**3.2. Interstitial nephrocalcinosis**

The presence of crystals in the renal interstitium is defined as interstitial nephrocalcinosis (**Figure 5A**). Although the causal role of aberrant epithelial tissue in crystal adhesion—dem‐ onstrated in renal cell lines *in vitro*, in animal models, in kidney transplant patients, and in neonates—may account for intratubular crystal formation and retention [138–140], the spe‐ cific pathogenic mechanisms leading to interstitial crystal formation and deposition are still unclear [7].

Translocation of intratubular crystals and/or *de novo* interstitial calcification have been pro‐ posed as causative factors (**Figure 5B**).

## *3.2.1. Translocation of intratubular crystals to the interstitium*

Crystal translocation can be induced by transcytosis (**Figure 5B**), a process during which small intraluminal crystals are internalized within apical vesicles (with or without the mediation of a receptor) and transferred across the cell wall to the basolateral side, where they are released into the interstitial extracellular environment [12]. Apical endocytosis of small crystals has been well described [141–144], but there is little evidence of any basolateral release of crystals into the interstitium. It has been suggested that these crystals probably disintegrate into lyso‐ somes [142, 143, 145]. Very recently, however, Chiangjong et al. [146] demonstrated that, after exposure to CaOx crystals, renal tubular epithelial cells secrete more crystal‐binding protein (enolase‐1 [147]) into the basolateral compartment; the authors suggested that this protein could in turn promote CaOx crystal invasion through the renal interstitium. The transloca‐ tion of crystals into the interstitium is associated with inflammation, attracting leukocytes, monocytes, and macrophages that—according to some—would then remove the crystalline material [148].

An alternative mechanism of transepithelial crystal translocation was described using the term "exotubulosis" (**Figure 5B**) in an *in vivo* study conducted by De Bruijn et al. [149, 150]. These authors demonstrated that crystals adhering to the inside wall of the tubule can be overgrown by tubular epithelial cells adjacent to the site of adhesion of the crystals. After pro‐ liferation and migration, the tubular epithelial cells cover the crystals and differentiate into a new mature epithelium, with its basement membrane on top of the crystals and its apical side directed toward the lumen, thereby restoring the epithelial integrity of the affected tubule, and translocating the crystals into the interstitium.

For a long time, translocation was the only explanation for the advent of mineral deposits in the interstitium [151, 152], but crystals can also form *de novo* in the interstitium.

#### *3.2.2. De novo interstitial crystal formation*

It has been claimed that these crystal deposits start in the interstitium around the thin limbs of the loop of Henle (below the basement membrane) and give rise to subepithelial calcifications better known as Randall's plaques [153].

Nobody knows, however, whether this *de novo* crystal formation is due merely to a chem‐ ically driven supersaturation or whether cells are involved too. For some time, the most accredited hypothesis advanced to explain the onset of interstitial nephrocalcinosis was purely physicochemical, relating to spontaneous Ca2 PO4 crystallization in the interstitium as a result of calcium and phosphate oversaturation in this milieu. Evidence has been produced of a lower expression and defective barrier and fence functions of the tight junction in renal tubular epithelial cells exposed to CaOx crystals. This could lead to intercellular (paracel‐ lular) migration of intratubular COM crystals, and of calcium, oxalate, and phosphate ions to the interstitium to initiate tubulointerstitial injury, inflammation, and interstitial nephro‐ calcinosis [154–157].

Dysregulation of calcium homeostasis in the renal interstitium (and probably on a systemic level too) may have a key role in the pathogenesis of nephrocalcinosis. Bushinsky DA [154] proposed a sequence of events that could lead to an increased supersaturation and subse‐ quent crystal formation. "*Following ingestion and absorption of dietary calcium, the renal‐filtered load of calcium would increase, resulting in increased tubular calcium concentration. The medullary countercurrent mechanism would concentrate the calcium extracted from the TAL into the hypertonic papilla. The vasa recta, also with an increased calcium concentration, would fail to readily remove calcium from the interstitium. The increased serum calcium would stimulate the calcium receptor and decrease reabsorption of water in the collecting duct, further concentrating the interstitium. Vectorial proton transport into the collecting duct would alkalinize the interstitium. The pH of the vasa recta would also increase following gastric proton secretion, the so‐called alkaline tide, resulting in less bicarbonate removal from the medullary interstitium. The increased pH would decrease the solubility of CaP complexes. Perhaps an extracellular matrix protein, specific to the papillary interstitium, could provide a site promoting heterogeneous nucleation, which occurs with a lower degree of supersatura‐ tion than homogeneous nucleation.*"

Estimates of tubular fluid supersaturation based on data obtained in the rat suggest that CaP supersaturation often occurs in the thin limbs of the loop of Henle [158], where tubular fluid is saturated even under normal circumstances. In humans, this condition could drive the precip‐ itation of CaP deposits at interstitial sites, in the inner medulla—known as Randall's plaques when they become extensive enough to be macroscopically visible [4, 6, 158, 159]. Randall's plaques have been proposed as a nidus for the development of the most common variety of CaOx stones [4, 160].

