Effect of Vitamin B2-Deficient Diet on Hydroxyproline- or Obesity-Induced Hyperoxaluria in Mice.

SCOPE: Hyperoxaluria is a major cause of kidney stone disease. Around half of the oxalate in mammals is supplied from the diet and the other half is endogenously synthesized from glyoxylate. Reduction of hepatic glycolate oxidase (GO) activity is one approach to reduce endogenous production of oxalate. However, there are currently few effective dietary approaches to reduce hepatic GO activity. METHODS AND RESULTS: In the present study, it is investigated whether restriction of dietary vitamin B2 (VB2) can reduce hepatic GO activity and oxalate excretion in mice with hyperoxaluria induce by hydroxyproline (Hyp) or obesity. It is found that VB2 restriction significantly reduces hepatic GO activity in both the Hyp- and obesity-induced model of hyperoxaluria in mice. However, VB2 restriction reduces urinary oxalate excretion only in the Hyp-treated mice and not the obese mice. This difference could be due to the contribution of endogenous oxalate production that manifests as increased hepatic GO activity in Hyp-treated mice but not obese mice. CONCLUSION: Together these results suggest that VB2 restriction could be a new dietary approach to improve hyperoxaluria when endogenous production of oxalate is increased.

Development and characterization of oxaceprol-loaded poly-lactide-co-glycolide nanoparticles for the treatment of osteoarthritis.

Oxaceprol is well-defined therapeutic agent as an atypical inhibitor of inflammation in osteoarthritis. In the present study, we aimed to develop and characterize oxaceprol-loaded poly-lactide-co-glycolide (PLGA) nanoparticles for intra-articular administration in osteoarthritis. PLGA nanoparticles were prepared by double-emulsion solvent evaporation method. Meanwhile, a straightforward and generally applicable high performance liquid chromatography method was developed, and validated for the first time for the quantification of oxaceprol. To examine the drug carrying capacity of nanoparticles, varying amount of oxaceprol was entrapped into a constant amount of polymer matrix. Moreover, the efficacy of drug amount on nanoparticle characteristics such as particle size, zeta potential, morphology, drug entrapment, and in vitro drug release was investigated. Nanoparticle sizes were between 229 and 509 nm for different amount of oxaceprol with spherical smooth morphology. Encapsulation efficiency ranged between 39.73 and 63.83% by decreasing oxaceprol amount. The results of Fourier transform infrared and DSC showed absence of interaction between oxaceprol and PLGA. The in vitro drug release from these nanoparticles showed a sustained release of oxaceprol over 30 days. According to cell culture studies, oxaceprol-loaded nanoparticles had no cytotoxicity with high biocompatibility. This study was the first step of developing an intra-articular system in the treatment of osteoarthritis for the controlled release of oxaceprol. Our findings showed that these nanoparticles can be beneficial for an effective treatment of osteoarthritis avoiding side effects associated with oral administration.

Total flavonoids of Desmodium styracifolium attenuates the formation of hydroxy-L-proline-induced calcium oxalate urolithiasis in rats.

Desmosium styracifolium (D. styracifolium), which is considered as a Chinese herbal medicine, has been reported to treat the kidney stone diseases. However, the potential phytochemically active components and the underlying mechanisms associated with its efficacy in targeting urolithiasis remain to be elucidated. This study aims to investigate the anti-urolithiatic effect of total flavonoids of D. styracifolium (TFDS) on calcium oxalate (CaOx) renal stones in Sprague-Dawley rats. Animal models of CaOx urolithiasis were established in male Sprague-Dawley rats by adding 5% w/w hydroxy-L-proline (HLP) in regular rat chow. The TFDS orally at 100, 400 mg/kg, respectively, were administered along with HLP for 28 days. At the end of 28 days of treatment, urine and serum samples were collected for crystalluria determination and various biochemical analysis. Kidney tissues were isolated and processed for antioxidant parameters measurement and histopathological examinations. HLP-induced hyperoxaluria alone reliably caused CaOx nephrolithiasis in rats. We showed that TFDS significantly reduced crystalluria and CaOx crystal deposits in the kidney sections as compared to untreated HLP group. Also, TFDS was observed to decrease urinary oxalate excretion, alleviate the pro-acidosis condition, improve the impaired renal functions and renal epithelial cell injury. Moreover, TFDS protected against the oxidative stress changes via reducing MDA content, increasing CAT and GSH-Px activities in renal homogenate, as well as attenuating the expression of MCP-1, OPN and TGF-ß proteins. These results indicated that TFDS had beneficial effect on inhibition of CaOx formation in the rat kidney probably through a combination of antioxidant, anti-inflammatory, urine alkalinizing activities, and lowering the concentration of urinary stone-forming constituents. Thus, TFDS might have clinical implications in preventing oxidative renal cell injury and, ultimately, kidney stone formation. The data provide a rationale for the medicinal use of TFDS in nephrolithiasis and identify this agent as a potential source of new antiurolithic drugs.

