Two distinct phenotypes of calcium oxalate stone formers could imply different long-term risks for renal function.

Endoscopic and biopsy findings have identified two distinct phenotypes among individuals with calcium oxalate (CaOx) kidney stones. One phenotype exhibits normal renal papillae but shows interstitial mineral deposition, known as Randall's plaque. The other phenotype presents with collecting duct plugging and a higher incidence of loss of papilla tissue mass. With Randall's plaque, renal papilla injury involves the loss of small patches of calcified tissue (Randall's plaque detaching with the stone), which likely results in damage to only a few nephrons. In contrast, collecting duct mineral plugs are very large, causing obstruction to tubular flow. Since each terminal collecting duct drains thousands of nephrons, ductal plugs could lead to the degeneration of many nephrons and a significant loss of renal glomeruli. New visualization techniques for immune cells in papillary biopsies have revealed that the Randall's plaque phenotype is marked by the accumulation of macrophages around the plaque regions. In contrast, preliminary data on the plugging phenotype shows collecting duct damage with mineral plugs, increased T-lymphocytes throughout the papilla, and tubulitis, characterized by T-cell infiltration into nearby collecting duct epithelium. This suggests that while some CaOx stone formers may have some papillary inflammation but with minimal damage to nephrons, others suffer from obstruction to flow for many nephrons that may also include destructive inflammation in the renal tissue. We propose that the long-term risks for loss of renal function will be greater for CaOx stone formers with the plugging phenotype.

Two distinct phenotypes of calcium oxalate stone formers could imply different long-term risks for renal function.

Endoscopic and biopsy findings have identified two distinct phenotypes among individuals with calcium oxalate (CaOx) kidney stones. The first type has normal renal papillae but shows interstitial mineral deposition, known as Randall's plaque. The other phenotype presents with collecting duct plugging and a higher incidence of loss of papilla tissue mass. With Randall's plaque, renal papilla injury involves the loss of small patches of calcified tissue (Randall's plaque detaching with the stone), which likely results in damage to only a few nephrons. In contrast, collecting duct mineral plugs are very large, causing obstruction to tubular flow. Since each terminal collecting duct drains thousands of nephrons, ductal plugs could lead to the degeneration of many nephrons and a significant loss of renal glomeruli. New visualization techniques for immune cells in papillary biopsies have revealed that the Randall's plaque phenotype is marked by the accumulation of macrophages around the plaque regions. In contrast, preliminary data on the plugging phenotype shows collecting duct damage with mineral plugs and increased T-lymphocytes throughout the papilla. These regions also show tubulitis, i.e., T-cell infiltration into nearby collecting duct epithelium. This suggests that while some CaOx stone formers may have some papillary inflammation but with minimal damage to nephrons, others suffer from obstruction to flow for many nephrons that may also include destructive inflammation in the renal tissue. We propose that CaOx stone formers with the plugging phenotype will have a higher long-term risk for loss of renal function.

Metabolomic profiles and pathogenesis of nephrolithiasis.

PURPOSE OF REVIEW: Kidney stone disease is caused by supersaturation of urine with certain metabolites and minerals. The urine composition of stone formers has been measured to prevent stone recurrence, specifically calcium, uric acid, oxalate, ammonia, citrate. However, these minerals and metabolites have proven to be unreliable in predicting stone recurrence. Metabolomics using high throughput technologies in well defined patient cohorts can identify metabolites that may provide insight into the pathogenesis of stones as well as offer possibilities in therapeutics. RECENT FINDINGS: Techniques including 1H-NMR, and liquid chromatography paired with tandem mass spectroscopy have identified multiple possible metabolites involved in stone formation. Compared to formers of calcium oxalate stones, healthy controls had higher levels of hippuric acid as well as metabolites involved in caffeine metabolism. Both the gut and urine microbiome may contribute to the altered metabolome of stone formers. SUMMARY: Although metabolomics has offered several potential metabolites that may be protective against or promote stone formation, the mechanisms behind these metabolomic profiles and their clinical significance requires further investigation.

Primary hyperoxaluria: the adult nephrologist's point of view.

