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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.

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.

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., Graphical abstract

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.

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.

Idiopathic hypercalciuria (IH) is a common familial trait among patients with calcium nephrolithiasis. Previously, we have demonstrated that hypercalciuria is primarily due to reduced renal proximal and distal tubule calcium reabsorption. Here, using measurements of the clearances of sodium, calcium, and endogenous lithium taken from the General Clinical Research Center, we test the hypothesis that patterns of segmental nephron tubule calcium reabsorption differ between the sexes in IH and normal subjects. When the sexes are compared, we reconfirm the reduced proximal and distal calcium reabsorption. In IH women, distal nephron calcium reabsorption is decreased compared to normal women. In IH men, proximal tubule calcium reabsorption falls significantly, with a more modest reduction in distal calcium reabsorption compared to normal men. Additionally, we demonstrate that male IH patients have lower systolic blood pressures than normal males. We conclude that women and men differ in the way they produce the hypercalciuria of IH, with females reducing distal reabsorption and males primarily reducing proximal tubule function.

Calcium nephrolithiasis in children is increasing in prevalence and tends to be recurrent. Although children have a lower incidence of nephrolithiasis than adults, its etiology in children is less well understood; hence treatments targeted for adults may not be optimal in children. To better understand metabolic abnormalities in stone forming children, we compared chemical measurements and the crystallization properties of 24-hour urine collections from 129 stone formers matched to 105 non-stone forming siblings and 183 normal, healthy children with no family history of stones; all aged 6 to 17 years. The principal risk factor for calcium stone formation was hypercalciuria. Stone formers have strikingly higher calcium excretion along with high supersaturation for calcium oxalate and calcium phosphate, and a reduced distance between the upper limit of metastability and supersaturation for calcium phosphate, indicating increased risk of calcium phosphate crystallization. Other differences in urine chemistry that exist between adult stone formers and normal individuals such as hyperoxaluria, hypocitraturia, abnormal urine pH and low urine volume were not found in these children. Hence, hypercalciuria and a reduction in the gap between calcium phosphate upper limit of metastability and supersaturation are crucial determinants of stone risk. This highlights the importance of managing hypercalciuria in children with calcium stones.

A main mechanism of idiopathic hypercalciuria (IH) in calcium stone-forming patients (IHSF) is postprandial reduction of renal tubule calcium reabsorption that cannot be explained by selective reduction of serum parathyroid hormone levels; the nephron site(s) responsible are not as yet defined. Using fourteen 1-h measurements of the clearances of sodium, calcium, and endogenous lithium during a three-meal day in the University of Chicago General Clinical Research Center, we found reduced postprandial proximal tubule reabsorption of sodium and calcium in IHSF vs. normal subjects. The increased distal sodium delivery is matched by increased distal reabsorption so that urine sodium excretions do not differ, but distal calcium reabsorption does not increase enough to match increased calcium delivery, so hypercalciuria results. In fact, urine calcium excretion and overall renal fractional calcium reabsorption both are high in IHSF vs. normal when adjusted for distal calcium delivery, strongly suggesting a distal as well as proximal reduction of calcium reabsorption. The combination of reduced proximal tubule and distal nephron calcium reabsorption in IHSF is a new finding and indicates that IH involves a complex, presumably genetic, variation of nephron function. The increased calcium delivery into the later nephron may play a role in stone formation via deposition of papillary interstitial apatite plaque.

Purpose: We define the relationships between urine inhibition of calcium oxalate crystal growth and age, gender, urine chemistries and stone formation among relatives of calcium stone forming patients.Materials and Methods: We collected 24-hour urine samples from 366 first degree relatives of calcium stone formers. Calcium oxalate crystal growth inhibition was studied using a constant amount of dialyzed urine protein in a seeded crystallization system. Standard stone risk measurements were also performed on the urine, including supersaturation for calcium oxalate, calcium phosphate and uric acid.Results: By multivariate analysis crystal growth inhibition is strongly inversely related to the amount of protein excreted per day, and the age of the subject. When corrected for protein excretion and age, urine proteins from nonstone forming male subjects inhibited crystal growth more strongly than those from corresponding female subjects. Among stone formers the sex difference was not present.Conclusions: Inhib...

