Pathomorphological changes in the tubulointerstitial compartment in primary glomerulopathies S. Kostadinova – Kunovska 1, G. Petrushevska 1, R. Jovanovic 1, L. Grchevska 2, M. Polenakovic 3 1. Institute of Pathology, Faculty of Medicine, Skopje, Macedonia 2. Clinic for Nephrology, Clinical Center, Skopje, Macedonia 3. Macedonian Academy of Sciences and Arts, Skopje, Macedonia

 The tubulointerstitial compartment of the kidney makes about 80% of the total kidney volume. §The tubular segments comprise the major part of the nephron and are structurally and functionally heterogenous.  The interstitium is the extravascular, intertubular space comprised of cells and extracellular matrix.

Cells  Fibroblasts  Macrophages  Dendritic cells Extracellular matrix  Collagen fibers ( I, III, VI )  Proteoglycans  Glycoproteins (fibronectin, laminin)  Interstitial liquid §The relative interstitial volume varies in different compartments of the kidney. l Cortex: 7 - 9% l Medulla: %

l Structural support to the nephrons and blood vessels l Exchange processes between the tubular and vascular elements l Regulation of the glomerular filtration through the tubuloglomerular feed-back l Production and regulation of the extracellular matrix l Production of local and systemic hormones, etc. §Functions of the interstitium

l Group of diseases characterized by histological and functional changes of the tubules and interstitium, responsible for the progression of renal diseases of different ethiologies  Primary  Secondary – associated with primary disease of the other renal compartments §Tubulointerstitionephritis (TIN)

l Acute  Interstitial edema  Leukocyte infiltration  Focal tubular necrosis l Chronic  Interstitial fibrosis  Mononuclear inflammatory infiltrate  Tubular atrophy §Morphology

l Membranous nephropathy l Membranoproliferative glomerulonephritis l IgA nephropathy l Focal segmental sclerosis l Diffuse proliferative glomerulonephritis l Lupus nephritis l Diabetic nephropathy, ….. §TIN associated with glomerulonephritis

l Phase I: Glomerular destruction and spreading of the damage to the tubulointerstitium l Phase II: Tubular cells (mediators) l Phase III:Activation of fibroblasts - fibrosis Schena FP. Molecular biology studies for the progression of renal damage in human glomerulonephritis. Mac Medical Review 1999;53:37-9. §Pathogenesis of TIN associated with glomerulonephritis:

l Glomerular destruction Alpers CE. The Kidney. In: Kumar V, Abbas AK, Fausto N (Eds). Robbins and Cotran Pathologic Basis of Disease, 7th edition. Philadelphia: Elsevier Saunders; 2005:

Rastaldi MP. Epithelial-mesenchymal transition and its implications for the development of renal tubulointersitial fibrosis. J Nephrol. 2006;19: §Pathogenesis of TIN associated with glomerulonephritis: l Tubular destruction

 Epithelial-mesenchymal transition  Cells lose their epithelial phenotype and acquire new, mesenchymal one  Mediators from the inflammatory infiltrate: EGF, FGF-2, TGF-b 1.  Basic events (Liu, 2004):  Epithelial cells lose their adhesive properties  De novo expression of Vimentin and α-SMA  Disruption of the tubular basement membrane  Migration of cells to the interstitium - myofibroblasts that synthetise collagen §Pathogenesis of TIN associated with glomerulonephritis: l Tubular destruction

 T lymphocytes  CD4  CD8  B lymphocytes  Macrophages §Pathogenesis of TIN associated with glomerulonephritis: l Interstitial inflammatory infiltrate

 Activated resident fibroblasts  Myofibroblasts  Accumulation of matrix proteins  Collagens I, III, V, VI, XV and fibronectin  Modified collagen type IV and laminin §Pathogenesis of TIN associated with glomerulonephritis: l Interstitial fibrosis

 Destruction of the renal interstitium  Tubular atrophy  Obliteration of peritubular capillaries  Atubular glomeruli  Consequent reduction of the glomerular filtration rate §Pathogenesis of TIN associated with glomerulonephritis: l Interstitial fibrosis

l 50 renal biopsies with previously diagnosed primary glomerulopathy l At least 10 glomeruli, cortical tubulointerstitium and arteriolar segments l Clinical data for evaluation of the renal function (serum creatinine and proteinuria) l Control group of 20 samples from kidneys without pathological changes in the tubulointerstitial compartment (nephrectomised), without significant difference in the patients` age. §MATERIAL

l Standard histochemical stainings for evaluation of the kidney biopsies:  HeEo  PAS  Trichrom Masson  Silvermethenamine Jones l Immunohistochemical stainings (single and double):  Cytokeratin AE1/AE3  Cytokeratin 7  E-cadherin  Collagen IV  CD43  CD20  CD68  CD34  Ki-67  HLA-DR  Vimentin   -SMA §METHODS

l Morphometric analysis:  Presence of the interstitial fibrosis on the staining with Trichrom-Masson, expressed as % of the scanned view field  Number of T-cells, B-cells and macrophages on 10 selected view fields on adequate immunohistochemical stainings  Determination of the proliferative index in the tubulointerstitial compartment on the Ki-67 staining, expressed as number of cells per 10 high power view fields.