High Sodium-Induced Oxidative Stress and Poor Anticrystallization Defense Aggravate Calcium Oxalate Crystal Formation in Rat Hyperoxaluric Kidneys.

Enhanced sodium excretion is associated with intrarenal oxidative stress. The present study evaluated whether oxidative stress caused by high sodium (HS) may be involved in calcium oxalate crystal formation. Male rats were fed a sodium-depleted diet. Normal-sodium and HS diets were achieved by providing drinking water containing 0.3% and 3% NaCl, respectively. Rats were fed a sodium-depleted diet with 5% hydroxyl-L-proline (HP) for 7 and 42 days to induce hyperoxaluria and/or calcium oxalate deposition. Compared to normal sodium, HS slightly increased calcium excretion despite diuresis; however, the result did not reach statistical significance. HS did not affect the hyperoxaluria, hypocalciuria or supersaturation caused by HP; however, it increased calcium oxalate crystal deposition soon after 7 days of co-treatment. Massive calcium oxalate formation and calcium crystal excretion in HS+HP rats were seen after 42 days of treatment. HP-mediated hypocitraturia was further exacerbated by HS. Moreover, HS aggravated HP-induced renal injury and tubular damage via increased apoptosis and oxidative stress. Increased urinary malondialdehyde excretion, in situ superoxide production, NAD(P)H oxidase and xanthine oxidase expression and activity, and decreased antioxidant enzyme expression or activity in the HS+HP kidney indicated exaggerated oxidative stress. Interestingly, this redox imbalance was associated with reduced renal osteopontin and Tamm-Horsfall protein expression (via increased excretion) and sodium-dependent dicarboxylate cotransporter NaDC-1 upregulation. Collectively, our results demonstrate that a HS diet induces massive crystal formation in the hyperoxaluric kidney; this is not due to increased urinary calcium excretion but is related to oxidative injury and loss of anticrystallization defense.

Dietary hydroxyproline induced calcium oxalate lithiasis and associated renal injury in the porcine model.

BACKGROUND AND PURPOSE: We previously reported hyperoxaluria and calcium oxalate calculi in adult pigs (sows) fed hydroxyproline (HP). The purpose of this study was to grossly and histopathologically characterize intrarenal effects in this model. METHODS: In the swine facility at our campus, we maintained 21 gestating sows, of which 15 received daily treatment (5% HP mixed with dry feed) and 6 received no treatment (controls). Nine were sacrificed at 21 d (three control, six HP). All kidneys were extracted and examined grossly and for radiographic evidence of stones (GE CT scanner, 80kV, 400MA, 1 sec rotation, 0.625 mm slices). Papillary and cortical samples were processed for histologic analysis. RESULTS: Kidneys from treated sows showed significant calculi distributed within the renal papilla on CT, appeared mottled in the renal cortex and papillary areas, and had less distinct corticomedullary borders. Tiny crystals and mucinous debris lined the papillary tips, calices, and pelvis in kidneys from four of six treated sows, and multiple stones were noted at the papillary tips. Hematoxylin and eosin stain revealed crystals in collecting tubules and papillary tips in treated kidneys and none in controls. Yasue staining confirmed crystals in proximal periglomerular tubules of treated but not control animals. Tubular dilation and inflammatory/fibrotic changes were identified in kidneys from treated animals; none of these changes were evident in control kidneys. CONCLUSIONS: We report renal damage as a result of dietary-induced hyperoxaluria in adult sows. Specifically, we found crystalluria in proximal periglomerular tubules and collecting ducts, with tubular damage at all segments.

