Ekstracelularni matriks kod primarnih glomerulonefritisa

Čužić, Snježana (2007) Ekstracelularni matriks kod primarnih glomerulonefritisa. PhD thesis, Sveučilište u Zagrebu.

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Abstract

Aim: The extracellular matrix is a complex superstructure surrounding cells composed of various proteins. In the kidney, ECM proteins are part of basement membranes, glomerular mesangium, interstitium and vessel walls. Their role is not only mechanical support of the cells, but also modification of cell phenotype and mediation of cellular response. The composition of glomerular and interstitial extracellular matrix (ECM) in glomerulonephritis (GN) is changing. Chronic renal insufficiency develops only in those cases of glomerulonephritis in which the lumina of postglomerular capillaries in the renal cortex are narrowed due to expansion of extracellular matrix (ECM).The aim of this study was to elucidate the distribution of extracellular matrix proteins in various forms of primary GN and to compare the composition of interstitial ECM proteins with clinical data in various forms of primary glomerulonephritis (GN). Methods: We examined renal biopsies from 55 GN patients, while normal renal tissue was obtained from nephrectomy due to small renal tumour. Specimens fixed in B5 and/or Dubosque fixative were analysed by immunohistochemistry (APPAP-method) using a monoclonal antibodies against tenascin (TN), fibronectin (FN), collagen I, III and IV (COI, COIII, COIV) (DAKO). The same fixatives were used for controle renal tissue. Clinical data were: proteinuria and creatinine level, obtained by standard laboratory methods, immunotherapy and ACE-genotype. Results: In normal renal tissue glomerular mesangium is TN, FN and weakly COI positive. Glomerular and tubular basal membrane stained with COIV antibody. FN is found around TBM. There was a weak COI expression in interstitium. Intensive TN staining was found in medulla interstitium only. The wall of larger blood vessels was TN, FN and COI positive. Irrespectably of histological form of GN foci of glomerular sclerosis were TN, FN, COI, COIII and COIV positive. In focal glomerulosclerosis mesangium, apart from sclerotic foci, was FN negative. There was simetimes de novo COIII and COIV staining of mesangium in GN with mesangial proliferation. Fibrous cresscents showed TN, FN, COI, COIII and COIV staining. Thickened Bowman’s capsule expressed COI, COIV and TN. At sites of interstitial inflammation there was a de novo TN expression. At sites of interstitial fibrosis FN and COIV were invariably present. TN was found at sites of interstitial fibrosis in 20 of 33 cases with interstitial fibrosis, mainly in biopsies with concomitant interstitial inflammation. COIII staining was found at sites of fibrosis only in 5 biopsies. COI was expressed more intesively in interstitium when inflammation or fibrosis were present. Fifty patients had proteinuria at time of biopsy and 33 of them continued to have one after follow up period. Patients with persistent proteinuria had more often interstitial fibrosis with elevated content of COI and COIV. Presence of fibrin/fibrinogen deposits in interstium of patients with nephrotic proteinuria is negative prognostic factor (p=0,035). Those patients did not respond well to therapy. There were 12 patients with elevated creatinine at biopsy with similar composition of ECM as patients with proteinuria. Majority of patients with interstitial fibrosis had ACE-genotyp II although this genotype wasn’t the most common in the cochort. Conclusions: Our study showed that persistant proteinuria could be linked to interstitial fibrosis and fibrin deposits.

