**2.8. Regulation of urinary acidification**

Hereditary forms of hypomagnesemia include rare, genetically determined disorders that may affect renal magnesium handling either primarily or secondarily. **Table 5** summarizes

**Gene Protein Disorder Mode of inheritance**

Bartter syndrome type 1 AR

Bartter syndrome type 2 AR

AR

AR

AR

AD

with hypercalciuria and nephrocalcinosis

with hypercalciuria and nephrocalcinosis with ocular

Hypercalciuric hypercalcemia AD

Hypomagnesemia 2, renal AD

Hypomagnesemia 4, renal AR

with secondary hypocalcemia

hypomagnesemia

impairment

SLC12A1 Sodium‐potassium‐

KCNJ1 Potassium channel

CASR Calcium‐sensing receptor (CasR)

ATPase

(Pro‐EGF)

KCNA1 Kv1.1 Potassium channel Myokymia 1 with

FXYD2 Gamma subunit Na/K/

EGF Epidermal growth factor

chloride transporter

CLCNKB Chloride channel ClC‐Kb Bartter syndrome type 3 AR BSND Barttin Bartter syndrome type 4 AR

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

CLDN16 Claudin‐16, tight junction Familial hypomagnesemia

CLDN19 Claudin‐19, tight junction Familial hypomagnesemia

TRPM6 TRPM6 cation channel Hypomagnesemia 1, intestinal

SLC12A3 NaCl cotrasporter (NCCT) Gitelman syndrome AR

KCNJ10 Kir4.1 potassium channel SESAME syndrome AR HNF1B HNF1β transcription factor HNF1B nephropathy AD

(NKCC2)

(ROMK1)

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

the spectrum of underlying genetic defects [31].

*Note:* AD = autosomal dominant; AR = autosomal recessive.

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

**2.7. Pyrophosphaturia**

One of the main functions of the kidney is to keep the systemic acid‐base chemistry constant. The kidney has evolved so that it can regulate blood acidity by means of three key func‐ tions: (1) by reabsorbing the HCO3− filtered through the glomeruli to prevent its excretion in the urine; (2) by generating a sufficient quantity of new HCO3− to compensate for the loss of HCO3− due to dietary metabolic H+ loads and loss of HCO3− in the urea cycle; and (3) by excreting HCO3− (or metabolizable organic anions) following a systemic base load [40]. For the kidney to be able to perform these functions, various types of cell throughout the nephron have to respond to changes in acid‐base chemistry by modulating specific ion transport and/or metabolic processes in a coordinated fashion, such that the urine and renal vein chemistry is adjusted appropriately. The kidney contributes to acid‐base homeostasis by recovering filtered bicarbonate in the proximal tubule. Distally, intercalated cells of the collecting duct generate new bicarbonate, which is consumed by the titration of non‐volatile acid [41].

The renal tubular acidosis (RTA) syndromes encompass a disparate group of tubular trans‐ port defects that share the inability to secrete hydrogen ions (H<sup>+</sup> ). This inability results in fail‐ ure to excrete acid in the form of ammonium (NH4<sup>+</sup> ) ions and titratable acids or to reabsorb some of the filtered bicarbonate (HCO3− ). Either situation coincides with a drop in plasma bicarbonate levels, leading to chronic metabolic acidosis. Much of the morbidity of RTA syn‐ dromes is attributable to the systemic consequences of chronic metabolic acidosis, including growth retardation, bone disease, and kidney stones [42].

Dysfunction of the proximal tubules, where approximately 90% of the bicarbonate is reab‐ sorbed, leads to proximal RTA [43], whereas malfunctioning of the intercalated cells in the collecting ducts accounts for all known genetic causes of distal RTA (dRTA).

Inherited proximal RTA is a rare disorder that may be inherited as an autosomal recessive or dominant trait [44]. The more common autosomal recessive form has been associated with mutations in the basolateral sodium bicarbonate cotransporter NBCe1, encoded by the SLC4A4 gene. Mutations in this transporter lead to a reduced activity and/or trafficking, thus, disrupting the normal bicarbonate reabsorption process in the proximal tubules [45]. As an isolated defect of bicarbonate transport, proximal RTA is rare. It is more often associated with Fanconi syndrome, which features urinary wastage of solutes such as phosphate, uric acid, glucose, amino acids, and low‐molecular‐weight proteins, as well as bicarbonate. The distal acidification mechanisms remain intact, however, and acid urine can still be produced. The clinical phenotype is of a metabolic acidosis with hypokalemia; metabolic bone disease is common, but nephrocalcinosis and nephrolithiasis are rare [46].

In contrast, 80% of cases of distal RTA (dRTA) are associated with medullary nephrocalcino‐ sis. The molecular basis underlying primary dRTA is a defective functioning of alpha inter‐ calated cells [41]. The molecular defects behind proximal and distal RTA are listed in **Table 6**.

The clinical signs and symptoms of dRTA can vary, depending on the underlying mutation: patients may reveal a mild metabolic acidosis after the incidental detection of kidney stones, or they may have severe health issues with failure to thrive and growth retardation in chil‐ dren, rickets, severe metabolic acidosis, and nephrocalcinosis. Kidney stones in dRTA consist of CaP due to the release of Ca and Pi from bone to buffer the acidosis, leading to hypercalci‐ uria and consequent CaP precipitation due to an alkaline pH [47].