In adults, primary hyperoxaluria (PH) does not always present as obviously as in children, leading to delayed or even missed diagnosis. When diagnosed in adulthood, PH usually progresses at a slower rate and the focus is on the prevention of recurrent kidney stones as much as it is on the preservation of renal function. The most tragic presentation is when the diagnosis is made after primary non-function of a renal graft for treating previously unknown renal disease. Recurrent stones, nephrocalcinosis and features of systemic oxalosis can all be presenting features. For these reasons, consideration should be given to screening for this rare condition, using biochemical and/or genetic means, but being careful to exclude common differential diagnoses. Such efforts should be synchronized with diagnostic methods for other rare kidney diseases.

Stone Morphology Distinguishes Two Pathways of Idiopathic Calcium Oxalate Stone Pathogenesis.

Introduction: About 1 in 11 Americans will experience a kidney stone, but underlying causes remain obscure. The objective of the present study was to separate idiopathic calcium oxalate stone formers by whether or not they showed positive evidence of forming a stone on Randall's plaque (RP). Materials and Methods: In patients undergoing either percutaneous or ureteroscopic procedures for kidney stone removal, all stone material was extracted and analyzed using micro-CT imaging to identify those attached to RP. Twenty-four-hour urine samples were collected weeks after the stone removal procedure and patients were off of medications that would affect urine composition. The endoscopic video was analyzed for papillary pathology (RP, pitting, plugging, dilated ducts, and loss of papillary shape) by an observer blinded to the data on stone type. The percent papillary area occupied by RP and ductal plugging was quantified using image analysis software. Results: Patients having even one stone on RP (N = 36) did not differ from non-RP patients (N = 37) in age, sex, BMI, or other clinical characteristics. Compared with the non-RP group, RP stone formers had more numerous, but smaller, stones, more abundant papillary RP formation, and fewer ductal plugs, both by quantitative measurement of surface area (on average, three times more plaque area, but only 41% as much plug area as in non-RP patients) and by semiquantitative visual grading. Serum and blood values did not differ between RP and non-RP stone formers by any measure. Conclusions: Growth of many small stones on plaque seems the pathogenetic scheme for the RP stone-forming phenotype, whereas the non-RP phenotype stone pathogenesis pathway is less obvious. Higher papillary plugging in non-RP patients suggests that plugs play a role in stone formation and that these patients have a greater degree of papillary damage. Underlying mechanisms that create these distinctive phenotypes are presently unknown.

Collagen fibrils and cell nuclei are entrapped within Randall's plaques but not in CaOx matrix overgrowth: A microscopic inquiry into Randall's plaque stone pathogenesis.

Calcium oxalate (CaOx) stones can grow attached to the renal papillary calcification known as Randall's plaque. Although stone growth on Randall's plaque is a common phenomenon, this mechanism of stone formation is still poorly understood. The objective of this study was to investigate the microenvironment of mature Randall's plaque, explore its molecular composition and differentiate plaque from CaOx overgrowth using multimodal imaging on demineralized stone sections. Fluorescence imaging showed consistent differences in autofluorescence patterns between Randall's plaque and calcium oxalate overgrowth regions. Second harmonic generation imaging established the presence of collagen only in regions of decalcified Randall's plaque but not in regions of CaOx overgrowth matrix. Surprisingly, in these stone sections we observed cell nuclei with preserved morphology within regions of mature Randall's plaque. These conserved cells had variable expression of vimentin and CD45. The presence of nuclei in mature plaque indicates that mineralization is not necessarily associated with cell death. The markers identified suggest that some of the entrapped cells may be undergoing dedifferentiation or could emanate from a mesenchymal or immune origin. We propose that entrapped cells may play an important role in the growth and maintenance of Randall's plaque. Further characterization of these cells and thorough analyses of the mineralized stone forming renal papilla will be fundamental in understanding the pathogenesis of Randall's plaque and CaOx stone formation.

Evidence for abnormal linkage between urine oxalate and citrate excretion in human kidney stone formers.

