XIST Inhibition Attenuates Calcium Oxalate Nephrocalcinosis-Induced Renal Inflammation and Oxidative Injury via the miR-223/NLRP3 Pathway.

The roles of the lncRNA X inactive specific transcript (XIST) in many diseases, including cancers and inflammatory sickness, have been previously elucidated. However, renal calculus remained poorly understood. In this study, we revealed the potential effects of XIST on kidney stones that were exerted via inflammatory response and oxidative stress mechanisms. We established a glyoxylate-induced calcium oxalate (CaOx) stone mouse model and exposed HK-2 cells to calcium oxalate monohydrate (COM). The interactions among XIST, miR-223-3p, and NOD-like receptor protein 3 (NLRP3) and their respective effects were determined by RNAs and protein expression, luciferase activity, and immunohistochemistry (IHC) assays. Cell necrosis, reactive oxygen species (ROS) generation, and inflammatory responses were detected after silencing XIST, activating and inhibiting miR-223-3p, and both knocking down XIST and activating miR-223-3p in vitro and in vivo. The XIST, NLRP3, caspase-1, and IL-1ß levels were notably increased in kidney samples from glyoxylate-induced CaOx stone model mice. XIST knockdown significantly suppressed the inflammatory damage and ROS production and further attenuated oxalate crystal deposition. miRNA-223-3p mimics also exerted the same effects. Moreover, we verified the interactions among XIST, miRNA-223-3p and NLRP3, and the subsequent effects. Our results suggest that the lncRNA XIST participates in the formation and progression of renal calculus by interacting with miR-223-3p and the NLRP3/Caspase-1/IL-1ß pathway to mediate the inflammatory response and ROS production.

Calcium oxalate crystals and oxalate induce an epithelial-to-mesenchymal transition in the proximal tubular epithelial cells: Contribution to oxalate kidney injury.

TGF-ß1 is the main mediator of epithelial-to-mesenchymal transition (EMT). Hyperoxaluria induces crystalluria, interstitial fibrosis, and progressive renal failure. This study analyzed whether hyperoxaluria is associated with TGF-ß1 production and kidney fibrosis in mice and if oxalate or calcium oxalate (CaOx) could induce EMT in proximal tubule cells (HK2) and therefore contribute to the fibrotic process. Hyperoxaluria was induced by adding hydroxyproline and ethylene glycol to the mice's drinking water for up to 60 days. Renal function and oxalate and urinary crystals were evaluated. Kidney collagen production and TGF-ß1 expression were assessed. EMT was analyzed in vitro according to TGF-ß1 production, phenotypic characterization, invasion, cell migration, gene and protein expression of epithelial and mesenchymal markers. Hyperoxaluric mice showed a decrease in renal function and an increase in CaOx crystals and Ox urinary excretion. The deposition of collagen in the renal interstitium was observed. HK2 cells stimulated with Ox and CaOx exhibited a decreased expression of epithelial as well as increased expression mesenchymal markers; these cells presented mesenchymal phenotypic changes, migration, invasiveness capability and TGF-ß1 production, characterizing EMT. Treatment with BMP-7 or its overexpression in HK2 cells was effective at preventing it. This mechanism may contribute to the fibrosis observed in hyperoxaluria.

Effect of calcium oxalate on renal cells as revealed by real-time measurement of extracellular oxidative burst.

Calcium oxalate is one of the main constituents of kidney stones and has a proved deleterious effect on renal cells that is mediated by oxidative stress. However, the subcellular source of this oxidative stress, and whether it is extending to the extracellular space or not, is still disputed. Therefore, an electrochemical superoxide biosensor was constructed, positioned above A6 renal cells, and used to measure in real-time the extracellular oxidative burst following addition of calcium oxalate crystals. It was observed that A6 cells do secrete superoxide into their extracellular space in few minutes after encountering calcium oxalate crystals. The amount of released superoxide peaks at about 20 min. Superoxide is cleared away from the extracellular space after approximately 3h. Superoxide secretion depends on the presence of superoxide-scavenging enzyme superoxide dismutase, the age of the cells, the amount of calcium oxalate crystals, and the temperature. Moreover, the effect of calcium oxalate crystals was mimicked by phorbol 12-myristate 13-acetate. The developed sensing system can be a useful tool for biologists investigating nephrolithiasis at cellular level.

Influence of a low- and a high-oxalate vegetarian diet on intestinal oxalate absorption and urinary excretion.

