Pathomorphological changes in the tubulointerstitial compartment

in primary glomerulopathies

S. Kostadinova – Kunovska1, G. Petrushevska1, R. Jovanovic1, L. Grchevska2, M. Polenakovic3

1. Institute of Pathology, Faculty of Medicine, Skopje, Macedonia2. Clinic for Nephrology, Clinical Center, Skopje, Macedonia3. 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. Cortex: 7 - 9% Medulla: 30 - 40%

Structural support to the nephrons and blood vessels

Exchange processes between the tubular and vascular elements

Regulation of the glomerular filtration through the tubuloglomerular feed-back

Production and regulation of the extracellular matrix

Production of local and systemic hormones, etc.

Functions of the interstitium

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)

Acute Interstitial edema Leukocyte infiltration Focal tubular necrosis

Chronic Interstitial fibrosis Mononuclear inflammatory infiltrate Tubular atrophy

Morphology

Membranous nephropathy Membranoproliferative glomerulonephritis IgA nephropathy Focal segmental sclerosis Diffuse proliferative glomerulonephritis Lupus nephritis Diabetic nephropathy, …..

TIN associated with glomerulonephritis

Phase I: Glomerular destruction and spreading of the damage to the tubulointerstitium

Phase II: Tubular cells (mediators)

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:

Pathogenesis of TIN associated with glomerulonephritis: 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; 2005:955-1021.

Rastaldi MP. Epithelial-mesenchymal transition and its implications for the development of renal tubulointersitial fibrosis. J Nephrol. 2006;19:407-12.

Pathogenesis of TIN associated with glomerulonephritis: Tubular destruction

Epithelial-mesenchymal transition Cells lose their epithelial phenotype and acquire new,

mesenchymal one Mediators from the inflammatory infiltrate: EGF, FGF-2,

TGF-b1.

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: Tubular destruction

T lymphocytes CD4 CD8

B lymphocytes

Macrophages

Pathogenesis of TIN associated with glomerulonephritis: 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: 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: Interstitial fibrosis

50 renal biopsies with previously diagnosed primary glomerulopathy

At least 10 glomeruli, cortical tubulointerstitium and arteriolar segments

Clinical data for evaluation of the renal function (serum creatinine and proteinuria)

Control group of 20 samples from kidneys without pathological changes in the tubulointerstitial compartment (nephrectomised), without significant difference in the patients` age.

MATERIAL

Standard histochemical stainings for evaluation of the kidney biopsies: HeEo PAS Trichrom Masson Silvermethenamine Jones

Immunohistochemical stainings (single and double):Cytokeratin AE1/AE3Cytokeratin 7E-cadherinCollagen IVCD43 CD20

CD68CD34Ki-67HLA-DRVimentin-SMA

METHODS

METHODS

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.

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)

@eni, 30%

Ma`i, 70%

35

15

Female, 30%Female, 30%

Male, 70%Male, 70%

RESULTS Analysed group

Average age: 55,2 (SD=14,41; min.=27; max.=76)

RESULTS Control group

@eni, 35%

Ma`i, 65%

7

13

Female, 35%Female, 35%

Male, 65%Male, 65%

RESULTS Entities

IgAN; (22); 44%

EGN; (3); 6%

MGN; (4); 8%

MSGN; (5); 10%

FSGS; (7); 14%

MPGN; (9); 18%

RESULTS Tubules

Toluidine blue; x1000 PASM; x1000

RESULTS Tubules

Collagen IV; x400

E-cadherin; x400Cytokeratin 7; x400

RESULTS Tubules

Ki-67; x400

Mean ±SE ±SD

IgANMSGN

MPGNFSGS

MGNEGN

Kontrola

Dijagnoza

-10

0

10

20

30

40

50

Ki-6

7

Analysed group: 6.62 cells/10 HPFControl group: 0.08 cells/10 HPF

p<0.05

RESULTS Tubular epithelial cells

Vimentin; x400

RESULTS Tubular epithelial cells

CK7 / Vimentin; x400

SMA; x400

RESULTS Tubular epithelial cells

HLA-DR; x400

RESULTS Interstitial inflammatory infiltrate

Toluidine blue; x1000

PASM; x1000

RESULTS Interstitial inflammatory infiltrate

CD43; x400 Analysed group: 68.3 cells/HPFControl group: 6.15 cells/HPF

p<0.01

Mean ±SE ±SD

IgANMSGN

MPGNFSGS

MGNEGN

Kontrola

Dijagnoza

-20

0

20

40

60

80

100

120

140

160

180

200

220

240

CD

43

RESULTS Interstitial inflammatory infiltrate

T cells

CD20; x400

Mean ±SE ±SD

IgANMSGN

MPGNFSGS

MGNEGN

Kontrola

Dijagnoza

-20

0

20

40

60

80

100

120

CD

20

Analysed group: 24.98 cells/HPFControl group: 1.15 cells/HPF

p<0.01

RESULTS Interstitial inflammatory infiltrate

B cells

CD68; x400 Analysed group: 27.16 cells/HPFControl group: 1.95 cells/HPF

p<0.01

Mean ±SE ±SD

IgANMSGN

MPGNFSGS

MGNEGN

Kontrola

D ija gnoza

-10

0

10

20

30

40

50

60

70

80

CD

68

RESULTS Interstitial inflammatory infiltrate

Macrophages

Analysed group: 114.48 cells/HPFControl group: 9.2 cells/HPF

p<0.01

Mean ±SE ±SD

IgANMSGN

MPGNFSGS

MGNEGN

Kontrola

Dijagnoza

-50

0

50

100

150

200

250

300

350

400

Vku

pen

broj

na

infla

mat

orni

kle

tki

vo in

ters

tici

umotT cells

58.26%

B cells 18.62%

Macrophages 22.92%

RESULTS Interstitial inflammatory infiltrate

Total inflammatory infiltrate

RESULTS Fibroblasts

SMA; x400Vimentin; x400

Mean ±SE ±SD

IgANMSGN

MPGNFSGS

MGNEGN

Kontrola

Dijagnoza

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

Fibr

oza

(%

)

Analysed group: 18.75%Control group: 5.32%

p<0.01

> 9% in 49/50 cases (98%)

RESULTS Interstitial fibrosis

RESULTS 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 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.