BACKGROUND: Animal models have demonstrated an interactive relationship between the epithelial anion exchanger SLC26A6 and transporter NaDC-1 that regulates citrate and oxalate homeostasis. This relationship is a potential mechanism to protect against kidney stones as higher urine oxalate is accompanied by higher urine citrate but it has not been explored in humans. METHODS: We examined 24-h urine data on 13,155 kidney stone forming patients (SF) from separate datasets at the University of Chicago and Litholink, a national laboratory, and 143 non-kidney stone forming participants (NSF) to examine this relationship in humans. We used multivariate linear regression models to examine the association between oxalate and citrate in all study participants and separately in SF and NSF. RESULTS: Higher urinary oxalate was associated with higher urinary citrate in both SF and NSF. In NSF, the multivariate adjusted urine citrate excretion was 3.0 (1.5-4.6) (mmol)/creatinine (mmol) per oxalate (mmol)/creatinine (mmol). In SF, the multivariate adjusted urine citrate excretion was 0.3 (0.2-0.4) (mmol)/creatinine (mmol) per oxalate (mmol)/creatinine (mmol). CONCLUSIONS: Higher urinary oxalate excretion was associated with higher urinary citrate excretion and this effect was larger in non-kidney stone forming participants compared with those who form kidney stones.

Demineralization and sectioning of human kidney stones: A molecular investigation revealing the spatial heterogeneity of the stone matrix.

The molecular mechanisms by which kidney stones grow are largely unknown. Organic molecules from the urine combine with mineral crystals to form stones, but analysis of the stone matrix has revealed over a thousand different proteins, with no clues as to which are important for stone growth. Molecules that are present in every layer of a stone would be candidates for having an essential function, and thus the analysis of the stone matrix at a microscopic level is necessary. For this purpose, kidney stones were demineralized, sectioned, stained, and imaged by microscopy, using micro CT for precise orientation. Histological staining demonstrated heterogeneity in the density of adjacent layers within stones. Additional results also showed brilliant and unique autofluorescence patterns in decalcified nephroliths, indicating heterogeneous organic composition in adjacent layers. Regions of calcium oxalate (CaOx) stones were dissected using laser microdissection (LMD) for protein analysis. LMD of broad regions of demineralized CaOx stone sections yielded the same proteins as those found in different specimens of pulverized CaOx stones. These innovative methodologies will allow spatial mapping of protein composition within the heterogeneous stone matrix. Proteins that consistently coincide spatially with mineral deposition would be candidates for molecules essential for stone growth. This kind of analysis will be required to assess which of the thousand proteins in the stone matrix may be fundamental for stone growth.

Multimodal imaging reveals a unique autofluorescence signature of Randall's plaque.

Kidney stones frequently develop as an overgrowth on Randall's plaque (RP) which is formed in the papillary interstitium. The organic composition of RP is distinct from stone matrix in that RP contains fibrillar collagen; RP in tissue has also been shown to have two proteins that are also found in stones, but otherwise the molecular constituents of RP are unstudied. We hypothesized that RP contains unique organic molecules that can be differentiated from the stone overgrowth by fluorescence. To test this, we used micro-CT-guided polishing to expose the interior of kidney stones for multimodal imaging with multiphoton, confocal and infrared microscopy. We detected a blue autofluorescence signature unique to RP, the specificity of which was also confirmed in papillary tissue from patients with stone disease. High-resolution mineral mapping of the stone also showed a transition from the apatite within RP to the calcium oxalate in the overgrowth, demonstrating the molecular and spatial transition from the tissue to the urine. This work provides a systematic and practical approach to uncover specific fluorescence signatures which correlate with mineral type, verifies previous observations regarding mineral overgrowth onto RP and identifies a novel autofluorescence signature of RP demonstrating RP's unique molecular composition.

Racial Differences in Risk Factors for Kidney Stone Formation.