Patients with idiopathic hypercalciuria (IH) have decreased renal calcium reabsorption, most marked in the postprandial state, but the mechanisms are unknown. We compared 29 subjects with IH and 17 normal subjects (N) each fed meals providing identical amounts of calcium. Urine and blood samples were collected fasting and after meals. Levels of three candidate signalers, serum calcium (SCa), insulin (I), and plasma parathyroid hormone (PTH), did not differ between IH and N either fasting or fed, but all changed with feeding, and the change in SCa was greater in IH than in N. Regression analysis of fractional excretion of calcium (FECa) was significant for PTH and SCa in IH but not N. With the use of multivariable analysis, Sca entered the model while PTH and I did not. To avoid internal correlation we decomposed FECa into its independent terms: adjusted urine calcium (UCa) and UFCa molarity. Analyses using adjusted Uca and unadjusted Uca parallel those using FECa, showing a dominant effect of SCa with no effect of PTH or I. The effect of SCa may be mediated via vitamin D receptor-stimulated increased abundance of basolateral Ca receptor, which is supported by the fact PTH levels also seem more responsive to serum Ca in IH than in N. Although our data support an effect of SCa on FECa and UCa, which is more marked in IH than in N, it can account for only a modest fraction of the meal effect, perhaps 10–20%, suggesting additional mediators are also responsible for the exaggerated postprandial hypercalciuria seen in IH.

The most common metabolic abnormality found in calcium (Ca) kidney stone formers is idiopathic hypercalciuria (IH). Using endogenous lithium (Li) clearance, we previously showed that in IH, there is decreased proximal tubule sodium absorption, and increased delivery of Ca into the distal nephron. Distal Ca reabsorption may facilitate the formation of Randall's plaque (RP) by washdown of excess Ca through the vasa recta toward the papillary tip. Elevated Ca excretion leads to increased urinary supersaturation (SS) with respect to calcium oxalate (CaOx) and calcium phosphate (CaP), providing the driving force for stone growth on RP. Thiazide (TZ) diuretics reduce Ca excretion and prevent stone recurrence, but the mechanism in humans is unknown. We studied the effect of chronic TZ administration on renal mineral handling in four male IH patients using a fixed three meal day in the General Clinical Research Center. Each subject was studied twice: once before treatment and once after 4–7 mo of daily chlorthalidone treatment. As expected, urine Ca fell with TZ, along with fraction of filtered Ca excreted. Fraction of filtered Li excreted also fell sharply with TZ, as did distal delivery of Ca. Unexpectedly, TZ lowered urine pH. Together with reduced urine Ca, this led to a marked fall in CaP SS, but not CaOx SS. Since CaOx stone formation begins with an initial CaP overlay on RP, by lowering urine pH and decreasing distal nephron Ca delivery, TZ might diminish stone risk both by reducing CaP SS, as well as slowing progression of RP.

The process of kidney stone formation depends on an imbalance between excretion of water and insoluble stone-forming salts, leading to high concentrations that supersaturate urine and inner medullary collecting duct (IMCD) fluid. For common calcium-containing stones, a critical mechanism that has been proposed for integrating water and calcium salt excretions is activation of the cell surface calcium-sensing receptor (CaSR) on the apical membranes of IMCD cells. High deliveries of calcium into the IMCD would be predicted to activate CaSR, leading to reduced membrane abundance of aquaporin-2, thereby limiting water conservation and protecting against stone formation. We have tested this hypothesis in 16 idiopathic hypercalciuric calcium stone formers and 14 matched normal men and women in the General Clinical Research Center. Subjects were fed identical diets; we collected 14 urine samples at 1-h intervals during a single study day, and one sample overnight. Hypercalciuria did not increase urine volume, so urine calcium molarity and supersaturation with respect to calcium oxalate and calcium phosphate rose proportionately to calcium excretion. Thus CaSR modulation of urine volume via IMCD CaSR activation does not appear to be an important mechanism of protection against stone formation. The overnight period, one of maximal water conservation, was a time of maximal stone risk and perhaps a target of specific clinical intervention.

The papillary surface epithelium (PSE) covers the renal papilla in mammalian kidneys and serves as a diffusion barrier between the urine on the apical surface and the interstitium on the basolateral surface. The PSE also plays a physiological role in transport of solutes between the urine and interstitium both by active transport and paracellular pathways. Permeability of the PSE may be affected by alterations in specific transporters, components of intercellular tight junctions, cell surface glycosaminoglycans and urine composition. In idiopathic calcium oxalate (CaOx) stone formers, apatite deposits known as Randall’s plaque form in the papillary interstitium and lodge beneath the PSE. The presence of plaque may perturb the normal function of the PSE, possibly by provoking the up‐regulation of pro‐inflammatory cytokines such as TNFα in the interstitium. Disruption of the epithelial barrier may lead to increased permeability and exposure of the plaque matrix to urine constituents, followed by loss of the...

