| Part Ⅰ Intravoxel Incoherent Motion Diffusion-Weighted Imaging for Noninvasive Assessment of Renal Fibrosis in a Rat Model of doxorubicin-induced NephropathyObjective(s):To investigate the potential of intravoxel incoherent motion diffusion-weighted imaging(IVIM-DWI)in assessment of renal histopathological changes observed during renal fibrogenesis in a rat model of doxorubicin nephropathy(DN).Methods:Thirty-five(n=7)adult male Sprague-Dawley(SD)rats recruited in this study underwent baseline MRI followed by injection of adriamycin,and then follow-up MRI on weeks 1,3,5,and 8 after molding.Hematoxylin and eosin,periodic acid-Schiff(PAS),and masson’s trichrome staining was performed to evaluate the severity of the glomerular injury and tubulointerstitial lesions,and area of fibrosis of each rats.IVIM parameters of renal cortex and medulla were measured.Changes in each parameter with time were analyzed and correlated with pathological damage and histopathological fibrosis scores.Results:1.There was a significant tendency for the average apparent diffusion coefficient(ADC),true diffusion coefficient(D),perfusion fraction(f)values of renal cortex(CO),outer medulla(OM)and inner medulla(IM)on the DN to increase at week 1 and 3 and to derease at week 5 and 8(P<0.05).ADC,D,f values had a marked increase at the 3 week,there was a significant increase in the three values when compared with the values of baseline(P<0.0125)except the f value in OM.The changes of the three values in other time didn’t achieve statistical significance when compared with the values of baseline(P>0.0125).Pseudo diffusion coefficient(D*)values tended to be fluctuant with time(P>0.05).2.There was a continuous increase in the area of fibrosis in DN(P<0.001).There was a significant difference in the area of fibrosis with time(P<0.005)except the area of fibrosis between the 5 week and 8 week.3.There was a continuous glomerular injury and tubulointerstitial lesions,and increase in the area of fibrosis in DN(P<0.001).The glomerular injury and tubulointerstitial lesions appeared since week 3,and was progressively aggravated at the week 5 and week 8(P<0.005).4.Spearman correlation analysis revealed that there were negative correlations between partial of the IVIM parameters and glomerular injury,tubulointerstitial lesion and area of fibrosis(P<0.05).Negative correlations were found between f of CO and glomerular injury(r=-0.468,P<0.001),between f of CO and tubulointerstitial lesion(r=-0.452,P<0.001),between f of CO and area of fibrosis(r=-0.406,P<0.001).Conclusion(s):1.IVIM-DWI parameters shows a sensitive biomarker for changes in difusion and microperfusion of DN,which has a great potential in noninvasive assessment of renal pathology of DN;2.IVIM-DWI might be dynamically feasible for noninvasive evaluation of renal pathology and fibrosis of CKD,especially in detection of renal fibrosis at an early stage.Part Ⅱ Intra-arterial Renal Infusion of Bone Marrow-derived Mesenchymal Stem Cells inhibit TLR4-NF-κB-NLRP3-mediated Cell Death-Pyroptosis in Doxorubicin Nephropathy RatsObjective(s):Doxorubicin nephropathy(DN)is a well-known model of chronic kidney disease(CKD),Doxorubicin(Dox)-induced nephrotoxicity involves increased oxidative stress,apoptosis and necrosis.But it remains unclarified whether Dox induces the specific inflammatory-mediated form of cell death called pyroptosis in DN.And Bone marrow-derived mesenchymal stem cells(BMMSCs)have therapeutic potential in improving the structure and function of DN.However,whether BMMSCs regulate pyroptosis in DN is also not clear.Our study was undertaken to explore whether doxorubicin(Dox)induces pyroptosis and determine the potential of bone marrow-derived mesenchymal stem cells(BMMSCs)in inhibiting pyroptosis in a rat kidney model in vivo.Methods:Forty adult male Sprague-Dawley(SD)rats were randomized into four groups(n=10/group):(1)doxorubicin nephropathy(Dox)group rats received doxorubicin nephropathy and treated with phosphate buffer;(2)mesenchymal stem cells(BMMSCs)group rats received doxorubicin nephropathy and received a single injection of BMMSCs intra-arterial infusion via right renal artery via a carotid arterial cut-down approach after eight weeks from second doxorubicin administration;(3)control(Con)group rats received equivalent DEME medium treatment;and(4)normal(Nor)group rats received no treatment.For each group,biochemical examination from blood and urine samples(including blood urea nitrogen,serum creatinine and proteinuria).and morphologic analysis(hematoxylin and eosin,periodic acid-Schiff,masson trichrome and transmission electron microscopy included)were assessed.Blood