Multiple Cytokine Receptors Modified By FUT8 Involved In Tubule-microvascular Crosstalk Of Renal Interstitial Fibrosis In Proteinuria Model

Persistent proteinuria is one of the most common symptoms of chronic kidney disease(CKD)that contributes to progressive renal interstitial fibrosis(RIF)and final renal function failure.Unfortunately,a considerable proportion of patients have persistent proteinuria throughout CKD.Thus,prevention of RIF induced by proteinuria should be examined.Emerging evidence has shown that microvascular injury is a key event in promoting RIF progression.However,few studies have investigated how proteinuria causes injury to the peritubular microvessels.This may be partly due to the unavailability of a suitable experimental model.Conventional nonfluid-state cell culture models fail to reproduce the structural and functional characteristics of the renal interstitial microenvironment.System-level analysis of renal interstitial disease mechanisms largely relies on animal studies.However,animal studies have failed to monitor the process of proteinuria-induced peritubular microvascular injury in real time and obtain detailed intercellular cell communication in animal models.Recent technological advances have led to a considerable progress in the field of biomimetics.Microfluidic organ-on-a-chip models have successfully monitored spatially and temporally the physiological and pathological processes under biomimetic flow conditions.In our previous study,we established a functional renal tubule-on-a-chip model.We used it to illustrate the transition of human proximal tubule cells to myofibroblasts and their real-time migration into the interstitial space.However,peritubular microvessels were not included in the previous device,which was an indispensable structure of renal interstitial microenvironment.Therefore,in the current study,we established a microfluidic device that emulates the microenvironment consisting of renal tubules and peritubular microvessels,allowing the successful coculture of PTECs,ECs,and pericytes under microfluidic conditions.A flow of non-inactivate healthy human serum(HHS)was added to the renal tubule channel to simulate human proteinuria and to explore the changes in ECs and pericytes in the peritubular microvascular channel.Our previous studies observed that FUT8-catalyzed core fucosylation(CF)could modify the transforming growth factor β receptors(TGF-βRs)and platelet-derived growth factor receptors(PDGFRs)and diminish FUT8-attenuated pericyte activation and renal fibrosis in a unilateral ureteral obstruction(UUO)rodent model.Thus,we aimed to investigate the role of FUT8 in the progression of proteinuria-induced RIF in our chip and in FUT8 knockout heterozygous mice.Objective: 1.To design and construct a bionic renal tubule-interstitium-peritubular tubular microvessel microfluidic chip;2.to explore the injury effect of proteinuria on peritubular vessels based on the microfluidic chip of "renal tubulointerstitial and peritubular vessels".3.To elucidate the mechanism of proteinuria induced peritubular vascular injury;4.To explore and clarify the regulatory role and mechanism of core fucosylation modification in the process of perivascular injury caused by proteinuria.Method: 1.The microfluidic chip template was prepared by soft etching technology,and the irreversible sealing method was used to process PDMS chips.The renal tubular epithelial cells,vascular endothelial cells and perivascular cells were co-cultured to construct a high-throughput bionic "renal tubule-interstitium-perivascular microvessel" microfluidic chip.Optical inverted microscope was used to observe the growth status of cells in the chip,and transmission electron microscope was used to observe the intercellular junction,and immunofluorescence was used to characterize the cell biomarkers;FITC-albumin and FITC-glucose were used to incubate renal tubular epithelial cells to investigate the albumin and glucose reabsorption functions of renal tubular epithelial cells.2.Based on the renal tubule-interstitium-microvessel microfluidic chip,using 5% healthy serum stimulus renal tubular epithelial cells for 24 h,48 h to build proteinuria model.Using immunofluorescence to investigate the cell biomarkers of renal tubular epithelial cells,endothelial cells and pericytes respectively;TUNEL staining was used to investigate the apoptosis of three kinds of cell;laser confocal microscope was used to observe the migration of three kinds of cells which were labeled with live cell dye.3.Human cytokine antibody arrays were used to detect the cytokines of medium from renal tubule and vascular channels.Furthermore,we screened out the key cytokines whose receptors were modified by core fucosylation.Immunofluorescence was used to test the expression of corresponding receptors of the key cytokines in