| Objective: Wnts compose a family of signaling proteins that play an essential role in kidney development, but their expression in adult kidney is thought to be silenced. Aberrant regulation of Wnt signaling has been implicated in the pathogenesis of many human diseases in diverse types of tissues. Acute renal failure (ARF) is a common disease. Many disparate etiologies can lead to ARF via a mechanism involving reduced total or segmental renal blood flow with resultant ischemic injury. The damaged kidney epithelium, however, in contrast to heart or brain, can be completely restored in structure and function.Despite the kidney has great capcity for self-repair, many patients with acute kidney injury are left with chronic kidney disease (CKD)or end atage kidney disease(ESRD) due to failure of repair mechanisms.However, progress in understanding the mechanisms of disease progression, we are still left with limited therapies or only supportive treatment in the management of kidney disease in humans.It's a highlight spot to investigate the mechanism and new therapies of AKI, to counteract the progression of CKD. Here, We plan to observe the expression and regulation of Wnt canonical signaling and their agonists in normal and postinjury kidney after ischemina reperfusion injury, to analyze how indicating activation of the canonical pathway of Wnt signaling affect a repairment following IRI. The research will reveal the discovery that a critical role of hyperactive Wnt/β-catenin signaling in the pathogenesis of repair postinjury kidney and present a novel target for therapeutic intervention of acute kidney diseases.Methods:(1) Transgenic mice were identified by analysis of genomic DNA from tail biopsies by PCR. We induced unilateral ischemia reperfusion acute kidney injury (IRI)in BATgal Wnt pathway reporter mice and sacrified mice on 0hour, 3th or 5th day and collected kidneys and blood.To understand the changes of location and expression of Wnt/β-catenin signaling, wholemount or frozen section was detected by X-gal staining. BAT-gal expression identifies novel sites of Wnt signaling.(2) We made IRI in wild type C57BL/6 (WT) mice and collected kidneys and blood on day 2 and 5th,7th day after injury compare with 0 hour. The kidney can be assessed functionally by measuring plasma creatinine in ELISA. Renal pathological changes were measured by PAS stain; and kidney injury can be assessed quantitatively using an injury score. The protein expressions of the Wnt4 and co-receptor Lrp6 were assessed by Western blot. This C2 recombinant form of Dkk2 was administered from 12hours to 48hours to WT mice which had kidney injury; Kidneys were harvested on day 7 after injury and assessed quantitatively for injury and the level of pLrp6 in cortex.(3) We induced unilateral ischemia reperfusion acute kidney injury (IRI)in BATgal Wnt pathway reporter mice and sacrified mice on 3th or 5th day and collected kidneys. To understand which cells respond to activated Wnt/β-catenin signaling and to konw the sources and localization of Wnts in vivo, we use specific and workable antibodies to detect many types cell and Wnt signaling in immunohistochemical&immunofluorescence co-stainning studies, as X-gal/CD31(marker of endothelium),X-gal/PDGFβ(marker of pericytes),X-gal/α-SMA(marker of fibroblast/smooth muscle cells),X-gal/F4/80(marker of macrophages),X-gal/LTL(marker of proximal tubule),X-gal/NKCC2(marker of henle loop),X-gal/DBA(marker of collecting duct) respectively.(4) The study population included hospitalization patients who underwent biopsy of kidney had minimal change disease(MCD) or MCD&ATN(acute tubular necrosis). All patients admitted in the study were taken blood for measuring serum creatinine and clinical characteristics were investigated. Paraffin sections of kidney tissue were stained by PAS, srius red to assess the degree of tubule-interstitial injury. We also detected Wnt4 andβ-cateninin proteins in immunohistochemical co-stainning studies; proliferation and apoptotic changes were identified by staining with Ki67, Tunel in immunofluorescence study.Results:(1) Wholemount images of adult kidneys of BAT-gal mice show lacZ staining restricted to the papilla of the normal adult kidney but injury induced Wnt activity in both cortex and medulla of kidneys on d3 and d5 during repair, following injury. The control WT kidney stainedidentically shows minimal staining. It noted an injury-induced enhancement of the Wnt pathway response.