OBJECTIVE: To compare quantitatively the effect of a low- and a high-oxalate vegetarian diet on intestinal oxalate absorption and urinary excretion. SUBJECTS AND METHODS: Eight healthy volunteers (three men and five women, mean age 28.6+/-6.3) were studied. Each volunteer performed the [(13)C(2)]oxalate absorption test thrice on a low-oxalate mixed diet, thrice on a low-oxalate vegetarian diet and thrice on a high-oxalate vegetarian diet. For each test, the volunteers had to adhere to an identical diet and collect their 24-h urines. In the morning of the second day, a capsule containing [(13)C(2)]oxalate was ingested. RESULTS: On the low-oxalate vegetarian diet, mean intestinal oxalate absorption and urinary oxalate excretion increased significantly to 15.8+/-2.9% (P=0.012) and 0.414+/-0.126 mmol/day (P=0.012), compared to the mixed diet. On the high-oxalate vegetarian diet, oxalate absorption (12.5+/-4.6%, P=0.161) and urinary excretion (0.340+/-0.077 mmol/day, P=0.093) did not change significantly, compared to the mixed diet. CONCLUSIONS: A vegetarian diet can only be recommended for calcium oxalate stone patients, if the diet (1) contains the recommended amounts of divalent cations such as calcium and its timing of ingestion to a meal rich in oxalate is considered and (2) excludes foodstuffs with a high content of nutritional factors, such as phytic acid, which are able to chelate calcium.

Tea and coffee as the main sources of oxalate in diets of patients with kidney oxalate stones.

We analyzed nutritional habits of 22 stone formers with special regard to oxalate content as one of the main nutritional lithogenic factors associated with kidney stones. Daily dietary oxalate intake was 354 +/- 261 mg and 406 +/- 265 mg in men and women respectively. These values were much higher than those found by other researches. The main sources of oxalate in diets were regular tea and coffee (80-85%). Only 15-20% of oxalate was derived from other plant foods. Patients did not adhere to high fluid diet and, what is more, as common beverage they chose rich-oxalate black tea. Patients' daily intake of calcium was low and didn't exceed 520 mg. Vitamin C consumption was higher than Polish Dietary Reference Intake (DRI) and vitamin B6 lower than DRI. In the management of stone patients, to lower the risk of recurrence, appropriate diet (according to the type of stone) should be provided by dietitian.

The role of diet in the prevention of common kidney stones.

Current dietary recommendations for patients who form kidney stones are discussed. Focusing on the most common kidney stone types, calcium oxalate and uric acid, the rationale for dietary changes are described based on the renal and urine physiology.

Near-infrared fluorescence imaging of microcalcification in an animal model of breast cancer.

RATIONALE AND OBJECTIVES: At present, there is no animal model of breast cancer that forms reproducible microcalcification. The aim of this study was to develop a straightforward, reproducible model system that could be used to develop multimodality contrast agents for the identification of breast cancer microcalcification. METHODS: The R3230 mammary adenocarcinoma cell line was implanted in the mammary fat pad of female Fischer 344 rats (two rats with two implanted tumors and two rats with a single implanted tumor). After growth to 1-2 cm in diameter, tumors were implanted with 100 microm hydroxyapatite crystals (positive control) or calcium oxalate crystals (negative control). Twenty-four hours after crystal implantation, rats were injected intravenously with a previously described near-infrared fluorescent bisphosphonate derivative known as Pam78, and the tumors were imaged using a reflectance optical imaging system. RESULTS: Tumors implanted with hydroxyapatite displayed bright, focal, near-infrared fluorescence in the area of crystal implantation. Control tumors, grown in the same animal and implanted with calcium oxalate, did not display any near-infrared fluorescence, even along the needle track used for crystal implantation. CONCLUSIONS: A simple and rapid animal model of focal calcification in breast cancer tumors has been developed and validated. The model used Pam78, a near-infrared fluorescent contrast agent specific for hydroxyapatite. The potential usefulness of the model for developing similar contrast agents for magnetic resonance and other imaging modalities is discussed.

Urinary oxalic acid excretion differs after oral loading of rats with various oxalate salts.