Effect of NADPH oxidase inhibition on the expression of kidney injury molecule and calcium oxalate crystal deposition in hydroxy-L-proline-induced hyperoxaluria in the male Sprague-Dawley rats.

BACKGROUND: Renal calcium oxalate (CaOx) crystal deposition is associated with epithelial injury and movement of inflammatory cells into the interstitium. We have proposed that oxalate (Ox)- and CaOx crystal-induced injury is most likely caused by reactive oxygen species (ROS) produced by activation of membrane nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. METHODS: Present study was undertaken to determine the effect of NADPH oxidase inhibitor apocynin on the expression of kidney injury molecule-1 (KIM-1) and renal CaOx crystal deposition in rats with hyperoxaluria. We also investigated the urinary excretion of KIM-1, osteopontin (OPN) and monocyte chemoattractant protein-1 (MCP-1) and renal expression of OPN and ED-1. Male Sprague-Dawley rats were fed a diet containing 5% hydroxyl-L-proline (HLP) and 4 mmol apocynin to drink for 28 days. Urine was collected on Days 7, 14, 21 and 28. After that, rats were sacrificed and their kidneys processed for various microscopic and molecular investigations. RESULTS: HLP consumption produced heavy deposits of CaOx crystals. Renal expression of KIM-1 and OPN and urinary excretion of KIM-1, OPN, H(2)O(2) and MCP-1 was significantly increased. ED-1-positive cells migrated into renal interstitium. Apocynin treatment caused significant reduction of crystal deposits, injured and dilated tubules; renal expression of KIM-1, OPN and ED-1 and urinary excretion of KIM-1, OPN, MCP-1 and H(2)O(2). Apocynin had no effect on the urinary excretion of Ox. CONCLUSIONS: This is the first study of urinary excretion and renal expression of KIM-1 in association with renal CaOx crystal deposition, experimental or clinical. The results indicate that NADPH oxidase inhibition leads to reduction in KIM-1 expression and urinary excretion as well as renal CaOx crystal deposition. KIM-1 is an important marker of renal epithelial injury. The results provide further support to our proposal that renal epithelial injury is critical for crystal retention and that injury is in part caused by the production of ROS with the involvement of NADPH oxidase.

Improved methodology to induce hyperoxaluria without treatment using hydroxyproline.

The use of hydroxyproline (HP) to generate hyperoxaluria in the rat is a problem because it is impossible to separate the effect of oxalate on renal injury from the effects of HP and the large array of metabolic intermediates formed when HP is converted to oxalate. Previously, the Dahl salt-sensitive (SS) and Brown Norway (BN) rat strains were studied to determine genetic control of resistance or susceptibility to HP-induced renal injury and crystal deposition. To develop a better model to induce hyperoxaluria without causing injury from HP metabolites, animals were fed a diet containing various levels of added oxalate (0, 1, 2, 3, or 5%). After 5 weeks rats were killed and the kidneys were removed for microscopic evaluation of tubule changes and crystal deposition. The 3 and 5% oxalate-fed groups had a substantial increase in urine oxalate, about 50 and 140 µmol/g body weight over controls, respectively. Both the SS and BN 3% oxalate-fed animals showed only slightly elevated tubule area and no crystal deposition. However, BN animals fed 5% oxalate had a dramatic increase in their percent tubule areas compared to control BN rats and treated SS rats. Crystal deposition in the kidneys was only observed in the 5% oxalate-fed groups. The BN kidneys demonstrated a threefold higher crystal deposition compared to oxalate-fed SS rats. We conclude that oxalate-supplemented food is a better method of producing hyperoxaluria in the rat than using HP which may introduce metabolic intermediates injurious to the kidney.

Hydroxyproline-induced hyperoxaluria using acidified and traditional diets in the porcine model.