BACKGROUND AND OBJECTIVES: Incidence of kidney stone disease is rising. It is not known whether mechanisms of stone formation differ across racial groups. Our objective was to identify differing lithogenic risk factors across racial groups in idiopathic nephrolithiasis. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We conducted a retrospective cohort study evaluating metabolic risk factors in black and age-matched white idiopathic stone formers at our tertiary referral center. We compared serum and urine metabolic risk factors pre- and post-treatment across racial groups using analysis of covariance. Generalized linear modeling was used to build regression models for risk of stone formation in both groups. RESULTS: Among 117 black and 172 white stone formers, urine volume was lower in black stone formers (1.4±0.8 versus 2.0±0.8 L/d, P<0.001). Urine calcium was lower in black stone formers (116±70 versus 217±115 mg/d, P<0.001). Supersaturations for calcium oxalate were similar among the groups, whereas calcium phosphate supersaturation was higher in white stone formers, and uric acid supersaturation was higher in black stone formers. Electrolyte free water clearance was significantly lower in black stone formers (207±780 versus 435±759 ml/d, P=0.02). In the subgroup of 77 black patients and 107 white patients with post-treatment evaluations, urine volume rose significantly and similarly in both groups. Urine sodium was unchanged in whites but increased in blacks by 40 mmol/d (95% confidence interval, 32 to 48 mmol/d). Electrolyte free water clearance remained lower in black stone formers (385±891 versus 706±893 ml/d, P=0.02). Post-treatment supersaturations were similar across the groups except for calcium phosphate, which improved with treatment in whites. CONCLUSIONS: Black stone formers have lower 24-hour urine calcium excretion and urine volume. Increases in urine volume with treatment were associated with increased solute, but not free water, excretion in black stone formers.

A Precision Medicine Approach Uncovers a Unique Signature of Neutrophils in Patients With Brushite Kidney Stones.

INTRODUCTION: We have previously found that papillary histopathology differs greatly between calcium oxalate and brushite stone formers (SF); the latter have much more papillary mineral deposition, tubular cell injury, and tissue fibrosis. METHODS: In this study, we applied unbiased orthogonal omics approaches on biopsied renal papillae and extracted stones from patients with brushite or calcium oxalate (CaOx) stones. Our goal was to discover stone type-specific molecular signatures to advance our understanding of the underlying pathogenesis. RESULTS: Brushite SF did not differ from CaOx SF with respect to metabolic risk factors for stones but did exhibit increased tubule plugging in their papillae. Brushite SF had upregulation of inflammatory pathways in papillary tissue and increased neutrophil markers in stone matrix compared with those with CaOx stones. Large-scale 3-dimensional tissue cytometry on renal papillary biopsies showed an increase in the number and density of neutrophils in the papillae of patients with brushite versus CaOx, thereby linking the observed inflammatory signatures to the neutrophils in the tissue. To explain how neutrophil proteins appear in the stone matrix, we measured neutrophil extracellular trap (NET) formation-NETosis-and found it significantly increased in the papillae of patients with brushite stones compared with CaOx stones. CONCLUSION: We show that increased neutrophil infiltration and NETosis is an unrecognized factor that differentiates brushite and CaOx SF and may explain the markedly increased scarring and inflammation seen in the papillae of patients with brushite stones. Given the increasing prevalence of brushite stones, the role of neutrophil activation in brushite stone formation requires further study.

In Vivo Renal Tubule pH in Stone-Forming Human Kidneys.

Introduction: There is evidence that patients with a history of ileostomies, who produce acidic urine and form uric acid or calcium oxalate stones, may plug some collecting ducts with calcium phosphate (CaP) and urate crystals. This is a paradoxical finding as such minerals should not form at an acid pH. One possible explanation is the presence of acidification defects due to focal damage to inner medullary collecting duct and Bellini duct (BD) cells. We sought to further investigate this hypothesis through direct measurement of ductal pH in dilated BDs in patients with ileostomies undergoing percutaneous nephrolithotomy (PCNL) for stone removal. Methods: After obtaining institutional review board approval, we used a fiber-optic pH microsensor with a 140-µm-diameter tip to measure intraluminal pH from the bladder, saline irrigant, and dilated BDs of patients undergoing PCNL. Results: Measurements were taken from three patients meeting inclusion criteria. Measured pH of bladder urine ranged from 4.97 to 5.58 and pH of saline irrigant used during surgery ranged from 5.17 to 5.75. BD measurements were achieved in 11 different BDs. Mean intraductal BD pH was more than 1 unit higher than bulk urine (6.43 ± 0.22 vs 5.31 ± 0.22, p < 0.01). Conclusions: This is the first evidence for focal acidification defects within injured/dilated BDs of human kidneys producing highly acidic bulk phase urine. These results may help explain the paradoxical finding of CaP and urate plugs in dilated ducts of patients with stone-forming diseases characterized by highly acidic urine.

Evidence for disordered acid-base handling in calcium stone-forming patients.