BACKGROUND: To compare urinary oxalate excretion after the oral administration of oxalic acid, disodium oxalate, or calcium oxalate in rats. METHODS: Male Wistar rats were divided into four groups of six rats each and were intravenously hydrated with normal saline, and then were administered normal saline (control group), 10 mg of oxalic acid, equimolar disodium oxalate, or equimolar calcium oxalate via a gastrostomy. Urine specimens were collected just before administration and at hourly intervals up to 5 h afterwards. The urinary oxalate, calcium, magnesium and phosphorus levels were measured. RESULTS: Urinary oxalate excretion peaked at 1-2 h after administration of oxalic acid or equimolar disodium oxalate, while administration of calcium oxalate only caused a small increase of urinary oxalate excretion. Cumulative urinary oxalate excretion during 5 h was 1.69 +/- 0.10 mg (mean +/- SD; 17%), 1.43 +/- 0.13 mg (13%), and 0.22 +/- 0.03 mg (2%) after the administration of oxalic acid, disodium oxalate, and calcium oxalate, respectively. Urinary calcium excretion showed a decrease in the oxalic acid and disodium oxalate groups, while urinary magnesium or phosphorus excretion did not change significantly. CONCLUSION: The upper gastrointestinal tract seems to be the major site of oxalic acid absorption and only free oxalate is absorbed irrespective of whether it is the sodium salt or not. After binding to calcium in the gut, oxalic acid absorption seems to be inhibited in the presence of calcium and this means that calcium oxalate is poorly absorbed (at least in the upper gastrointestinal tract).

The influence of a high-oxalate/low-calcium diet on calcium oxalate renal stone risk factors in non-stone-forming black and white South African subjects.

OBJECTIVE: To evaluate the influence of a high-oxalate/low-calcium diet on calcium oxalate stone risk factors in both black South Africans (who are largely immune to kidney stones) and white South Africans (in whom stones are more common). SUBJECTS AND METHODS: Urinary and dietary variables were examined in 11 black and 11 white South African men. None of the subjects had had a kidney stone or any metabolic illness. Their normal domestic food intake was assessed using a semiquantitative food frequency questionnaire. Subjects were given a standardized high-oxalate/low-calcium diet for 3 days; 24-h urine samples were collected before the protocol and during the final day. The samples were analysed using routine modern laboratory techniques. The urine analysis data were used to calculate the Tiselius risk index and the relative urinary supersaturations of calcium oxalate, uric acid and calcium phosphate. RESULTS: Urine analysis showed an intriguing anomaly; black subjects had significantly higher urinary pH and oxalate values than whites (6.50 vs 6.21 and 0.23 vs 0.14 mmol/24 h, respectively), while their urinary citrate was lower (1.47 vs 3.69 mmol/24 h). In addition, the Tiselius risk index and relative supersaturation of calcium oxalate were higher in black subjects. These results are contrary to those which might have been reasonably expected when comparing stone-free and stone-prone groups. After the dietary protocol, the only urinary variable which changed significantly was urinary oxalate, which increased by 57% in whites. CONCLUSION: Factors which are conventionally used to assess stone risk (pH, oxaluria, citraturia, relative supersaturation) are not helpful in identifying why South African blacks are relatively immune to stones. We suggest that relatively lower oxalate absorption rates may be a physiological feature of this racial group.

High-calcium intake abolishes hyperoxaluria and reduces urinary crystallization during a 20-fold normal oxalate load in humans.

BACKGROUND: The aim of the study was to test whether increasing dietary calcium intake lowers intestinal oxalate absorption and thereby prevents hyperoxaluria and urinary crystallization during a 20-fold normal oxalate load in healthy subjects. METHODS: Fourteen healthy male volunteers (age 23-44 years, BMI 21.5-27.7 kg/m2) collected 24-h urines while on free-choice diet as well as on two standardized diets. The latter contained 2545 kcal, 2500 ml of mineral water, 102 g of protein, 13.6 g of sodium chloride and 2220 mg of oxalate (approximately 20-fold content of an average diet). Subjects were studied twice while on the standardized diet, once while eating a normal amount of calcium (1211 mg/day, oxalate-rich diet), and once while eating 3858 mg of calcium/day (calcium and oxalate-rich diet). RESULTS: Compared with the free-choice diet (322+/-36 micromol/d), UOx x V increased to 780+/-72 micromol/d on the oxalate-rich diet (P=0.001) and fell again to 326+/-31 micromol/d on calcium and oxalate-rich diet (P=0.001 vs oxalate-rich diet). Urinary glycolate (a metabolic precursor of Ox) always remained below the upper limit of the normal range and did not change between different diets, indicating that changes in UOX x V reflect respective variations in intestinal absorption of Ox. Uca x V was 4.60+/-0.45 mmol/d on the free-choice diet and 3.20+/-0.32 mmol/d on the oxalate-rich diet (P=0.011 vs free-choice diet); it increased to 7.28+/-0.74 mmol/d on the calcium- and oxalate-rich diet (P=0.001 vs free-choice and oxalate-rich diets). As indicated by the AP (CaOx) index (Tiselius), urinary supersaturation did not vary significantly between the three diets. In freshly voided morning urines (studied in 8/14 subjects) on the oxalate-rich diet, CaOx crystals or crystal aggregates of up to 80 microm diameter were found in 5/8 urines, whereas this never occurred on the free-choice diet and only t once on the calcium- and oxalate-rich diet. CONCLUSION: . Increasing calcium intake while eating Ox-rich food prevents dietary hyperoxaluria and reduces CaOx crystallization in healthy subjects. This further illustrates that dietary counseling to idiopathic calcium-stone formers should ensure sufficient calcium intake, especially during oxalate-rich meals.

