Long-Term Gene Therapy with Thrombospondin 2 Inhibits TGF-β Activation, Inflammation and Angiogenesis in Chronic Allograft Nephropathy

We recently identified Thrombospondin-2 (TSP-2) as a regulator of matrix remodelling and inflammation in experimental kidney disease by using TSP-2 null mice and successfully proved TSP-2 overexpression as a therapeutic concept in a short term glomerulonephritis model in the rat. In this current study, we investigated if long-term TSP-2 overexpression is also capable to ameliorate the progression of chronic kidney disease in the setting of the chronic allograft nephropathy F344-Lewis model in the rat. Two weeks after renal transplantation, two rat thigh muscles were transfected once only with either a TSP-2 overexpressing plasmid (n = 8) or a luciferase-expressing plasmid as control (n = 8). Rats were monitored for renal function, histological changes and gene expression in the graft for up to 30 weeks after transplantation. Unexpectedly, only in the TSP-2 treated group 2 rats died before the end of the experiment and renal function tended to be worsened in the TSP-2 group compared to the luciferase-treated controls. In addition, glomerular sclerosis and tubular interstitial injury as well as cortical fibronectin deposition was significantly increased in the TSP-2 treated kidneys despite reduced TGF-β activation and marked anti-inflammatory (macrophages, T-cells and B-cells) effects in this group. Long-term TSP-2 therapy impaired repair of renal endothelium, as demonstrated by significant higher glomerular and peritubular endothelial rarefaction and reduced endothelial cell proliferation in the transplanted kidneys from TSP-2 treated rats compared to controls. This TSP-2 effect was associated with decreased levels of renal VEGF but not VEGF1 receptor. In conclusion, despite its anti-inflammatory and TGF-β activation blocking effects, TSP-2 gene therapy did not ameliorate but rather worsened experimental chronic allograft nephropathy most likely via its anti-angiogenic properties on the renal microvasculature

Long-Term Gene Therapy with Thrombospondin 2 Inhibits TGF-b Activation, Inflammation and Angiogenesis in Chronic Allograft Nephropathy

We recently identified Thrombospondin-2 (TSP-2) as a regulator of matrix remodelling and inflammation in experimental kidney disease by using TSP-2 null mice and successfully proved TSP-2 overexpression as a therapeutic concept in a short term glomerulonephritis model in the rat. In this current study, we investigated if long-term TSP-2 overexpression is also capable to ameliorate the progression of chronic kidney disease in the setting of the chronic allograft nephropathy F344-Lewis model in the rat. Two weeks after renal transplantation, two rat thigh muscles were transfected once only with either a TSP-2 overexpressing plasmid (n = 8) or a luciferase-expressing plasmid as control (n = 8). Rats were monitored for renal function, histological changes and gene expression in the graft for up to 30 weeks after transplantation. Unexpectedly, only in the TSP-2 treated group 2 rats died before the end of the experiment and renal function tended to be worsened in the TSP-2 group compared to the luciferase-treated controls. In addition, glomerular sclerosis and tubular interstitial injury as well as cortical fibronectin deposition was significantly increased in the TSP-2 treated kidneys despite reduced TGF-b activation and marked anti-inflammatory (macrophages, T-cells and B-cells) effects in this group. Long-term TSP-2 therapy impaired repair of renal endothelium, as demonstrated by significant higher glomerular and peritubular endothelial rarefaction and reduced endothelial cell proliferation in the transplanted kidneys from TSP-2 treated rats compared to controls. This TSP-2 effect was associated with decreased levels of renal VEGF but not VEGF1 receptor. In conclusion, despit

Long-term gene therapy with thrombospondin 2 inhibits TGF-β activation, inflammation and angiogenesis in chronic allograft nephropathy.

We recently identified Thrombospondin-2 (TSP-2) as a regulator of matrix remodelling and inflammation in experimental kidney disease by using TSP-2 null mice and successfully proved TSP-2 overexpression as a therapeutic concept in a short term glomerulonephritis model in the rat. In this current study, we investigated if long-term TSP-2 overexpression is also capable to ameliorate the progression of chronic kidney disease in the setting of the chronic allograft nephropathy F344-Lewis model in the rat. Two weeks after renal transplantation, two rat thigh muscles were transfected once only with either a TSP-2 overexpressing plasmid (n = 8) or a luciferase-expressing plasmid as control (n = 8). Rats were monitored for renal function, histological changes and gene expression in the graft for up to 30 weeks after transplantation. Unexpectedly, only in the TSP-2 treated group 2 rats died before the end of the experiment and renal function tended to be worsened in the TSP-2 group compared to the luciferase-treated controls. In addition, glomerular sclerosis and tubular interstitial injury as well as cortical fibronectin deposition was significantly increased in the TSP-2 treated kidneys despite reduced TGF-β activation and marked anti-inflammatory (macrophages, T-cells and B-cells) effects in this group. Long-term TSP-2 therapy impaired repair of renal endothelium, as demonstrated by significant higher glomerular and peritubular endothelial rarefaction and reduced endothelial cell proliferation in the transplanted kidneys from TSP-2 treated rats compared to controls. This TSP-2 effect was associated with decreased levels of renal VEGF but not VEGF1 receptor. In conclusion, despite its anti-inflammatory and TGF-β activation blocking effects, TSP-2 gene therapy did not ameliorate but rather worsened experimental chronic allograft nephropathy most likely via its anti-angiogenic properties on the renal microvasculature