In stone formers (SFs) with idiopathic hypercalciuria, urine pH governs the mineral phase of stones. Calcium phosphate (CaP) SFs have higher urine pH than calcium oxalate (CaOx) SFs. Normal women have higher urine pH than men on fixed diets, accompanied by greater absorption of food alkali. Female CaP and male CaOx SFs have similar urine pH as same sex normal individuals, but male CaP and female CaOx SFs may have abnormal acid-base handling. We studied 25 normal individuals (13 men and 12 women), 17 CaOx SFs (11 men and 6 women), and 15 CaP SFs (8 men and 7 women) on fixed diets. Urine and blood samples were collected under fasting and fed conditions. Female CaOx SFs had lower urine pH and lower alkali absorption, fed, compared with normal women; their urine NH4 was higher and urine citrate excretion lower than in normal women, consistent with their higher net acid excretion. Male CaOx SFs had higher urine citrate excretion and higher serum ultrafilterable citrate levels than normal men. Both male and female CaP SFs had higher urine pH fasting than same sex normal individuals, but only men were higher in the fed period, and there were no differences from normal in gut alkali absorption. CaP SFs of both sexes had higher urine NH4 and lower urine citrate than same sex normal individuals. The lower urine pH of female CaOx SFs seems related to decreased gut alkali absorption, while the higher pH of CaP SFs, accompanied by higher urine NH4 and lower urine citrate, suggests a proximal tubule disorder.

Discrepancy Between Stone and Tissue Mineral Type in Patients with Idiopathic Uric Acid Stones.

Objectives: To describe the papillary pathology found in uric acid (UA) stone formers, and to investigate the mineral form of tissue deposits. Materials and Methods: We studied eight UA stone formers treated with percutaneous nephrolithotomy. Papillae were imaged intraoperatively using digital endoscopy, and cortical and papillary biopsies were taken. Biopsies were analyzed by light microscopy, micro-CT, and microinfrared spectroscopy. Results: As expected, urine pH was generally low. UA supersaturation exceeded one in all but one case, compatible with the stone material. By intraoperative imaging, the renal papillae displayed a heterogeneous mixture of plaque and plugging, ranging from normal to severe. All patients had mineral in ducts of Bellini and inner medullary collecting ducts, mainly apatite with lesser amounts of urate and/or calcium oxalate in some specimens. Papillary and cortical interstitial tissue injury was modest despite the tubule plugging. No instance was found of a stone growing attached to either plaque or plugs. Conclusions: UA stone formers resemble those with ileostomy in having rather low urine pH while forming tubule plugs that contain crystals that can only form at pH values above those of their bulk urine. This discrepancy between tissue mineral deposits and stone type suggests that local tubular pH exceeds that of the bulk urine, perhaps because of localized tubule injury. The manner in which UA stones form and the discordance between tubule crystals and stone type remain open research questions.

Mechanisms for falling urine pH with age in stone formers.

One of the main functions of the kidney is to excrete an acid load derived from both dietary and endogenous sources, thus maintaining the pH of other fluids in the body. Urine pH is also of particular interest in stone formers, since it determines the presence of either calcium phosphate or uric acid content in stones. Others have noted in epidemiological studies a rise in incidence of low pH-dependent uric acid stones with age, coinciding with a decrease in the incidence of high pH-dependent phosphate stones. Taken together, these trends are suggestive of a longitudinal decline in urine pH in stone-forming patients, and, if true, this could explain the observed trends in stone incidence. We studied 7,891 stone formers, all of whom collected a 24-h urine sample and matching serum. Multivariate modeling revealed that urine pH did indeed fall with age and particularly between the ages of 20 and 50 yr old in both men and women. We sought to explain this trend through the inclusion of traditionally understood determinants of urine pH such as urinary buffers, estimates of glomerular filtration, and dietary acid load, but these, taken together, accounted for but a small fraction of the pH fall. Gastrointestinal anion absorption was the strongest predictor of urine pH in all age groups, as we have previously reported in middle-aged normal men and women. However, we found that, despite a decreasing urine pH, gastrointestinal anion absorption increased monotonically with age. In fact, after adjustment for gastrointestinal anion absorption, urine pH declined more markedly, suggesting that bicarbonate-producing anion absorption is regulated in a manner that offsets the decline of urine pH.