l Semi-thin sections:  DURCUPAN  1 µm sections  Light-microscopic analysis of the changes of the tubules, tubular basal membrane and the interstitium §METHODS Toluidine blue PASM

Average age: 41,8 (SD=14,4; min.=15; max.=80) Female, 30% Male, 70% §RESULTS l Analysed group

Average age: 55,2 (SD=14,41; min.=27; max.=76) §RESULTS l Control group 7 13 Female, 35% Male, 65%

§RESULTS l Entities

§RESULTS l Tubules Toluidine blue; x1000PASM; x1000

§RESULTS l Tubules Collagen IV; x400

E-cadherin; x400Cytokeratin 7; x400 §RESULTS l Tubules

Ki-67; x400 Analysed group: 6.62 cells/10 HPF Control group: 0.08 cells/10 HPF p<0.05 §RESULTS l Tubular epithelial cells

Vimentin; x400 §RESULTS l Tubular epithelial cells CK7 / Vimentin; x400

 SMA; x400 §RESULTS l Tubular epithelial cells HLA-DR  ; x400

§RESULTS l Interstitial inflammatory infiltrate Toluidine blue; x1000

PASM; x1000 §RESULTS l Interstitial inflammatory infiltrate

CD43; x400 Analysed group: 68.3 cells/HPF Control group: 6.15 cells/HPF p<0.01 §RESULTS l Interstitial inflammatory infiltrate  T cells

CD20; x400 Analysed group: cells/HPF Control group: 1.15 cells/HPF p<0.01 §RESULTS l Interstitial inflammatory infiltrate  B cells

CD68; x400 Analysed group: cells/HPF Control group: 1.95 cells/HPF p<0.01 §RESULTS l Interstitial inflammatory infiltrate  Macrophages

Analysed group: cells/HPF Control group: 9.2 cells/HPF p<0.01 T cells 58.26% B cells 18.62% Macrophages22.92% §RESULTS l Interstitial inflammatory infiltrate  Total inflammatory infiltrate

§RESULTS l Fibroblasts  SMA; x400 Vimentin; x400

Analysed group: 18.75% Control group: 5.32% p<0.01 > 9% in 49/50 cases (98%) §RESULTS l Interstitial fibrosis

§RESULTS l Correlations  T cells / morphologic parameters of tubular damage (HLA-DRα, Vimentin, α SMA, Ki-67) R = 0.32 – 0.48 (p<0.01; p<0.05)  B cells / morphologic parameters of tubular damage (HLA-DRα, Vimentin, α SMA, Ki-67) R = 0.38 – 0.45 (p<0.01)  Macrophages / morphologic parameters of tubular damage (HLA-DRα, Vimentin, α SMA, Ki-67) R = 0.3 – 0.47 (p<0.01)  Total inflammatory infiltrate / morphologic parameters of tubular damage (HLA-DRα, Vimentin, α SMA, Ki-67) R = 0.35 – 0.45 (p<0.01; p<0.05)

§RESULTS l Correlations  Fibrosis / morphologic parameters of tubular damage (HLA-DRα, Vimentin, α SMA, Ki-67) R = 0.38 – 0.59 (p<0.01)  Fibrosis / interstitial inflammatory infiltrate (CD20, CD43, CD68, Total) R = 0.33 – 0.45 (p<0.05)  Fibrosis / serum creatinine R = 0.54 (p<0.01)  Serum creatinine / morphologic parameters of tubular damage (HLA-DRα, Vimentin, α SMA, Ki-67) R = 0.36 – 0.45 (p<0.01; p<0,05)  Serum creatinine / interstitial inflammatory infiltrate (CD20, CD43, CD68, Total) R = 0.44 – 0.47 (p<0.01)

§CONCLUSIONS  In 98% (49/50) of the analysed cases there is tubular atrophy and fibrosis in the interstitium (>9%).  In 90% of the analysed cases there is mononuclear inflammatory infiltrate in the interstitium, mainly with focal distribution, composed of:  T cells (average 58.26%),  B cells (average 18.62%)  Macrophages (average 22.92%)

§CONCLUSIONS  The correlation analyses provide arguments that histological abnormalities in the interstitium are closely related to each other and have impact on the kidney function.  Serum creatinine concentrations correlate to the interstitial fibrosis, to the degree of the tubular damage, as well as to the interstitial inflammatory infiltrate, due to which it can be used as prognostic parameter.