Endogenous Ouabain Protects Against Tubular Injury In Mice With Oxalate Nephropathy By Triggering Amphiregulin Release

Nephrolithiasis is one of the common obstructive nephropathies.There is a close connection between patients suffered one or more times of nephrolithiasis and the high risk of end-stage renal disease(ESRD).One of the major risk factors of renal dysfunction caused by nephrolithiasis is renal tubular epithelial cell injury.Calcium oxalate crystals are thought to be involved in renal tubular epithelial cell damage.Renal tubular epithelial cell injury will provide effective nucleation sites for early tiny crystals,thus triggering a cascade of stone formation.Therefore,elimination of the injury of renal tubular epithelial cells resulted from crystals may be a strategy for preventing the formation of calculus from the source,thereby alleviating renal dysfunction caused by oxalate nephropathy ultimately.Endogenous ouabain(EO)is an endogenous digoxin like substance(EDLS)found in mammalian tissues(hypothalamus,adrenal gland)and body fluids(blood,urine and cerebrospinal fluid).Traditionally,EO is believed involved in the regulation of the cardiovascular system.Recent studies have shown that EO is also associated with pregnancy and acute kidney injury after cardiac surgery.However,the relationship between EO and kidney stone disease has not been reported.This study showed that the level of serum EO was significantly elevated in mice with glyoxylate-induced oxalate nephropathy,suggesting a possible link between EO and progression of nephrolithiasis.To validate this hypothesis,EO specific antagonist rostafuroxin was administrated in mice with oxalate nephropathy,and the results showed that rostafuroxin can further aggravate renal dysfunction,calcium deposition in the kidney,renal tubules damage and kidney cell apoptosis caused by calcium oxalate.Similarly,bilateral adrenalectomy and ouabain Ab application in mice to eliminate the effects of EO can exacerbate renal dysfunction,calcium oxalate deposition and tubular damage caused by glyoxylic acid.The above results indicate that EO may have protective effects on renal tubular injury.To further investigate the mechanism of EO in renal tubular injury,the effects of exogenous ouabain on renal tubular injury caused by calcium oxalate were studied in renal tubular epithelial cell line HK-2 and mice with oxalate nephropathy.The results showed that exogenous low-dose ouabain alone exerted to promote proliferation of HK-2 cells,and it was able to reverse cell damage caused by calcium oxalate monohydrate.In oxalate nephropathy,exogenous low-dose ouabain was shown to alleviate renal dysfunction,the amount and distribution of calcium oxalate in the kidney,injury and apoptosis of kidney cells.However,the dose of exogenous ouabain used in this study did not produce significant side effects on cardiovascular systems of mice.Since exogenous low-dose ouabain exerted protective effects on cell damage induced by calcium oxalate and renal tubular injury in mice with oxalate nephropathy,what is the underlying molecular mechanism.In vitro and in vivo studies have shown that low-dose exogenous ouabain obviously promoted the expression and secretion of amphiregulin(Areg)in HK-2 cells and mouse kidney.On the contrary,calcium oxalate and glyoxylic acid treatment reduced Areg protein expression in HK-2 cells and mice kidneys respectively.Areg overexpression in mice evidently improved tubular damage in oxalate nephropathy.To further clarify the relationship between the protective effects of exogenous ouabain and Areg expression,mice with oxalate nephropathy were given a tail injection of Areg sh RNA adenovirus while receiving exogenous ouabain treatment.The results showed that Areg gene knockdown acted to antagonize the shielding effects of exogenous ouabain on renal tubular injury induced by calcium oxalate.The above results were also verified in vitro.Experiments in Areg knockout and bone marrow transplanted mice further confirmed that the protective effects of exogenous ouabain on renal tubular damage in oxalate nephropathy largely depend on the expression of Areg in renal parenchymal cells.Considering the nature of the secreted characteristic of Areg and the fact that exogenous ouabain promote the secretion of Areg,we investigated the role of secreted Areg in renal tubular injury induced by calcium oxalate.In HK-2 cells,using conditioned medium collected from the cells which Areg was interfered by si RNA and Areg neutralizing antibody to antagonize secreted Areg in the medium,we found that the improvement of calcium oxalate-induced cell damage of HK-2 by exogenous ouabain was dependent on the secreted form of Areg.In vivo studies,intraperitoneal injection of Areg neutralizing antibody to counteract the effects of secreted Areg in mouse body fluids can also antagonize the protective effects of exogenous ouabain on renal tubules in mice with oxalate nephropathy.At the same time,our studies demonstrated that exogenous ouabain activates Areg gene expression at a transcriptional level by promoting the binding of ATF2 and CREB to the promoter region of Areg(-117～-34 bp).Areg is one of the many ligands known for epidermal growth factor receptor(EGFR).This study showed that the protective effects of exogenous ouabain on renal tubular injury caused by calcium oxalate are associated with its effects on the promotion of Areg expression and secretion.Subsequently,the secreted form of Areg binds to the EGFR and activates the EGFR/ERK signaling pathway as an autocrine or a paracrine form,thereby promoting cell proliferation and triggering self repair of renal tubular injury caused by calcium oxalate.Furthermore,low-dose exogenous ouabain can also alleviate renal dysfunction,tubular damage in mouse models of acute kidney injury caused by cisplatin and glycerol.Moreover,it can effectively improve weight loss,renal dysfunction,tubular injury and renal interstitial fibrosis in diabetic mice.Summary of the full text,EO can act as a hormone like substance to sense external stimuli in the condition of kidney stones.When deposited in the kidney,calcium oxalate crystals cause damage to the renal tubules and promote secretion of EO from adrenal cortex.Subsequently,the released EO promotes the expression of Areg protein in the renal tubular epithelial cells and its secretion to extracellular regions,thus binding with the EGF receptor on the cell or adjacent cell membrane in an autocrine or paracrine manner,subsequently activating the EGFR/ERK signaling pathway.Activation of this pathway will promote the proliferation of renal tubular epithelial cells to resist outward stimuli and start the self repair process after injury,thereby reducing renal tubular damage caused by calcium oxalate,which also exists in other acute and chronic renal injuries that can cause tubular damage.Ouabain and its chemical analogues have the potential to be developed as an effective treatment for tubular injury caused by nephrolithiasis.