#### *3.2.3. Randall's plaques*

**3.2. Interstitial nephrocalcinosis**

posed as causative factors (**Figure 5B**).

*3.2.1. Translocation of intratubular crystals to the interstitium*

and translocating the crystals into the interstitium.

*3.2.2. De novo interstitial crystal formation*

better known as Randall's plaques [153].

unclear [7].

material [148].

The presence of crystals in the renal interstitium is defined as interstitial nephrocalcinosis (**Figure 5A**). Although the causal role of aberrant epithelial tissue in crystal adhesion—dem‐ onstrated in renal cell lines *in vitro*, in animal models, in kidney transplant patients, and in neonates—may account for intratubular crystal formation and retention [138–140], the spe‐ cific pathogenic mechanisms leading to interstitial crystal formation and deposition are still

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

Translocation of intratubular crystals and/or *de novo* interstitial calcification have been pro‐

Crystal translocation can be induced by transcytosis (**Figure 5B**), a process during which small intraluminal crystals are internalized within apical vesicles (with or without the mediation of a receptor) and transferred across the cell wall to the basolateral side, where they are released into the interstitial extracellular environment [12]. Apical endocytosis of small crystals has been well described [141–144], but there is little evidence of any basolateral release of crystals into the interstitium. It has been suggested that these crystals probably disintegrate into lyso‐ somes [142, 143, 145]. Very recently, however, Chiangjong et al. [146] demonstrated that, after exposure to CaOx crystals, renal tubular epithelial cells secrete more crystal‐binding protein (enolase‐1 [147]) into the basolateral compartment; the authors suggested that this protein could in turn promote CaOx crystal invasion through the renal interstitium. The transloca‐ tion of crystals into the interstitium is associated with inflammation, attracting leukocytes, monocytes, and macrophages that—according to some—would then remove the crystalline

An alternative mechanism of transepithelial crystal translocation was described using the term "exotubulosis" (**Figure 5B**) in an *in vivo* study conducted by De Bruijn et al. [149, 150]. These authors demonstrated that crystals adhering to the inside wall of the tubule can be overgrown by tubular epithelial cells adjacent to the site of adhesion of the crystals. After pro‐ liferation and migration, the tubular epithelial cells cover the crystals and differentiate into a new mature epithelium, with its basement membrane on top of the crystals and its apical side directed toward the lumen, thereby restoring the epithelial integrity of the affected tubule,

For a long time, translocation was the only explanation for the advent of mineral deposits in

It has been claimed that these crystal deposits start in the interstitium around the thin limbs of the loop of Henle (below the basement membrane) and give rise to subepithelial calcifications

the interstitium [151, 152], but crystals can also form *de novo* in the interstitium.

Randall demonstrated that interstitial crystals are located at, or adjacent to, the papillary tip [161]. These crystals in the papillary interstitium are composed not of CaOx (the most com‐ mon solid phase found in patients with nephrolithiasis), but of CaP [162, 163], that then eroded into the urinary space, serving as a heterogeneous nucleation surface for CaOx. Randall con‐ cluded that renal stones originated as a slow deposition/crystallization of urinary salts (CaOx, CaP, uric acid) on a lesion of the renal papilla—a picture confirmed and extended in patients with idiopathic CaOx nephrolithiasis [4, 5, 164] (**Figure 6**).

**Figure 6.** Mechanism of stone growing on Randall's plaque. The plaque appears in the interstitial tissue within the renal papilla, with no crystals present in any tubular lumens. The plaque is composed of calcium phosphate (CaP) in the mineral form of apatite. Papillary epithelium is lost, and the plaque can be exposed to urinary fluid in the renal calyx. The resulting calcium oxalate stone may grow and the plaque keeps the stone from flowing out with the urine, and the insolubility of the calcium oxalate makes the stone quite. Stones that are formed on Randall's plaques are released from the papilla in the renal calyx.