Diet and nephrolithiasis.

Pharmacologic therapy of recurrent nephrolithiasis continues to be the mainstay of the strategy to prevent recurrence. This approach persists even in the face of increasing evidence of a marked benefit of mere entry into a nonpharmacologic diet and fluid modification protocol at a clinic specializing in the evaluation and therapy of recurrent nephrolithiasis (the "Stone Clinic effect"). This review examines the role of diet in the pathogenesis of various forms of nephrolithiasis and the effectiveness of dietary therapy in preventing new stone formation. Recent and older evidence support a primary role for modification of diet, particularly diet protein and sodium intake, in the prevention of recurrent nephrolithiasis.

Oxalate: effect on calcium absorbability.

Absorption of calcium from intrinsically labeled Ca oxalate was measured in 18 normal women and compared with absorption of Ca from milk in these same subjects, both when the test substances were ingested in separate meals and when ingested together. Fractional Ca absorption from oxalate averaged 0.100 +/- 0.043 when ingested alone and 0.140 +/- 0.063 when ingested together with milk. Absorption was, as expected, substantially lower than absorption from milk (0.358 +/- 0.113). Nevertheless Ca oxalate absorbability in these women was higher than we had previously found for spinach Ca. When milk and Ca oxalate were ingested together, there was no interference of oxalate in milk Ca absorption and no evidence of tracer exchange between the two labeled Ca species.

The current role of medical treatment of nephrolithiasis: the impact of improved techniques of stone removal.

To document more clearly the effect of selective medical treatment on the course of nephrolithiasis, we surveyed 103 consecutive patients being followed in our stone clinic. Of the patients who initially had existing stones within the kidneys 69 per cent experienced no symptoms while undergoing medical therapy, compared to 96 per cent of those who had no existing stones. In all subjects studied new stone formation was reduced in more than 95 per cent and no new stones were formed in more than 75 per cent. An operation for newly formed calculi was necessary in only 2 per cent of the patients on medical therapy, whereas 58 to 69 per cent of the patients required an operation for new stones before beginning medical treatment. We believe that selective medical treatment has an important role in the over-all management of nephrolithiasis. Appropriate medical therapy may decrease significantly the number of new stones formed and may obviate the need for a repeat stone operation. Therefore, medical treatment should be an important adjunct to percutaneous nephrostolithotomy and extracorporeal shock wave lithotripsy.

Calcium oxalate urolithiasis in the rat: is it a model for human stone disease? A review of recent literature.

Calcium oxalate stone disease is the most common human urinary stone disease in the Western Hemisphere. To understand different aspects of the disease, calcium oxalate urolithiasis in the rat is used as a model. Spontaneous calcium oxalate urolithiasis is very rare in rats. Thus the disease is experimentally induced and the rats are generally made hyperoxaluric either by administration of excess oxalate, exposure to the toxin ethylene glycol, or various nutritional manipulations. All the experimental models show renal injury associated with crystal deposition. Calcium oxalate crystals are in most cases intraluminal in renal tubules and often attached to the basal lamina of the denuded epithelium. Rat renal papillary tips and fornices appear to be the preferential sites for the deposition of large calcium oxalate calculi. Where urinary supersaturation of calcium oxalate has been studied the crystal forming rat urines are shown to have higher urinary supersaturation of calcium oxalate than their controls. Oxalate metabolism in the rat is nearly identical to that in humans. Thus, in a number of respects, experimental calcium oxalate urolithiasis in the rat is similar to calcium oxalate stone disease in man.