Clinicopathological Study On Correlation Between Slit2/Robo1Signaling Pathway And Glomerular Vascular Lesion In Henoch-Sch?Nlein Purpura Nephritis

Background and objective:Vasculitis is the main pathological feature of Henoch-Sch?nlein purpura nephritis(HSPN).Glomerular vasculopathy is essential for the pathogenesis and progression of HSPN.Therefore,accurate grading of HSPN glomerular vasculopathy will be of great help in the treatment options and prognosis of the disease.The classification of HSPN is still based on the 1974 International Study of Kidney Diseases in Childhood(ISKDC)pathological grading standard,which is based on the extent of mesangial hyperplasia,crescent formation and membrane proliferative lesions.Although the Nephrology Group of the Pediatrics Branch of the Chinese Medical Association in 2000 made a slight modification on this basis,which glomerular segmental lesions(hardening,adhesion,thrombosis,necrosis,etc.)were added in addition to the crescentic lesions in the grade III,none of the above methods can fully reflect the degree of glomerular vascular disease in HSPN,because vascular damage is an important cause of the progression in HSPN.The ISKDC classification criteria have a limited role for predicting clinical prognosis of HSPN.Further improvement of ISKDC classification,for example,adding important grading parameters such as vascular endothelial cell damage and inflammatory cell infiltration,will help to improve the accuracy of pathological grading and clinical prognosis.Recent studies have found that the Slit2/Robo1 signaling pathway plays an important role in regulating vascular inflammation and angiogenesis.As the most popular Slit protein,Slit2 and its cognate receptor Robo1 have been detected in human glomeruli,renal tubules and renal interstitium.Activation of Slit2/Robo1 signaling promotes endothelial cell migration and inhibits chemokine-induced leukocyte infiltration,inhibits platelet aggregation,and prevents thrombosis.Previous studies have suggested that in the inflammatory model of rapidly progressive glomerulonephritis,external administration of Slit2 can effectively reduce leukocyte infiltration,crescent formation and improve renal insufficiency,suggesting that Slit2 may be an inhibitor of vascular inflammation.We have previously discovered that the Slit2/Robo1 signaling pathway is involved in early abnormal angiogenesis in diabetic nephropathy,but whether it is also involved in vascular inflammation and angiogenesis after vascular injury in HSPN has not been reported.The study firstly analyzed the pathological features of HSPN glomerular vascular lesions by collecting clinical data and renal histopathological specimens of HSPN patients,to establish a glomerular vascular lesion percentage scoring system(GVLPS),and to explore the correlation of GVLPS grading and ISKDC grading,and the relationship between GVLPS grading and 24h urinary protein quantification,glomerular filtration rate(eGFR),urinary red blood cell count and serum albumin(ALB),to understand the effectiveness of the GVLPS.Then we analyzed the relationship between Slit2/Robo1 signaling protein and degree of inflammatory cell infiltration by investigating the localization and expression of the Slit2/Robo1 signaling pathway in HSPN glomeruli,and the feature of inflammatory cell infiltration(CD20 positive cells and CD68 positive cells)in HSPN glomeruli,to determine whether the Slit2/Robo1 signaling pathway is involved in the development of glomerular vasculopathy in HSPN,and propose a new mechanism for the development of HSPN.Methods:The research method of this subject consists of two parts:Part ?:A preliminary study on the pathological grade of glomerular vascular lesions in HSPN.1.Clinical and renal histopathological data of 140 patients diagnosed HSPN with typical skin purpura,hematuria and duration of less than one year,from the Department of Nephrology,Xinqiao Hospital,Army Medical University,were collected.Twenty renal histoma resections or renal tumor resections away from the lesions of renal tissue specimens were used as a control group.2.Renal tissue specimens were stained with HE,PAS and Masson,and immunofluorescence staining of IgA,IgG,IgM,etc,and standard electron microscopy sections were made.The histopathological features of all kidney specimens were observed by light microscopy and transmission electron microscopy,and the kidney specimens were classified according to the ISKDC classification standard.3.The GVLPS(Glomerular Vascular Lesion Percentage Score)grading standard is established.The GVLPS is defined as:endothelial cell proliferation,hyalinosis,cellulose necrosis,subendothelial hyaline