformed crystals in a seed growth assay [131, 132], but there is also evidence to suggest that OPN bound to the surface of cells may enhance crystal attachment [102, 103]. In addition, the inhibitory effect of OPN on CaOx aggregation *in vitro* can be switched to an aggregation promoting effect if its net negative charge is neutralized by polyarginine [133].

Tamm‐Horsfall glycoprotein (THP) is the most abundant of the urinary proteins under normal circumstances [134]. THP coats CaOx crystals and prevents their adhesion to cul‐ tured epithelia, but there are few *in vivo* studies on how it would affect their aggregate, once it anchored to the epithelia [134, 135]. Another protein, called urinary prothrombin fragment 1, has been isolated from the matrix of crystals formed by adding oxalate to urine [136]. This is an effective inhibitor of CaOx crystal growth and aggregation, *in vivo* as well as *in vitro* [137].
