The Inhibitory Effect Of Apocynin On Melamine-related Urolithiasis And Its Related Mechanism

Background: In 2007, more than 4,000 dogs and cats that consumed melamine-contaminated feed showed renal failure in America. In 2008, due to long-term consumption of melamine-contaminated powdered infant formula, a total of 294,000 infants underwent examinations and treatment; of which 51,900 infants were hospitalized and 4 died, mainly because melamine causes urinary stones and renal failure. In 2008, the number of infants with urolithiasis treated in the outpatient department of our hospital was markedly increased. These children were typically less than 3 years of age. The symptoms in these children included fever, cough, cough phlegm, crying, nausea, diarrhea, vomiting, decreased or no urine output, and hematuria. Some children had cloudy urine or sediment in the urine. Ultrasound examinations were used to diagnose these children with urolithiasis. When we examined the medical histories of these children, we found that they had been fed the same brand of milk powder(infant formula produced by the Sanlu Group). Subsequently, the urology department of our hospital reported this issue to the Health Department of the Province in a timely manner. For this crucial social issue, we used epidemiological methods to study the risk factors of melamine-induced urinary calculi and obstructive acute renal injury in infants and explored clinical treatment programs including extracorporeal shock wave lithotripsy, surgical interventions, improved drug dissolution, and treatment of postrenal acute renal injury to establish a set of effective methods for the diagnosis and treatment of children with urolithiasis. We followed these children for 3 years and evaluated the safety and effectiveness of different treatments. We used physicochemical methods to determine the melamine content in the stones, plasma and urine specimen and qualitatively analyzed the stone form and composition. However, few domestic or international studies have focused on experimental research on melamine-related urinary stones and the establishment of cellular and animal models. Oxidative damage is considered an important mechanism of urinary stone formation. After establishment of cellular and animal models of melamine-related urinary stones, we examined the effects of the selective NADPH oxidase inhibitor apocynin on the two models to provide molecular biological evidence for apocynin treatment of melamine-related urinary stones.Objectives: After establishing cellular and animal models of melamine-related urinary stones, we measured oxidative stress(OS) markers, urolithiasis-associated protein expression, renal histopathological changes, crystallization, and nephrotoxicity. After prophylactic and therapeutic apocynin treatment, we assessed these indices to explore the pathogenesis of melamine-related urinary stones and to provide a theoretical basis for the pathogenesis of urinary calculi.Methods: In cellular experiments, we used a mixture of melamine and cyanuric acid(MCA)(600 μmol/L 1:1) to stimulate human kidney cortex proximal tubule epithelial cells(HK-2) to establish a cellular model of melamine-related urinary calculi. Then, we examined prophylactic or therapeutic administration of different concentrations of apocynin(0.1 or 0.2 mmol/L) on melamine and cyanuric acid-induced HK-2 cells. After a mixture of MCA was used to induce HK-2 cells for 24 hours, we collected cells and medium to detect OS markers including 8-isoprostane(8-IP), malondialdehyde(MDA), and superoxide dismutase(SOD). The urolithiasis-related proteins p-p38 and OPN were evaluated by Western blots.In animal experiments, a mixture of MCA(400 mg/day/kg; 1:1) was used to induce male SD rats to form melamine-related urinary stones.Apocynin(0.2 or 0.4g/kg/day) was prophylactically or therapeutically administered to rats with melamine-related urinary stones. At 28 days, after weighing the rats, we collected their urine, blood, and kidneys and measured the OS and nephrotoxicity(absolute kidney weight, relative kidney weight, urea nitrogen, and blood serum creatinine) levels. One kidney was fixed with 20% formaldehyde after removal for hematoxylin and eosin staining. Histological changes and crystal deposits in the kidney were observed under a light microscope. A laser-scanning confocal microscope was used to observe crystals in transparent tissue sections(without HE staining). The expression levels of the urolithiasis-associated proteins OPN and p-p38 were evaluated by immunohistochemistry and Western blots.Results: In the cell experiments, after induction with a mixture of MCA, the OS indicators and urolithiasis-associated protein expression levels of HK-2 cells were increased. Prophylactic apocynin treatment improved these indices(p<0.05). However, after therapeutic apocynin treatment of MCA-induced stone formation in HK-2 cells, the OS indicators were decreased, but the urolithiasis-associated protein expression levels were not significantly improved(p>0.05).In nephrolithic rats, the nephrotoxicity, histological changes, crystal deposits, OS indicators, and urolithiasis-associated protein expression levels were significantly increased(p<0.05). Prophylactic apocynin treatment improved these indices(p<0.05). However, after therapeutic apocynin treatment of nephrolithic rats, the OS indicators were reduced but the other indicators were not significantly improved(p>0.05).Conclusions: OS is an important mechanism of melamine-related urolithiasis.Apocynin treatment can be used to prevent melamine-related urolithiasis.