deposits,cellular crescents,fibrous crescents,segmental sclerosis or global sclerosis/number of total obtained glomeruli×100(%),and classified into histopathological grades(HG):HG1(<25%),HG2(?25%,<50%),HG3(?50%,<75%)and HG4(?75%,?100%).20 case of normal renal tissues were assigned as control groups.4.Correlation of GVLPS with ISKDC pathology classification,24h proteinuria,glomerular filtration rate(eGFR),urinary red blood cell counts and serum albumin(ALB)were analyzed.Part ?:A study on correlation between Slit2/Robo1 signaling pathway and glomerular vascular lesion in HSPN1.Immunohistochemical staining of Slit2 and Robo1 was implemented in renal tissue specimens,and the localization and expression of Slit2 and Robo1 were observed in glomeruli of HSPN.2.Immunohistochemical staining of CD20 and CD68 was implemented in renal tissue specimens,and the localization and expression of CD20 and CD68 were observed in glomeruli of HSPN.3.The relationship between Slit2/Robo1 expression and CD20~+/CD68~+expression was analyzed.Results:Part ?:1.From the control group to the HG1,HG2,HG3,and HG4 group,the serum creatinine,blood urea nitrogen,24h proteinuria,and urinary red blood cell counts of the patients were gradually increased with the increase of HG levels,and eGFR,serum albumin(ALB)gradually decrease as the HG grade increases.2.Histopathological examination showed that the main glomerular vascular pathological lesions of HSPN were endothelial cell proliferation,hyalinosis,cellulose necrosis,subendothelial hyaline deposits,cellular crescents,fibrous crescents,segmental sclerosis and global sclerosis,etc,IgA and IgM mesangial deposition were also discovered.And there was a gradual increase in the numbers of each vascular lesions from HG1 to HG4 group.3.There was an positively correlation between GVLPS and ISKDC grade,and GVLPS was positively correlated with 24h proteinuria and urinary red blood cell counts,but negatively correlated with eGFR and serum albumin.It was found that the GVLPS grade could better reflect the degree of vascular lesions,and its warning effect of disease severity was better than the ISKDC classification by analysis.Part ?:1.It was showed that the expression of Slit2 and Robo1 were discovered in glomeruli by immunohistochemistry.Slit2 was predominantly expressed in mesangial cells,endothelial cells and renal tubular cytoplasm,and Robo1 was mainly expressed in glomerular endothelial cells and renal tubular cytoplasm.Compared with the control,the expression of Slit2 was significantly increased from HG1 to HG2 group,and gradually decreased in HG3 and HG4group,and the HG4 group was less than the control group.The expression of Robo1 was significantly higher in all HG groups than in the control group.Similar to Slit2,Robo1expression increased from HG1 to HG2,and decreased in HG3 and HG4.2.The immunohistochemical results of CD20 and CD68 were analyzed.It was found that the expression of CD20~+and CD68~+cells in the control group were rare in glomerular,renal tubule and renal interstitial,while in the HSPN group,CD20~+and CD68~+cells were mainly expressed in capillary lumen and glomerular mesangial area,and the number of glomerular CD20~+and CD68~+cells significantly increased compared with the control group.The number of CD20~+cells was gradually increased from HG1 to HG3,but compared with HG3,the expression of CD20~+cells in HG4 was significantly reduced.Similar to CD20~+cells,the number of CD68~+cells was gradually increased from HG1 to HG3,but it was significantly decreased from HG3 to HG4.3.By analysis of the correlation between CD20~+/CD68~+cells and Slit2/Robo1expression,it was found that in the range of GVLPS<25%(HG1),the expression of Slit2 and Robo1 was positively correlated with the number of CD20~+and CD68~+cells,respectively.In the range of 25%?GVLPS<75%(HG2 and HG3),the expression of Slit2 and Robo1 was negatively correlated with the number of CD20~+and CD68~+cells,respectively.However,in the range of GVLPS?75%(HG4),there was a less correlation between the expression of Slit2/Robo1 and CD20~+/CD68~+cells.These results suggested that the GVLPS grade was only associated with the number of inflammatory cells in mild-to-moderate glomerular vasculopathy,and the correlation was weak when the glomerular sclerosis index was high.Conclusions:1.GVLPS grading can predict early glomerular vascular lesions in HSPN.2.GVLPS grading combined with ISKDC classification may improve the pathological diagnosis of HSPN.3.The Slit2/Robo1 signaling pathway is involved in the development of glomerular vascular lesions,and activating Slit2/Robo1 signaling may contribute to improve inflammatory cell infiltration in HSPN.