INTRODUCTION: Swine models have proven useful for many different disease processes, especially for urologic research. In this study, we sought to create a model of hyperoxaluria in the adult sow by feeding hydroxyproline (HP). The development of an adult porcine model for calcium oxalate stone disease would represent a significant contribution to stone research as previous animal models have been developed only for rats and baby pigs. METHODS: The experiment included a total of 12 multiparous, gestating sows (Large White x Landrace). Sows were randomly allotted to one of the two treatment groups. Treatments involved basal diets that were either control diet (CD) or acidogenic diet (AD). Urine was collected for 6 consecutive days. On days 1 and 2, each sow was fed 2 kg of the assigned basal diet (CD or AD). On days 3, 4, and 5, 200 g of L-hydroxyproline (Wilshire Technologies, Princeton, NJ) was added to each basal diet for all the 12 sows. The HP was evenly mixed with the basal diets before feeding. On day 6, each sow was fed the basal diet originally assigned without HP ( Fig. 1 ). Urine was collected for each entire 24-hour period to control for differences in the diurnal and postprandial variations in the renal handling of oxalate and glycolate. RESULTS: The addition of HP to the diet increased urinary oxalate excretion. Overall, there was a 192% (CD) and 187% (AD) increase in urinary oxalate between days 1 and 3. The increase peaked on day 3 and gradually returned to baseline by day 6. Student's paired t-test was performed and it confirmed that oxalate on days 3 and 5 was significantly different than baseline (p = 0.009 and p = 0.03, respectively). Urinary glycolate also increased as a result of adding HP to the diet. Overall, there was a 12,340% (CD) and 14,400% (AD) increase in urinary glycolate between days 1 and 3. The increase peaked on day 3 and then declined, although remained more than 10 x greater than baseline at day 6. Student's paired t-test confirmed that glycolate levels on days 3, 5, and 6 were significantly different than baseline (p < 0.001, p = 0.01, and p = 0.03, respectively). CONCLUSION: The role of oxalate in the formation of kidney stones cannot be understated. Medical prevention and management of calcium oxalate nephrolithiasis will require a comprehensive understanding of oxalate metabolism in humans. A model for human hyperoxaluria can be reliably created in the adult sow. Such a model is necessary to further our understanding of oxalate metabolism and ultimately aid in the prevention of calcium oxalate calculi.

Dietary oxalate and calcium oxalate nephrolithiasis.

PURPOSE: Patients with calcium oxalate kidney stones are advised to decrease the consumption of foods that contain oxalate. We hypothesized that a cutback in dietary oxalate would lead to a decrease in the urinary excretion of oxalate and decreased stone recurrence. We tested the hypothesis in an animal model of calcium oxalate nephrolithiasis. MATERIALS AND METHODS: Hydroxy-L-proline (5%), a precursor of oxalate found in collagenous foods, was given with rat chow to male Sprague-Dawley rats. After 42 days rats in group 1 continued on hydroxy-L-proline, while those in group 2 were given chow without added hydroxy-L-proline for the next 21 days. Food and water consumption as well as weight were monitored regularly. Once weekly urine was collected and analyzed for creatinine, calcium, oxalate, lactate dehydrogenase, 8-isoprostane and H(2)O(2). Urinary pH and crystalluria were monitored. Rats were sacrificed at 28, 42 and 63 days, respectively. Renal tissue was examined for crystal deposition by light microscopy. RESULTS: Rats receiving hydroxy-L-proline showed hyperoxaluria, calcium oxalate crystalluria and nephrolithiasis, and by day 42 all contained renal calcium oxalate crystal deposits. Urinary excretion of lactate dehydrogenase, 8-isoprostane and H(2)O(2) increased significantly. After hydroxy-L-proline was discontinued in group 2 there was a significant decrease in urinary oxalate, 8-isoprostane and H(2)O(2). Half of the group 2 rats appeared to be crystal-free. CONCLUSIONS: Dietary sources of oxalate can induce hyperoxaluria and crystal deposition in the kidneys with associated degradation in renal biology. Eliminating oxalate from the diet decreases not only urinary oxalate, but also calcium oxalate crystal deposits in the kidneys and improves their function.