vascular endothelial cells and pericytes;Proximity ligation assay was used to test the combination of the secreted proteins derived from epithelial cells and their corresponding receptors in endothelial cells and pericytes.4.FUT8 si RNA was used to knock down the expression of FUT8 of vascular endothelial cells and pericytes in vitro and reduce the level of core fucosylation,immunofluorescent staining was used to observe that the effect of FUT8-inhibition on the paracrine between renal tubule and peritubular microvessels and the downstream pathways of receptors in endothelial cells and pericytes.the effect of apoptosis.In vivo,FUT8+/-mice were used to verify the down-regulated effect of FUT8-inhibution on renal interstitial fibrosis and peritubular vascular injury induced by intraperitoneal injection of bovine serum albumin,as well as the regulation of the paracrine between renal tubules and peritubular microvessels.Results: 1.We designed a microfluidic chip consisting of eight renal tubule-vascular units to mimic the intricate structure and function of the renal tubules and peritubular microvessels.The media for tubules and vascular channels were injected through the inlets from the top PDMS layer to each unit in the second layer,flowed into the fusion channels,and were then collected from the respective outlets.Three layers were stacked vertically from the bottom to the top layer.PTECs,ECs,and pericytes grew well in the microfluidic chip and formed cell-cell junctions between two adjacent cells.Cellular biomarkers for PTECs(E-cadherin),ECs(CD-31),and pericytes(PDGFRβ)were then observed.Large cell populations exhibited a significant increase in the intensity of FITC-HSA or FITC-glucose after exposed to(FITC)-labeled human serum albumin(HSA)and FITC-labeled glucose.The FITCconjugated dextran diffused into the Matrigel and permeated from the tubule channel to the peritubular microvascular channel.2.PTECs showed time-dependent myofibroblast-like morphological changes,increased expression of myofibroblast markers(α-SMA),decreased expression of epithelial marker(E-cadherin),and migration into the Matrigel channel.PTECs displayed a significant increase in cell apoptosis following stimulation with 5% HHS.ECs and pericytes exhibited dramatic myofibroblast-like morphological changes.Furthermore,ECs depicted an increase in vimentin expression and a decrease in CD31 expression,while pericytes displayed a significant increase in α-SMA expression.These were in accordance with the morphological data.ECs and pericytes then migrated to the Matrigel as early as 24 h after HHS stimulation.The migration distance and number of pericytes were higher than those of ECs.ECs and pericytes also exhibited increased apoptosis.3.Cytokines such as VEGF-D,b FGF,TGF-β1,IFN-γ,Pl GF,leptin,IL-8,EGF,MCP-1,TIMP-1,PDGF-BB,TIMP-2,IL-6,ENA-78,and GRO were increased in the media collected from the tubule channels in the 48-h HHS stimulation group compared with that of the control and 24-h HHS stimulation groups.The corresponding receptor expression of cytokines in ECs and pericytes were observed and found that TGFβR,VEGFR1,integrin β1,and integrin α3β1 were expressed in ECs,while TGFβR,PDGFRβ,integrin β1,and integrin α3β1 were expressed in pericytes after HHS stimulation.significant combinations between the ligands and their receptors were observed in situ via the proximity ligation assay,which examined TGFβ-TGFβR1,PBGF-BB-PDGFRβ,Pl GF-VEGFR1,TIMP-1-integrinβ1,and TIMP-2-integrin α3β1.4.After FUT8 knockdown in ECs and pericytes,we observed that the ligand-receptor binding ability was reduced.We then investigated the downstream pathways of these receptors and detected that the levels of phosphorylated Smad2/3 and Erk1/2 in ECs and pericytes were significantly decreased in response to the FUT8 knockdown compared with those of the HHS treatment group.Our results revealed that there was an increase in the expression of CD31 and a decrease in the expression of vimentin in FUT8 knockdown group compared with their corresponding values in the HHS stimulation group.Similarly,α-SMA expression was downregulated in pericytes upon FUT8 knockdown compared with its corresponding value in the HHS stimulation group.Reduced apoptosis of ECs and pericytes was also observed after FUT8 knockdown.Conclusion: 1.We designed and constructed a biomimetic multiple-unit integrated tubule-vascular on-chip;2.Serum in renal tubules not only causes renal tubules injury,but also induces the injury of peritubular vascular endothelial cells and pericytes;3.Serum in renal tubules induced peritubular microvessel injury through a paracrine network;4.FUT8 could alleviate proteinuria-induced peritubular microvascular injury by disabling core fucosylated receptors and by blocking the complex paracrine network between renal tubules and peritubular microvessel.