PAS and desmin staining in renal grafts treated with TSP-2 gene therapy.

<p>Representative microphotographs from PAS staining of kidney grafts showing glomerular (A, control plasmid; B, TSP-2 therapy) and tubulointerstitial changes (C, control plasmid; D, TSP-2 therapy) are shown. Glomerular desmin staining using immunohistochemistry is shown as a marker of injured podocytes (E, control plasmid; F, TSP-2 therapy, brown staining).</p

Renal graft inflammation was reduced by TSP-2 gene therapy.

<p>Graft inflammation was evaluated by counting CD8a positive T-cells (A, within the glomeruli; B cortical), CD45R positive B-cells (C, glomerular; D, cortical), MHC II positive antigen presenting cells (E, glomerular; F, cortical) and ED-1 positive macrophages (G, glomerular; H, cortical) after staining by immunohistochemistry. Control (n = 8) vs. TSP-2 treated (n = 8); *p<0,02.</p

Capillary rarefaction in renal grafts treated with TSP-2 gene therapy.

<p>Representative pictures of renal grafts after immunohistologic staining for CD31 within the glomeruli (A) and tubulointerstitial compartment (B) of kidney-transplanted rats treated with the control plasmid. Renal grafts from rats treated with TSP-2 showed considerable rarefaction of capillaries in the glomeruli (C) and the tubulointerstitium (D). Capillary rarefaction was analyzed by computer-assisted image analysis in the glomeruli (E) or using a grid in the tubulointerstitium (F). CD31 mRNA was analyzed in glomerular extracts in grafts from control and TSP-2 treated rats using real-time PCR. Control (n = 8) vs. TSP-2 treated (E-F:n = 8; G:n = 6); *p<0,008.</p

Fibronectin and alpha-smooth muscle actin staining in renal grafts treated with TSP-2 gene therapy.

<p>Representative microphotographs from immunohistological staining of kidney grafts for active TGF-β (A, control plasmid; B, TSP-2 therapy, brown cytosolic staining), P-smad 2/3 (C, control plasmid; D, TSP-2 therapy, brown nuclear staining), PAI-1 (E, control plasmid; F, TSP-2 therapy, brown staining), fibronectin (G, control plasmid; H, TSP-2 therapy, brown staining) and alpha-smooth muscle actin (I, control plasmid; J, TSP-2 therapy, brown staining) are shown.</p

Expression of TSP-1 and TSP-2 in F344 Lewis rat renal allograft model.

<p>Renal TSP-1 and TSP-1 expression was evaluated in biopsies from rats treated with the overexpression plasmid for luciferase control or TSP-2 using immunohistochemistry. TSP-1 expression was localized in glomeruli and within and around the Bowman's capsule (A, brown staining) as well as in fibroblasts and atrophic tubular cells (B). A semiquantitative evaluation of TSP-1 expression revealed no significant differences between the two groups (C). Staining for endogenous TSP-2 showed marked expression within some tubules (F), while glomerular staining for TSP-2 is lacking (E). Endogenous TSP-2 expression shows a tendency to increased TSP-2 expression within the TSP-2 treated group compared to the controls. Control (n = 8) vs. TSP-2 treated (n = 8).</p

Immunohistochemistry of inflammatory cells in renal grafts after TSP-2 gene therapy.

<p>Representative microphotographs from immunohistological staining of kidney grafts for CD8a positive T-cells (A, control plasmid; B, TSP-2 therapy, brown staining), MHC II positive antigen presenting cells (C, control plasmid; D, TSP-2 therapy, brown staining) and CD45R positive B-cells (E, control plasmid; F, TSP-2 therapy, brown staining) are shown.</p

Renal injury was impaired by TSP-2 gene therapy.

<p>Injury in renal transplants was assessed by semiquantitative scoring of glomerulosclerosis (A) and tubular injury (B) in PAS-stained paraffin sections as well as FSGS-lesions (C). Podocyte damage was evaluated by semiquantitative scoring of desmin positive podocytes by immunohistochemistry (D). Control (n = 8) vs. TSP-2 treated (n = 8); *p<0,02.</p