Kidney stone formers have more renal parenchymal crystals than non-stone formers, particularly in the papilla region

Since the introduction of extracorporeal shock-wave lithotripsy in 1980, [1] fewer opportunities for open surgery have led to fewer chances for pathological investigations of kidney stone formation (KSF) using human kidney parenchymal tissue. Therefore, studies of human KSF tend to focus on urinary inorganic concentrations [2, 3] and epidemiological data, such as those investigating the relationship with diabetes [4] and metabolic syndrome [5]. Recent progress in endoscopic technology has directed attention to Randall’s plaque [6]; interstitial apatite crystal deposits beginning at the basement membranes of the thin loops of Henle seem to be sites of calcium oxalate (CaOx) stone formation [7‐9]. However, intraparenchymal events involving the kinetics of intratubular crystals have not been elucidated.

Previous basic studies using hyperoxaluric-animal and cell-culture models led to the detection of morphological and genetic events in the renal parenchyma via the detection of stone matrix protein, [10] completion of the human genome project, and technological progress related to recombinant gene analysis [11‐13]. In particular, the factors currently considered to affect calcium kidney stone formation are stone matrix proteins, cell injury caused by oxidative stress, monocyte/macrophage induction, and urinary stone inhibitors.

Osteopontin (OPN), the main component of stone matrix protein, is a glycoprotein present in human calcium-containing kidney stones [10] that may play an important role in crystal conversion to stones. OPN antisense-expressing cultured renal tubular cells demonstrate reduced aggregation of CaOx crystals and crystal-cell interactions [14] and OPN-knockout mice show reduced growth of renal crystals [12]. OPN is also involved in the formation of the organic layer of apatite plaque particles in the renal inner medulla of CaOx stone formers (SFs) [15]. Furthermore, renal tubular-cell injury caused by oxidative stress is essential for kidney stone formation [16]. Some studies have indicated that tubular-cell apoptosis caused by deviated free radicals and diminished superoxide dismutase (SOD) expression, [17] and collapsed organelles, including mitochondria and fragmented microvilli in the renal tubular lumen, lead to stone nidus formation [18]. We reported renal intratubular crystal elimination in a mouse model, increased expression of macrophage-related inflammatory genes in a DNA microarray analysis of stone-forming kidneys, [13] and phagocytosis of interstitial crystals by macrophages under transmission electron microscopy, [19] suggesting the kidney stone-preventive ability of macrophages by crystal processing. Moreover, Umekawa et al. [20] demonstrated that exposure to CaOx crystals promotes the expression of monocyte chemotactic protein-1 (MCP-1) and induces macrophage migration. Finally, Tamm–Horsfall protein (THP), a urinary inhibitor of stone formation, has been studied because THP-deficient mice demonstrate spontaneous calcium crystal formation [21].

The above experimental findings suggest that intratubular crystal formation involves several steps and that animal models may have the ability to eliminate the crystals. However, among the possible mechanisms of stone formation, these processes are thought to model Randall’s plug due to hyperoxaluria or cystinuria rather than Randall’s plaque. However, there is no definitive evidence to confirm this assumption. With the above in mind, we aimed to elucidate the intratubular crystal kinetics and processing in human kidneys using nephrectomized parenchymal tissues.

Kohri et al. [10] suggested that calcium kidney stone formation involves the expression of several stone matrix proteins, mainly OPN, in renal tubular cells, indicating that the phenomenon is inducible by both environmental and genetic factors [24]. However, their explanation has two major problems: (i) because kidney stone formation is asymptomatic, patients do not recognize its onset until colic pain occurs due to stone descent or chance detection by imaging studies; and (ii) due to the spread of extracorporeal shock wave lithotripsy, it has become difficult to extract tissues ethically from living kidneys, making it impossible to conduct detailed studies on kidney tissues, in contrast to the situation when open surgery was common. Due to the recent development of endoscopic instruments, morphological and pathological studies on Randall’s plaque have become more common. We recently conducted a genome-wide study of plaque tissue, resulting in the confirmation of inflammatory cytokine expression, increased immune cell number, and cellular apoptosis in renal papilla stone tissue [25]. However, these findings were limited to the renal papilla tissue and represent only the change in expression levels after stone formation. To resolve these problems, we enrolled patients with asymptomatic stones detected contingently by preoperative CT for the diagnosis of renal tumors and investigated renal parenchyma integrally using pathological whole-kidney samples. Specifically, we analyzed the crystal morphology and transmigration in addition to kidney stone-related gene and protein expression.

We found that SF group had a significantly higher smoking rate than NSFs. Słojewski et al. [26] did not detect significant correlations between smoking and kidney stone composition. Smoking is a significant, independent risk factor for atherosclerosis via the oxidative stress associated with mitochondrial damage [27]. Considering the similarity between kidney stone and atherosclerosis formation, [22] smoking might conceivably affect stone formation or crystal kinetics. The precise relationship between the risk of stone formation and smoking should be investigated in future studies.