Randall's plaque in stone formers originates in ascending thin limbs.

Randall's plaque, an attachment site over which calcium oxalate stones form, begins in the basement membranes of thin limbs of the loop of Henle. The mechanism of its formation is unknown. Possibly, enhanced delivery of calcium out of the proximal tubule, found in many stone formers, increases reabsorption of calcium from the thick ascending limb into the interstitium around descending vasa recta, which convey that calcium into the deep medulla, and raises supersaturations near thin limbs ("vas washdown"). According to this hypothesis, plaque should form preferentially on ascending thin limbs, which do not reabsorb water. We stained serial sections of papillary biopsies from stone-forming patients for aquaporin 1 (which is found in the descending thin limb) and the kidney-specific chloride channel ClC-Ka (which is found in the ascending thin limb). Plaque (which is detected using Yasue stain) colocalized with ClC-Ka, but not with aquaporin 1 (χ2 = 464, P < 0.001). We conclude that plaque forms preferentially in the basement membranes of ascending thin limbs, fulfilling a critical prediction of the vas washdown theory of plaque pathogenesis. The clinical implication is that treatments such as a low-sodium diet or thiazide diuretics that raise proximal tubule calcium reabsorption may reduce formation of plaque as well as calcium kidney stones.

Mechanism for higher urine pH in normal women compared with men.

Regulation of acid-base metabolism maintains the pH of body fluids within a tight range. Urine pH (UpH) is also regulated under normal conditions. Median pH of 24-h urines is ~6, but others have noted that UpH in women is higher than men, which has been attributed to differences in diet. If true, it would help to explain the fact that calcium phosphate stones, which form at higher urine pH, are much more common in women than in men. We studied 14 normal subjects (7 men and 7 women) fed identical meals in a Clinical Research Center. Urine and blood samples were collected during fasting and after meals. UpH of women (6.74 ± 0.11) exceeded that of men (6.07 ± 0.17) fed, but not fasting, and UpH rose significantly with meals in women but not men. Serum and urine total CO2 rose with meals in women but not men, and in women net acid excretion fell to zero during the fed period. In a general linear model adjusted for age, sex, and weight, net gastrointestinal anion uptake was the main predictor of UpH and was significantly higher in women (3.9 ± 0.6) than men (1.8 ± 0.7) in the fed period. Urine citrate, an anion absorbed by the gastrointestinal tract, was higher in women than men in the fed state, and fractional excretion of citrate was higher in women than men. The higher fed UpH in women is related to a greater absorption of food anions and raises 24-h UpH.

Evidence for a role of PDZ domain-containing proteins to mediate hypophosphatemia in calcium stone formers.

Background: Hypophosphatemia (HYP) is common among calcium stone formers (SFs) and in rare cases is associated with mutations in sodium-phosphate cotransporters or in Na+/H+ exchanger regulatory factor 1 (NHERF1), but the majority of cases are unexplained. We hypothesized that reduced sodium-phosphate cotransporter activity mediated via NHERF1 or a similar PDZ domain-containing protein, causes HYP. If so, other transport activities controlled by NHERF1, such as NHE3 and URAT1, might be reduced in HYP. Methods: To test this idea, we analyzed two large but separate sets of 24-h urine samples and paired serums of 2700 SFs from the University of Chicago and 11 073 SFs from Litholink, a national laboratory. Patients were divided into quintiles based on serum phosphate. Results: Males were more common in the lowest phosphate tiles in both datasets. Phosphate excretion did not vary across the quintiles, excluding diet as a cause of HYP. Tubule maximum (Tm) phosphate per unit glomerular filtration rate decreased and fractional excretion increased with decreasing phosphate quintiles, indicating reduced tubule phosphate reabsorption was responsible for HYP. Urine pH and serum chloride increased with decreasing serum phosphate, suggesting a coordinate change in NHE3 activity. Serum uric acid and Tm uric acid decreased significantly with decreasing serum phosphate, while uric acid excretion did not vary. Conclusion. HYP in SFs results from decreased tubule phosphate reabsorption and, being associated with related changes in other proximal tubule transporters, may arise from alterations in or signaling to PDZ-containing proteins.