Upregulation of CD80 on glomerular podocytes plays an important role in development of proteinuria following pig-to-baboon xeno-renal transplantation - an experimental study

Abstract

We have previously reported that co-transplantation of the kidney with vascularized donor thymus from α-1,3-galactosyltransferase gene knockout pigs with an anti-CD154 with rituximab-based regimen led to improved xenograft survival in baboons with donor-specific unresponsiveness. However, nephrotic syndrome emerged as a complication in which the glomeruli showed mild mesangial expansion with similarities to minimal change disease (MCD) in humans. Since MCD is associated with CD80 expression in glomeruli and elevated urinary excretion, we evaluated a potential role for CD80 in xenograft nephropathy. Study 1 confirmed high urinary CD80 excretion in nephrotic animals with renal xenografts showing CD80 expression in glomeruli. In Study 2, baboons receiving xenografts received CTLA4-Ig once a week from the second postoperative week or no CTLA4-Ig. The non-CTLA4-Ig group developed severe proteinuria with modest mesangial expansion with high urinary excretion of CD80 and documented CD80 expression in glomerular podocytes. All of the recipients in non-CTLA4-Ig groups had to be euthanized before POD 60. In contrast, CTLA4-Ig group showed a marked reduction in proteinuria and survived significantly longer, up to 193 days. These results demonstrate that anti-CD80 targeted therapy represents a promising strategy for reduction of proteinuria following renal xeno-transplantation with improved survival.

Keywords: CD80; kidney transplantation; large animal model; proteinuria; xenograft.

Figure 1

Time course of urinary CD80 excretion relative to albuminuria in three baboons with porcine xenografts. Shown is the time course in postoperative days for baboon 289 (a), baboon 314 (b), and baboon 324 (c). A narrowed time course in these baboons shows urinary CD80 expression tended to increase in serial samples prior to increases in urine albumin excretion (d). Dark bars represent CD80 excretion (y bar on right) while urine albumin excretion is shown as open bars (y bar for albuminuria on left). *No sample was available.

Figure 2

(a) Anti-pig non-Gal antibodies assessed by FCM. Line graphs show median fluorescence index that was calculated by intensity at each point postoperative day (30, 40, 50, 60, 70, 80, and 90)/pre-Tx level. All baboons had preformed anti-non-Gal Nabs, IgM, and IgG, prior to thymokidney (TK) Tx that were adsorbed by TKs after revascularization. Anti-pig antibody levels never exceeded above pre-Tx levels (index less than 1), indicating no elicited anti-pig antibodies, IgM and IgG, developed after TK Tx in both CTLA4-Ig-treated (a-1) and CTLA4-Ig-nontreated (a-2) groups. (b) Baboon recipient survival days after porcine TK Tx in CTLA4-Ig group (B393, B394, 14P5) and non-CTLA4-IG group (B363, B341, B366, B358).

Figure 3

(a) Proteinuria assessed by dipstick in CTLA4-Ig group (a-1) and non-CTLA4-Ig group (a-2). Urine protein excretion was assessed three times a week. Proteinuria is described based on the following scale: 1+; 30 mg/dl, 2+; 100 mg/dl, 3+; 500 mg/dl, 4+; >500 mg/dl. (b) Western blot analysis of CD80 with molecular weight markers reveals CD80 to be 53 KDa. Also shown is the urinary excretion of CD80 in three recipients of TK that were treated with multiple doses of CTLA4-Ig (b-1: B394, B393, 14P5, b-1) and four recipients of TK without CTLA4-Ig therapy (b-2: B363, B341, B366, B358, b-2). Non-CTLA4-Ig-treated recipients consistently showed high urinary CD80 excretion following transplantation and greater proteinuria (b-2). In contrast, long-term survivors of TK with CTLA4Ig had many days with absent or minimal CD80 secretion and less proteinuria (b-1) although transient CD80 excretion was seen when nonimmunological complications such as ureteral obstruction or catheter complications developed.

Figure 4

CD80 Expression in a Nephrotic baboon. Baboon 314 developed severe nephrotic syndrome. CD80 was present in the glomeruli (c) but was negative in a normal porcine glomerulus (negative control, b), whereas it was positive in porcine spleen (a) [400×, CD80 in red, DAPI counterstain shows nuclei in blue].

Figure 5

CD80 expression in glomeruli of Thymokidney Grafts. The excised thymokidney graft at POD35 (B358, serum creatinine 0.8 mg/dl) appeared histologically normal (PAS. a-1) but CD80 upregulation was observed (a-2. red). (a3) Showed an image with anti-pig synaptopodin Ab alone (a-3. green). Double staining with anti-pig synaptopodin Ab (green) showed many CD80-positive cells which were double stained for synaptopodin (white arrows in a-4). Double staining for CD80 and CD31 was also performed. Rare CD31+ capillary endothelial cells (green) express CD80 (red) (double-positive cells were indicated by white arrow in a-5) of the thymokidney. Blue staining was DAPI. In contrast, a biopsy performed at POD 121 from baboon 14P5 which was not nephrotic with normal renal function (serum creatinine 0.8 mg/dl) showed a normal-appearing glomerulus (PAS. b-1), with no upregulation of CD80 by both single immunofluorescence staining (b-2) or double immunofluorescence staining (anti-CD80/anti-pig synaptopodin Ab, Fig. 4b4; anti-CD80/anti-CD31 ab, b-5) [4009, CD80, red; synaptopodin or CD31, green; DAPI, blue].

Figure 6

CD80 expression on porcine peripheral blood mononuclear cell (PBMC) and podocytes. (a) Pig PBMCs were stimulated with lipopolysaccharide (LPS) (5 μg/ml) for 24 h. CTLA4-Ig (at 0.25–5 μg/ml) was added and incubated for 3 h before LPS stimulation. CD80 expression was evaluated with flow cytometry (a-1). The mean MFI value in each of the groups was shown in the bar graph (a-2). Pig PBMC expressed CD80 (red line in the histogram, and third bar from left in the bar graph). CTLA4 Ig markedly inhibited CD80 expression. (b) Although resting pig podocytes did not express CD80 (blue line in the histogram and the second bar in the bar graph), LPS (5 μg/ml) stimulation induced CD80 upregulation (b-1, 2, red in histogram, and third bar from left in the bar graph). Pretreatment of CTLA4-Ig completely suppressed CD80 expression (green line in the histogram, and the fourth bar in the bar graph).

Figure 7

Replication of baboon CMV in sera of recipients in Study 2. (a) No bCMV replication was observed in all three baboons that were treated with CTLA4 Ig. (b) Three of four baboons in non-CTLA4-Ig-treated group did not develop any replication of bCMV by POD 20, and the remaining animal had only 19.4 copies/ml of serum at POD 10 and 242.95 copies/ml of serum at POD 20.