Abstract in Croatian

Cilj: Klinička prognoza primarnog glomerulonefritisa, osim brzo-progredirajućeg GN, ovisi o stupnju fibroze bubrežnog intersticija. Svrha ovog rada bila je da usporedbom kliničke i histološke slike pokuša ponuditi nove histološke prognostičke čimbenike primarnih glomerulonefritisa. Postupci: U ovom radu su u skupini 55 bolesnika sa primarnim glomerulonefritisom uspoređivani klinički parametri oštećenja bubrežne funkcije, proteinurija i razina kreatinina u serumu u vrijeme biopsije bubrega i nakon perioda praćenja s histološkim promjenama intersticija. Histološke promjene intersticija promatrane su kroz promjene sastava ekstracelularnog matriksa. Bioptički materijal preuzet je za svjetlosnu mikroskopiju, imunofluorescenciju, elektronsku mikroskopiju i imunohistokemiju. Histološka dijagnoza glomerulonefritisa postavljena je prema kriterijima Svjetske zdravstvene organizacije (Churg J, Sobin LH: Renal disease. Classification and atlas of glomerular disease. Tokyo, New York: Igaku Shoin, 1982). Imunohistokemijski je određivano prisustvo i rasprostranjenost kolagena tip I (monoklonalno antitijelo dobiveno iz zeca, 1:25), kolagena tip III (monoklonalno antitijelo dobiveno iz miša, klon MC3-HA, 1:10), kolagena tip IV (monoklonalno antitijelo dobiveno iz miša, klon CIV 22, 1:10), fibronektina (poliklonalno antitijelo dobiveno iz zeca, 1:10) i tenascina (monoklonalno antitijelo dobiveno iz miša, klon TN2, 1:25). Imunofluorescencijom je određivano prisustvo i rasprostranjenost fibrina/fibrinogena (primarna antitijela konjugirana sa fluoresceinom). Intenzitet imunohistokemijske reakcije i imunofluorescencije određen je semikvanititativno («-« bez reakcije, «+» slabo pozitivna reakcija, «++»pozitivna reakcija) na 10 velikih vidnih polja (povećanje 400x). Rezultati: U normalnom bubrežnom tkivu je kolagen tip IV građevni element tubularne bazalne membrane. Kolagen I je slabo pozitivan, difuzno u intersticiju kore i medule bubrega. Fibronektin se u intersticiju nalazi uz samu tubularnu bazalnu membranu. Intersticij medule bubrega je intezivno tenascin pozitivan, a u intersticiju kore nema tenascina. Stjenke većih krvnih žila bubrega su tenascin, fibronektin i kolagen tip I pozitivne. U normalnom bubrežnom tkivu nema kolagena III, niti fibrina/fibrinogena. Nema razlike u odlaganju proteina ekstracelularnog matriksa u intersticij između različitih oblika primarnog glomerulonefritisa. Tijekom upale u sklopu primarnog glomerulonefritisa se u intersticij bubrežne kore odlaže de novo tenascin. Kod bolesnika s intersticijalnom upalom postoji statistički značajna razlika u prisutnosti tenascina (p=0,04). U intersticij bubrega se u slučajevima fibroze u sklopu primarnog glomerulonefirtisa povećano odlažu kolagena tip I i fibronektin, a de novo kolagen tip III i tip IV. Kod bolesnika sa intersticijalnom fibrozom postoji statistički značajna razlika u prisutnosti kolagena tip IV u odnosu na sve ostale promatrane proteine ekstracelularnog matriksa (p=0,024). Nema razlike u intenzitetu proteinurije ili razini serumskog kreatinina, niti u času biopsije ni na kraju praćenja, obzirom na prisutnosti tenascina, fibronektina, kolagena tip I, kolagena tip III i kolagena tip IV u intersticiju bubrega. Prisustvo fibrina/fibrinogena u bubrežnom intersticiju je loš prognostički čimbenik kod bolesnika koji u času biopsije imaju nefrotsku proteinuriju (p=0,035). Zaključak: Utvrđeno je da nema razlike u odlaganju proteina ekstracelularnog matriksa u intersticij između različitih oblika primarnog glomerulonefritisa. Tijekom upale u sklopu primarnog glomerulonefrritisa se u intersticij bubrežne kore odlaže de novo tenascin. U intersticij bubrega se u slučajevima fibroze u sklopu primarnog glomerulonefritisa pojačano odlažu kolagena tip I i fibronektin, a de novo kolagen tip III i tip IV. Također je pokazano da nema razlike u intenzitetu proteinurije ili razini serumskog kreatinina, niti u času biopsije ni na kraju praćenja, obzirom na prisutnosti tenascina, fibronektina, kolagena tip I, tip III i a tip IV u intersticiju bubrega. Prisustvo fibrina/fibrinogena u bubrežnom intersticiju je loš prognostički čimbenik kod bolesnika koji u času biopsije imaju nefrotsku proteinuriju (p=0,035). Time je pronađen novi histološki prognostički čimbenik primarnih glomerulonefritisa koji do sada nije bio opisan u literaturi.

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