(2) According to injury scores and creatinine assays, both of which are improving by 48 h, the phase of kidney repair begins on day 2 PAS-stained kidney sections showed injured tubular epithelia: desquamative or flattened epithelia,necrotic debris; and regenerating tubules on day7.Similar injury-induced enhancement of the Wnt pathway response was noted by detecting Wnt4 protein and phosphorylation of the canonical pathway receptor Lrp6, but was not detected in uninjury adult kidney. When systemic delivery of Dkk2, Wnt signaling in kidney cortex of day 5 post-I/R mice was assessed by detection of phosphorylated Lrp6 because canonical signaling requires phosphorylation of this coreceptor. pLrp6 was not detected in healthy adult kidney cortex but was robustly detected in kidney cortex following injury. At times piont, tubule injury scores were more severe in vehicle-treated mice.(3) X-gal staining imagines of kidney sections from injured BATgal mice on d2 revealed that Lacz was detected in kidney out-medulla and cortex, principal proportion of interstitial cells. By contrast, 5 days following injury there was marked upregulation of X-gal staining in both out-medulla and cortex. Double labeling of immohistological sections of injured kidney, Lacz was detected in kidney epithelial cells of proximal tubule and henle hoop, but was not detected in collecting duct; furthermore confirmed that interstitial cells, including endothelial cells, pericytes,and vascular smooth muscle cells but not macrophages and frobioblast showed a Wnt pathway response. The number ofβ-gal positive endothelial cells or pericytes within the cortex or the outer medullary region in 5 days postischemic kidneys was scored increased, but in d0 normal kidney Lacz-positive endothelial cells and pericytes was not seen.(4) Of the 40 patients admitted to observe, and 18 cases were males.MCD&AKI were diagnosed in 20 cases. The mean age was 31±12 years in MCD,38±15 years in MCD&AKI.PAS-stained kidney sections images from pateints with MCD and ATN were seen regenerating tubules, injured flattened epithelia, necrotic debris. Kidney tubular injury score markedly increased in MCD&ATN compared with MCD but not ATN(17.15% vs 3.84% P<0.01). It showed the significant difference in the apoptosis response(16.52±9.18 vs 5.14±3.28 P<0.001) and interstitial fibrosis area(1.39±1.25% vs 0.12±0.09% P<0.05),but the proliferation showed a modest but statistically significant persistent(32.18±19.82 vs 30.09±12.81 P>0.05). The area of theβ-catenin responses and Wnt4 protein was statistically significant persistent, and clearly correlation each other. They were correlation with the repair parameter of kidney injury.Conclusion: (1) In adult kidney, Wnt/β-catenin signaling only showed prominently in the papilla, but the cortex and medulla showed almost no staining. By contrast, IRI led to activation of the canonical pathway of Wnt signaling, there was marked upregulation of X-gal staining in both cortex and out-medulla.(2) The day 5–7 postinjury enhancement of the Wnt response suggested that theWnt pathway could be a component of the injury repair mechanism.Therapy with Dkk2 was functioning to augment the canonical pathway, therefore, Dkk2 functions to enhance canonical Wnt responses in regenerating kidney cortex.(3) Epithelial cells in post-injury kidney proximal tubule and henle hoop, but was not in collecting duct, showed a Wnt pathway response; interstitial cells as endothelial cells and pericytes but not macrophages are Lacz-positive. Epithelial cells are responsible for repairment after kidney injury together with endothelial cells, pericytes, macrophagesand vascular smooth muscle cells.(4) In human acute renal tubular necrosis, similar mechanism of IRI from rodent mice, indicating activation of the canonical pathway of Wnt signaling was connect with repair of acute injury kidney.
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