samples and renal specimen were collected for enzyme-linked immunosorbent assay(ELISA).Renal specimen were collected for western blot analysis(WB)and confocal immunofluorescence.ELISA were performed to detect proinflammatory cytokines(TNF-α,IL-1β,IL-18,MCP-1 and IL-6),profibrotic cytokines TGF-β1,α-SMA and VEGF.The expression of TLR4,NF-κB,NLRP3,caspase-1,TNF-α,MCP-1 and TGF-β1,alpha-smooth muscle actin(a-SMA)were measured by WB.Results:1.Dox group rats showed a significant increase in Scr,BUN and 24h proteinuria compared with normal and control group.Administration of BMMSCs significantly decreased Scr,BUN as compared with Dox group(P<0.05)except for 24h urine protein content(P>0.05).The Scr,BUN and proteinuria levels in normal group rats were not significantly different from control group rats(P>0.05);2.Kidneys from normal and control group did not exhibit glomerular tubulointerstitial injury.Compared with these two groups,staining with H&E demonstrated that the Dox group presented with serious renal injury,as evidenced by glomerular sclerosis,thicked glomerular basement membrane,dilated tubular lumen,tubular atrophy,tubulointerstitial fibrosis and serious infiltration of inflammatory cells,while compared with Dox rats,renal injury was moderate in BMMSCs-treated rats.Consistently,PAS staining indicated a marked decrease of PAS reaction in the brush border of some tubules,and a marked increase basement membranes of renal tubules and glomerular capillaries in Dox group,whereas increase of PAS reaction in the brush border of some tubules and decrease of PAS reaction in basement membranes of renal tubules and glomerular capillaries was detected in the Dox+BMMSCs group.MT staining also showed large fibrotic areas among the glomerular capillaries and in the interstitium in the Dox group,whereas deposition of collagen fibers was decreased distributing in the BMMSCs group;3.Ultrastructural results from the renal tissue in normal and control group showed the proximal and distal convoluted tubular cells showed euchromatic rounded nuclei,apical pinocytotic vesicles and luminal closely packed microvilli,mitochondria appeared elongated and lodged within regular basal infoldings,podocytes with euchromatic nuclei,primary and secondary foot processes,and glomerular blood capillaries were lined by fenestrated endothelium.Whereas,Dox-treated group showed thickening of both glomerular basement membranes,a podocyte with thickening of its foot processes and extensive confluent foot processes of the podocytes,numerous swollen mitochondria,irregular basal infoldings and multiple electron dense bodies was noticed.The Dox+BMMSCs group revealed partly regularly arranged primary and secondary processes of podocytes,most of the proximal and distal tubular cells having euchromatic nuclei,and partial elongated mitochondria within regular basal infoldings were also observed;4.Quantitative ELISA data revealed a significant increase(P<0.05)in the serum level of IL-1β,IL-18 and IL-6 in the Dox group versus normal and control group.And quantitative ELISA data of renal tissue revealed a significant increase(P<0.05)in the level of IL-1β,IL-18,TNF-α,IL-6,MCP-1,TGF-β1,α-SMA and VEGF in the Dox group versus normal and control group.Moreover,the above cytokines showed significant decrease(P<0.05)upon BMMSCs administration treatment as compared with Dox group(P<0.05),TNF-α excepted(P>0.05);5.WB data showed a significant increase in protein expression of TNF-α,MCP-1,and TGF-β1,α-SMA in Dox group and a significant decrease(P<0.05)upon treatment with BMMSCs as compared with Dox,However,no significant changes have been reported upon control group versus normal group;6.Immunofluorescent staining results revealed higher expression of TLR4,NLRP3 and caspase-1 in Dox rats compared with the normal and control groups;while a lower expression of TLR4,NLRP3,and caspase-1 in the BMMSCs group compared with Dox.To verify staining results,WB analysis showed that expression of TLR4,NF-κB,p NF-κB,NLRP3,caspase-1 and gasdermin-D was significantly increased(P<0.05)in the Dox rats as compared with the normal and control groups;while a significant decrease(P<0.05)in TLR4,NF-κB,p NF-κB,NLRP3,caspase-1 as well as asdermin-D protein expression upon treatment with BMMSCs as compared with Dox.Conclusion(s):1.Doxorubicin nephropathy may be achieved by pro-inflammatory role of doxorubicin through TLR4-NF-κB-NLPR3-mediated pyroptosis;2.BMMSCs represent a relevant therapeutic potential against doxorubicin-induced renal damage.3.BMMSCs may promote kidney repair by attenuating pyroptosis cell death by downregulating TLR4-NF-κB-NLRP3 inflammasome signaling pathway. |