#### **2.9. Macromoleculuria**


The formation of crystal aggregates involves interaction between crystals and urinary mac‐ romolecules (UMs) that serve as an adhesive. The number of UMs isolated in urine has been

**Table 6.** The inherited renal tubular acidoses.

steadily increasing, and they now form a large group of proteins and some glycosaminogly‐ cans [48, 49]. The main macromolecules involved in crystallization are summarized in **Table 7**.

Dysfunction of the proximal tubules, where approximately 90% of the bicarbonate is reab‐ sorbed, leads to proximal RTA [43], whereas malfunctioning of the intercalated cells in the

Inherited proximal RTA is a rare disorder that may be inherited as an autosomal recessive or dominant trait [44]. The more common autosomal recessive form has been associated with mutations in the basolateral sodium bicarbonate cotransporter NBCe1, encoded by the SLC4A4 gene. Mutations in this transporter lead to a reduced activity and/or trafficking, thus, disrupting the normal bicarbonate reabsorption process in the proximal tubules [45]. As an isolated defect of bicarbonate transport, proximal RTA is rare. It is more often associated with Fanconi syndrome, which features urinary wastage of solutes such as phosphate, uric acid, glucose, amino acids, and low‐molecular‐weight proteins, as well as bicarbonate. The distal acidification mechanisms remain intact, however, and acid urine can still be produced. The clinical phenotype is of a metabolic acidosis with hypokalemia; metabolic bone disease is

In contrast, 80% of cases of distal RTA (dRTA) are associated with medullary nephrocalcino‐ sis. The molecular basis underlying primary dRTA is a defective functioning of alpha inter‐ calated cells [41]. The molecular defects behind proximal and distal RTA are listed in **Table 6**. The clinical signs and symptoms of dRTA can vary, depending on the underlying mutation: patients may reveal a mild metabolic acidosis after the incidental detection of kidney stones, or they may have severe health issues with failure to thrive and growth retardation in chil‐ dren, rickets, severe metabolic acidosis, and nephrocalcinosis. Kidney stones in dRTA consist of CaP due to the release of Ca and Pi from bone to buffer the acidosis, leading to hypercalci‐

The formation of crystal aggregates involves interaction between crystals and urinary mac‐ romolecules (UMs) that serve as an adhesive. The number of UMs isolated in urine has been

SLC4A1 AE1 AD

cotransporter 1 (NBC1)

 ‐ ATPase AR with early onset hearing loss

loss

AR

AR

‐ ATPase AR with later onset hearing

**Disorder Gene Protein Mode of inheritance**

ATP6V1B1 β1 Subunit of H<sup>+</sup>

ATPV0A4 α4 Subunit of H<sup>+</sup>

Ca2 Carbonic anhydrase 2

(CAH2)

Distal RTA type1 SLC4A1 AE1 AR

Proximal RTA type2 SLC4A4 Sodium bicarbonate

collecting ducts accounts for all known genetic causes of distal RTA (dRTA).

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

common, but nephrocalcinosis and nephrolithiasis are rare [46].

uria and consequent CaP precipitation due to an alkaline pH [47].

**2.9. Macromoleculuria**

Combined Proximal and Distal RTA type3

PH, primary hyperoxaluria; AR, autosomal recessive.

**Table 6.** The inherited renal tubular acidoses.

Although the role of these proteins in stone formation is still far from clear, coating of the crystals by the urinary macromolecules seems to prevent crystal aggregation or at least delay it for long enough for the urine to transit through the kidney.

An inhibitory role has repeatedly been confirmed for osteopontin (OPN) [50–54], which is synthesized in the kidney and excreted in the urine in concentrations that suffice to inhibit CaOx crystallization. No naturally occurring mutations in the SSP1 gene encoding OPN have ever been reported in human diseases, but SSP1 polymorphisms have been associated with the risk of nephrolithiasis [55–57].

Tamm‐Horsfall protein (THP), also called uromodulin, is a kidney‐specific protein synthe‐ sized by cells in the TAL of the loop of Henle. It is the most abundant protein in human urine. It is a potent inhibitor of crystal aggregation *in vitro*, and its ablation *in vivo* predisposes one of the two existing mouse models to spontaneous intrarenal calcium crystallization, but there are still some key issues to clarify regarding the role of THP in nephrolithiasis. By conduct‐ ing a long‐range follow‐up of more than 250 THP‐null mice and their wild‐type controls, Liu et al. [58] demonstrated that renal calcification was a highly consistent phenotype of the THP‐null mice. The crystals consisted primarily of CaP in the form of hydroxyapatite. They were located in the interstitial space of the renal papillae more frequently than in the tubules (particularly in older animals), and there was no accompanying inflammatory cell infiltration. The interstitial deposits of hydroxyapatite observed in THP‐null mice strongly resemble the renal crystals found in human kidneys with idiopathic CaOx stones. In humans, a number of naturally occurring THP mutations are reportedly linked to autosomal dominant medullary


**Table 7.** Crystallization‐modulating macromolecules (Modified from Khan SR and Canals BK 2009 [13]).

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 these mutations to date, however [13].

#### **2.10. Medullary sponge kidney**

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 the spontaneous formation of Ca2 PO4 nodules very similar to those of calcifying vascular cells. They demonstrated that silencing the GDNF gene in a human renal cell line and cultivating the silenced cells in osteogenic conditions triggered the deposition of Ca2 PO4 . These results 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. The role of renal cells in nephrocalcinosis is discussed in the subsequent paragraphs.