We have investigated urine protein inhibitors of calcium oxalate crystallization to determine whether variations in these proteins are associated with kidney stone disease and whether protein measurements improve the identification of stone formers compared with conventional risk factors (RF). Using Western blotting, we studied variations in the electrophoretic mobility patterns and relative abundances of crystallization-inhibitory proteins in urine from 50 stone-forming (SF) and 50 non-stone-forming (NS) first-degree relatives of calcium SF patients, matched by gender and age. Standard urine chemistry stone risk measurements were also made. Multivariate discriminant analysis was used to test the association of these proteins with nephrolithiasis. Differences in form and abundance of several urine proteins including inter-α-trypsin inhibitor (ITI), prothrombin fragment 1 (PF1), CD59, and calgranulin B (calB) were found to be associated with stone formation. By multivariate discriminant analysis, measurements of forms of PF1, ITI, and calB in men and ITI and CD59 in women, classified 84% of men and 76% of women correctly by stone status. In contrast, standard urine chemistry RF identified only 70% of men correctly and failed to distinguish female SF from NS. Thus a small subset of protein measurements distinguished SF from NS far better than conventional RF in a population of relatives of calcium SF, illustrating the significant association of these proteins with stone disease. Variations in these proteins may serve as markers of stone disease activity or vulnerability to recurrence and may provide new insights into mechanisms of stone formation.

We define the relationships between urine inhibition of calcium oxalate crystal growth and age, gender, urine chemistries and stone formation among relatives of calcium stone forming patients.We collected 24-hour urine samples from 366 first degree relatives of calcium stone formers. Calcium oxalate crystal growth inhibition was studied using a constant amount of dialyzed urine protein in a seeded crystallization system. Standard stone risk measurements were also performed on the urine, including supersaturation for calcium oxalate, calcium phosphate and uric acid.By multivariate analysis crystal growth inhibition is strongly inversely related to the amount of protein excreted per day, and the age of the subject. When corrected for protein excretion and age, urine proteins from nonstone forming male subjects inhibited crystal growth more strongly than those from corresponding female subjects. Among stone formers the sex difference was not present.Inhibition of calcium oxalate crystal growth is influenced by a complex combination of gender, age, stone formation and assay conditions. The effect of daily protein excretion is most likely a consequence of using a fixed amount of urine protein per assay. The influence of age is significant and unexplained, with the urine of young people (less than 20 years) demonstrating a vigorous ability to inhibit crystallization. In addition, the urine of nonstone forming male relatives appears to have a greater ability to inhibit crystallization than that of nonstone forming female relatives. Further use of this assay in clinical investigations must take age and gender into proper account.

Little is known about the renal handling of oxalate in patients with idiopathic hypercalciuria (IH). To explore the role of tubular oxalate handling in IH and to evaluate whether differences exist between IH and normal controls, we studied 19 IH subjects, 8 normal subjects, and 2 bariatric stone formers (BSF) during a 1-day General Clinical Research Center protocol utilizing a low-oxalate diet. Urine and blood samples were collected at 30- to 60-min intervals while subjects were fasting and after they ate three meals providing known amounts of calcium, phosphorus, sodium, protein, oxalate, and calories. Plasma oxalate concentrations and oxalate-filtered loads were similar between patients (includes IH and BSF) and controls in both the fasting and fed states. Urinary oxalate excretion was significantly higher in patients vs. controls regardless of feeding state. Fractional excretion of oxalate (FEOx) was >1, suggesting tubular secretion of oxalate, in 6 of 19 IH and both BSF, compared with none of the controls ( P < 0.00001). Adjusted for water extraction along the nephron, urine oxalate rose more rapidly among patients than normal subjects with increases in plasma oxalate. Our findings identify tubular secretion of oxalate as a key mediator of hyperoxaluria in calcium stone formers, potentially as a means of maintaining plasma oxalate in a tight range.

Inter-alpha-trypsin inhibitor heavy-chain proteins bind to the protease inhibitor bikunin and to hyaluronan, stabilizes extracellular matrix in various tissues, and also inhibits calcium oxalate crystallization in vitro. In both normal and stone-forming patients, we found heavy chain 3 and hyaluronan in the interstitial matrix of the kidney. Osteopontin was found in the collecting duct, thin loop of Henle, and urothelial cells. In stone formers, heavy chain 3 was also present in collecting duct, thin loop, and interstitial cells. Heavy chain 3 and osteopontin colocalized in plaque matrix and urothelial cells. Within individual plaque spherules, heavy chain 3 was found in the matrix layer while osteopontin was located along the crystal-matrix interface. Bikunin was present only in the collecting duct apical membranes and the loop cell cytoplasm of stone formers colocalizing with osteopontin and heavy chain 3. Widespread heavy chain 3 was only present in stone formers, whereas osteopontin was similarly expressed in normal and stone-forming subjects except for its localization in plaques of the stone formers. This is consistent with studies linking inter-alpha-trypsin inhibitor components to human stone disease, although their role is still unclear. Heavy chain 3 may also play a role in stabilizing hyaluronan in the renal interstitial matrix.