SFs had significantly lower RBC, hemoglobin, and Ht values than NSFs. Renal ischemia via anemia could lead to renal tubular-cell injury, [28] implying that anemia might be involved in stone formation. However, patients with kidney stones have erythropoietin resistance caused by bone marrow oxalosis [29]. Furthermore, the increase in urine RBC, WBC, and bacterial counts may be the result of the erosion of the renal pelvic mucosa on Randall’s plaque in patients with stones [30]. Unfortunately, we did not consider the existence of plaque in this study.

The notable findings of this study are as follows: (i) regardless of kidney stone history, intratubular crystal deposits were detectable in the renal parenchymal tissues; (ii) the crystals transmigrated from the tubular lumen to the papillary interstitium; and (iii) SFs had a significantly higher number of crystal deposits in the renal papilla. Bergsland et al. [31] noted that SFs, especially those with idiopathic hypercalciuria, have higher urinary calcium molarity than NSFs and that the difference becomes significant at night. CaOx supersaturation but not calcium phosphate supersaturation is higher in SFs than in NSFs, which could also explain CaOx stone formation on papillary Randal’s plaques. CaOx crystal residues in the renal papilla could be another factor related to CaOx stone formation. Furthermore, Vervaet et al. [32] used hyperoxaluric rat model and human renal biopsy samples to indicate the gradual migration of intratubular crystals to the interstitium. In the hyperoxaluric mouse model we previously established, [11] intratubular crystal deposits were eliminated in about 6 days. The crystals were englobed and fragmented by macrophages and crystal deposits were undetectable in the renal papillary region at all time points. Boonla et al. [33] investigated MCP-1 and interleukin (IL)-6 messenger RNA expression in renal biopsy samples from SFs and extracted kidney samples from patients with renal cancer; they demonstrated relatively low MCP-1 and IL-6 expression levels in the cancerous samples compared to those in noncancerous tissues. In the present study, the significantly higher number of interstitial crystal deposits in the papilla of SFs and relatively high CD68 expression level in NSFs suggest some important roles of macrophages in kidney stone prevention. The OPN, SOD, and CD68 expression levels were similar to those indicated in previous basic studies: SFs had increased OPN expression in the renal tubular cells, tubular-cell injury by oxidative stress, and reduced migration of renal macrophages. In particular, the significantly lower THP expression level in SFs indicates that THP has a crucial role as a kidney stone-preventive factor in humans.

On the basis of our results, we hypothesize the phenomena of human renal intratubular crystal processing. First, crystal nidi are generated in the tubular lumen of the renal cortex because of a urinary supersaturated condition [2, 3]. Some oxidative stresses, such as anemia or smoking, and renal tubular-cell injuries cause collapse of mitochondria and microvilli with decreased SOD expression [16‐18]. Consecutively, OPN expression increases and THP downregulation induces crystal-cell interaction and the adaptation of aggregated crystals to the tubular epithelium [26]. Thereafter, the tubular epithelium disintegrates via apoptosis and crystal clusters transmigrate to the renal interstitium via the regenerating epithelium [32]. Tubular-cell injury increases the expression of MCP-1 or various chemokines, in turn inducing monocytes, their transmigration to the renal interstitium, and their differentiation into macrophages [20]. The interstitial crystals can then be removed by macrophages.

These calcification processes, including epithelial-cell injury via oxidative stress, the participation of OPN via inflammation, macrophage activity with phagocytosis, and processing and conversion of foam cells into calcified tissue, are similar to the processes of atherosclerosis formation [34]. SFs tended to have higher levels of aortic calcification. These outcomes suggest a new approach to kidney stone formation involving similar biomolecular processes to those involved in metabolic syndrome that are not related to kidney stone disease because of hyperuricemia, decreased urinary pH, or hypocitraturia caused by metabolic syndrome [35‐37].

Multivariate analysis indicated that the presence of renal papillary crystals was significantly and independently related to stone formation. This result represents all of the relationships discussed above. These findings suggest the possibility that the process of kidney stone formation depends on some renoprotective abilities related to the processing of crystals formed in the renal parenchyma, especially the renal papilla.

This study has some limitations that should be discussed. We could not clarify how cancer background, involving environmental and genetic factors, affected “true” kidney stone formation. Furthermore, because this study was conducted retrospectively, detailed analysis of stone component and urinary biochemistry could not be performed. Moreover, Randall’s plaques were not detectable in the study sample.

We identified similar phenomena to those detected in previous basic studies, such as crystal-cell interactions, increased OPN expression, decreased SOD and THP expression, and macrophage involvement in the human renal parenchyma. The new findings of this study were crystal formation in patients without kidney stones, crystal transmigration to the papillary interstitium, and crystal processing at the renal papilla regardless of stone formation. SFs may have reduced ability to eliminate renal parenchymal crystals than NSFs (especially in the papilla region), with associated gene expression changes.