| 1. BackgroundDiabetic nephropathy (DN) is the leading cause of end-stage renal disease Tubulointerstitial inflammation is crucial in promoting the development and progression of DN. Interleukin-1(IL-1) family cytokines have an initiating role in early stages of inflammation in DN. They have direct relationships with many chronic complications of diabetes. Pro-IL-1β and pro-IL-18, the precursor molecules of IL-1family cytokines, rely on caspase-1shearing action to exert their biological effects. The NOD-like receptor3(NLRP3) inflammasome, the intracellular receptor of dangerous related molecular pattern (DAMP), can activate caspase-1, regulate IL-1β and IL-18maturation and secretion, and trigger the inflammation of metabolic diseases such as diabetes, obesity, and gout. NLRP3knock-out mice are protected from renal tubule damage and renal interstitial inflammation in kidney unilateral ureteral occlusion (UUO) and ischemia-reperfusion models. The NLRP3inflammasome is activated in kidney tissue of streptozotocin-induced diabetic rats, and both quercetin and allopurinol can significantly reduce renal tissue inflammation and improve renal function by blocking NLRP3inflammasome activation. These data show that the NLRP3inflammasome plays a key role in the process of kidney sterile inflammation. However, the mechanism of NLRP3inflammasome activation in DN is still not clear.Activated purinergic P2X receptors mediate endogenous DAMPs such as adenosine triphosphate (ATP), biglycan, and amyloid protein, activate the NLRP3inflammasome, and regulate aseptic inflammation in hypersensitivity, acute pancreatitis and cancer. P2X receptors are ligand-gated ion channel receptors in the P2family. There are seven subtypes (P2X1-7), which are expressed in the kidney with the exception of P2X3. Renal tubule epithelial cells mainly express P2X4and P2X6. ATP is a wide range P2X receptor agonist. Increased extracellular ATP (eATP) induced by hyperglycemia, can activate P2X7receptor of glomerular mesangial cell, and up-regulate TGF-β and extracellular matrix expression in diabetes. Hyperglycemia can also rely on eATP, and cause retinal cell caspase-1activation and IL-1β secretion. The role of P2X4in diabetic nephropathy is still undefined. The purpose of this study is to investigate the role of ATP-P2X4signaling in NLRP3inflammasome activation and renal interstitial inflammation of DN and provide theoretical foundation for analyzing the pathogenesis of renal inflammation and finding new treatments for DN.2. Methods2.1PatientsA total of45patients with type2diabetic nephropathy were recruited for this study from the Department of Nephrology in Daping Hospital from January1,2011to June1,2012. The enrollment criteria were as follows:patients were40-70years old with a history of type2diabetes;24h urine protein was above150mg; renal biopsy pathology led to a diagnosis of diabetic nephropathy; and no fever with obvious infection lesions or high uric acid. All patients used insulin to control blood glucose, angiotensin antagonist and CCB to control blood pressure, and statins to control lipids. Patients abstained from traditional Chinese medicine or sulfonylureas for three months after renal biopsy. Normal kidney tissues from nephrectomies of renal hamartoma were collected to be used in the control group.Serum total cholesterol, LDL cholesterol, SCr,24h urinary protein excretion was measured by biochemical analysis. P2X4, NLRP3, IL-1β and IL-18expression in renal tubule was detected by immunofluorescence and immunohistochemistry. The relationship of P2X4, NLRP3and urinary IL-1β and IL-18were analysed.2.2Cell cultureHK-2cells were cultured in Dulbecco’s modified Eagle’s medium with low glucose. The cells were seeded at1.5x106cells/10cm diameter dish for2-3days at37℃in a humidified atmosphere containing5%CO2. HK-2cells were grown in media with normal glucose concentration, or high glucose concentration, or high mannitol concentrations for48h. HK-2cells were incubated in media with high glucose (35mM,48h) with and without apyrase, P2receptor antagonist suramin, P2X receptor antagonist TNP-ATP and P2X4selective antagonist5-BDBD.ATP levels were determined using an ATP luciferase-based bioluminescence assay kit in a luminometer. Intracellular K+concentration was measured by PBFI-AM. Cytosolic free Ca2+was indicated by Fluo-3/AM. NLRP3protein expression, cleavage of caspase-1and IL-1β were analyzed by Western Blot. Release of IL-1β and IL-18were detected by ELISA.2.3P2X4siRNA transfectionTransfection control cells were transfected with siRNA for unrelated genes or control siRNA, while experimental cells were transfected with siRNA directed against P2X4, with all transfections utilizing Lipofectamine2000according to the manufacturer’s instructions. Cells incubated with Lipofectamine2000were also used as a negative control. P2X4knockdown was confirmed by Western blot and quantitative reverse transcription-PCR (qRT-PCR).3. Results3.1P2X4expression related with renal interstitial inflammation in DNP2X4and NLRP3expression was upregulated in tubular epithelial cells in patients with type2DN. P2X4immunostaining score, as well as NLRP3immunostaining score, was positively correlated with urine IL-1β and IL-18levels. P2X4expression was co-localized with NLRP3, IL-1β, and IL-18expression in the DN group.3.2High glucose-induced NLRP3inflammasome activation is extracellular ATP-dependentAfter stimulation of high glucose, NLRP3expression, cleavage of caspase-1and IL-1β, and release of ATP, IL-1β and IL-18were enhanced in a dose and time dependent way. Apyrase significantly inhibited NLRP3expression, cleavage of caspase-1and IL-1β, and release of IL-1β and IL-18caused by high glucose.3.3High glucose caused ATP-P2X4signaling activationHigh glucose cause intracellular calcium concentration increase, and potassium ion concentration reduce. Apyrase significantly inhibited potassium concentration reduction and calcium concentration increase under high glucose condition. Suramin, TNP-ATP,5-BDBD can significantly inhibit potassium concentration reducing caused by high glucose. Moreover, TNP-ATP,5-BDBD can restrain intracellular calcium concentration increasing cause by high glucose as well.3.4P2X4receptor mediated NLRP3inflammasome activation caused by high glucose Suramin, TNP-ATP and5-BDBD all obviously inhibited NLRP3expression, cleavage of caspase-land IL-1β, and release of IL-1β and IL-18with high glucose stimulus. P2X4gene silencing inhibited intracellular potassium efflux and extracellular calcium influx induced by high glucose. The increases in NLRP3expression, cleavage of caspase-land IL-1β, and secretion of IL-1β and IL-18were similarly blunted.4. ConclusionATP-P2X4signaling mediates high glucose-induced activation of the NLRP3inflammasome, regulates IL-1family cytokine secretion, and plays a privotal role in the development of tubulointerstitial inflammation in diabetic nephropathy. |
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