Role of Glomerular Proteoglycans in IgA Nephropathy

Mesangial matrix expansion is a prominent feature of the most common form of glomerulonephritis, IgA nephropathy (IgAN). To find molecular markers and improve the understanding of the disease, the gene and protein expression of proteoglycans were investigated in biopsies from IgAN patients and correlated to clinical and morphological data. We collected and microdissected renal biopsies from IgAN patients (n = 19) and from healthy kidney donors (n = 14). Patients were followed for an average time of 4 years and blood pressure was according to target guidelines. Distinct patterns of gene expression were seen in glomerular and tubulo-interstitial cells. Three of the proteoglycans investigated were found to be of special interest and upregulated in glomeruli: perlecan, decorin and biglycan. Perlecan gene expression negatively correlated to albumin excretion and progress of the disease. Abundant decorin protein expression was found in sclerotic glomeruli, but not in unaffected glomeruli from IgAN patients or in controls. Transforming growth factor beta (TGF-β), known to interact with perlecan, decorin and biglycan, were upregulated both on gene and protein level in the glomeruli. This study provides further insight into the molecular mechanisms involved in mesangial matrix expansion in IgAN. We conclude that perlecan is a possible prognostic marker for patients with IgAN. In addition, the up-regulation of biglycan and decorin, as well as TGF-β itself, indicate that regulation of TGF-β, and other profibrotic markers plays a role in IgAN pathology

Molecular mechanisms behind the liver-induced acceptance of renal grafts in highly sensitized patients

Preformed antibodies directed at donor HLA are considered an absolute contraindication for kidney transplantation, because of the high risk of rejection. Thus, patients with high levels of HLA antibodies have little chance of receiving a kidney transplant. Recently it was demonstrated that an auxiliary liver graft from the same donor may protect a subsequently grafted kidney from these harmful antibodies. The aim of this thesis was to elucidate the mechanisms behind the kidney protection afforded by the auxiliary liver graft in highly sensitized patients. We focused on the activation of dendritic cells, because these cells, which reside in all peripheral tissues, play a key role in the initiation of an immune response. This thesis demonstrates that gene expression in the liver graft correlates with clinical outcome: In patients without an acute rejection episode, 14 out of 45 investigated immunological genes were significantly higher expressed in the liver graft 4h after reperfusion, compared with patients that experienced an acute rejection episode within the first month. This indicates that high- and low-risk patients can be identified within hours after transplantation. One gene of particular interest was indoleamine 2,3-dioxygenase (IDO), which is a tolerance-inducing enzyme previously found to play a key role in maintenance of semi-allogeneic pregnancy in mice. In our study, mRNA levels of IDO were strongly upregulated in patients after combined auxiliary liver-kidney transplantation and IDO expression in the liver graft correlated with clinical outcome. Furthermore, IDO activity was higher in patients after combined auxiliary liver-kidney transplantation and liver transplantation compared with patients undergoing kidney transplantation. Strongly increased serum levels of the anti-inflammatory cytokine interleukin (IL) 10 were also found after liver but not kidney transplantation. IL-10 has several immune inhibitory effects on dendritic cells. We found that IL-10 inhibited the production of chemokines MIG, IP-10 and I-TAC in monocyte-derived dendritic cells in vitro. When comparing different cytokine cocktails for dendritic cell maturation, we showed that MIG, IP-10 and I-TAC were essential for dendritic cell-mediated recruitment of natural killer (NK) cells. This is considered important for the initiation of a type 1 T helper cell response. We also showed that IL-10 treated dendritic cells, which expressed less of these chemokines, had reduced potential to activate NK cells. Thus, the liver provides IDO and IL-10, both of which have the ability to reduce the immunostimulatory ability of dendritic cells, giving them a tolerance-promoting profile. We therefore suggest that the protective effect of an auxiliary liver in presensitized patients may, at least in part, be mediated by the liver-specific expression of IDO and IL-10

Correlation of tubular atrophy/interstitial fibrosis (TA/IF) score and albumin excretion in urine, r = 0.51, p<0.05 (A), progress, non-significant (B) and glomerular filtration rate, r = −0.68, p<0.01 (C).

<p>Correlation of tubular atrophy/interstitial fibrosis (TA/IF) score and albumin excretion in urine, r = 0.51, p<0.05 (A), progress, non-significant (B) and glomerular filtration rate, r = −0.68, p<0.01 (C).</p

Summary of key pathological features according to the Oxford classification system.

<p>Mesangial score ≤0.5 (M0) or >0.5 (M1).</p><p>Segmental glomerulosclerosis, absent (S0) or present (S1).</p><p>Endocapillary hypercellularity, absent (E0) or present (E1).</p><p>TA/IF ≤25% (T0), 26–50% (T1), or >50% (T2).</p

Protein expression of perlecan in sections from control (A), a patient with IgAN (B), and a negative control (C).

<p>Protein expression of Perlecan in frozen biopsy sections from another set of patients with IgAN stained significantly stronger for perlecan (n = 45, 6.3±0.7%) than controls (n = 30, 9.4±0.7%) P<0.01. Magnification ×63.</p

Clinical characteristics of patients with IgAN at time of biopsy.

<p>Abbreviations; GFR, glomerular filtration rate; MAP, mean arterial pressure; BP, blood pressure; RAAS, Renin-angiotensin-aldosterone system.</p><p>*estimated GFR calculated using MDRD formula (ml/min/1.73 m²), the other GFRs presented in the table are determined using ⁵¹Cr-EDTA clearance.</p>†<p>calculated from tU-protein.</p

PAS staining of sclerotic glomeruli from a representative IgAN patient (A), and the consecutive section stained for decorin (B) shows overlap of the sclerotic area and decorin staining.

<p>PAS staining of glomeruli without sclerosis (C) and consecutive section showing no staining for decorin (D) in the same patient. Decorin is abundantly expressed in sclerotic glomeruli both in controls and patients with IgAN, but not in non-sclerotic glomeruli. Magnification ×63.</p

Protein expression of TGF-β in sections from control (A), patient with IgAN (B) and negative control (C).

<p>IgAN sections stained significantly stronger for TGF-β, and the average arbitrary unit score for IgAN (n = 83 glomeruli, 4.51±0.25) was higher than control (n = 19 glomeruli, 2.89±0.28) P<0.05 (D). In (B) there is some auto-fluorescence of red blood cells, this phenomenon can also be seen in the negative control, and were not included in the analysis of staining intensity of TGF-β. Magnification ×63.</p

The relative gene expression of perlecan in the glomeruli from patients with IgAN at time of the biopsy correlates inversely to the patient's albumin excretion in urine, n = 17, r = −0.58, p<0.05 (A) and disease progression, n = 18, r = −0.52, p<0.05 (C).

<p>A high expression of perlecan at the time of the biopsy correlates to a lower albumin excretion and slower progression of the disease, implicating that the relative gene expression of perlecan can be used as a prognostic molecular marker in IgAN. The relative gene expression of nephrin did not correlate to urinary albumin excretion, n = 17 (B), but correlated inversely to disease progression, n = 18, r = −0.47, P<0.05 (D). This suggests that even the smallest changes in gene expression of nephrin may be important for disease progression in IgAN.</p

Relative gene expression of proteoglycan core proteins and enzymes in glomeruli (A) and tubulo-interstitial compartment (B) of biopsies from IgAN patients compared to controls.

<p>Controls values are defined as 1, and higher values indicate up-regulation while lower values indicate down-regulation. Error bars represent SEM. *p<0.05, **p<0.01, ***p<0.001.</p