Neue Therapiemöglichkeiten der chronischen Transplantnephropathie

The chronic allograft nephropathy is the leading cause for graft loss after kidney transplantation and causes a progressive decline in renal function leading to end stage renal disease mostly within 10-15 years. The current immunosuppressive therapy after kidney transplantation is based on calcineurin inhibitors like cyclosporine A or tacrolimus. These drugs are nephrotoxic and profibrotic, and hereby stimulate the process resulting in chronic allograft nephropathy. To improve graft survival after kidney transplantation, it is absolutely necessary to test additional drugs regarding their effectiveness in preventing or inhibiting chronic allograft nephropathy. In this work the effects of the mTOR-inhibitor everolimus was investigated in the non-immunological chronic progressive nephron loss model of 5/6 nephrectomy in the rat. In general everolimus has anti-fibrotic, anti-proliferative and anti-angiogenetic actions. In the 5/6 nephrectomy model of the rat glomerular injury affects endothelial, mesangial cells and podocytes similarly. An mTOR-inhibition (after day 3) in the initial stage after model induction led to a delayed healing reaction of the glomerular damage and to an increase in glomerulosclerosis and interstitial fibrosis. Furthermore a raise in proteinuria and a decline in kidney function also occurred. In the second chapter of this work we investigated the influence of the mTOR-inhibitor sirolimus and the proteasome inhibitor bortezomib, alone or in combination, regarding the immunological rejection after kidney transplantation in the fischer-lewis transplantation model in the rat. Hereby, we focused on the chronic-humoral aspect of this chronic allograft model, where antibodies against epitopes of the donor kidney are produced. Sirolimus and bortezomib caused depletion of antibody-producing plasma cells in spleens and bone marrows ameliorating the chronic-humoral rejection. The treated animals showed histological improvements of glomerular, tubulointerstitial and vascular structures of the kidney. In addition to the plasma cells, other cell types of the immune system like cytotoxic T cells, T helper cells, macrophages, monocytes and B cells were also decreased by sirolimus and bortezomib demonstrating their activity against the humoral and cellular immunological response. Therefore, we demonstrate that treatment with everolimus in the situation of a complex glomerular injury can negatively influence the healing process and can lead to a decline in renal function. The effectiveness of everolimus depends on the severity of injury, the drug doses and the right time of treatment. In the situation of a chronic humoral or mixed rejection situation, sirolimus and bortezomib are potent inhibitors of an ongoing chronic rejection mainly via depletion of plasma cells and other immune cells.Die chronische Transplantatnephropathie ist die Hauptursache für den Verlust einer transplantierten Niere und führt von einer Funktionsverschlechterung bis hin zur Niereninsuffizienz, so dass das mittlere Transplantatüberleben 10-15 Jahre beträgt. Die derzeitige immunsuppressive Therapie nach einer Nierentransplantation basiert meist auf Calcineurin-Inhibitoren, wie Ciclosporin A oder Tacrolimus. Speziell die Calcineurin-Inhibitoren wirken nephrotoxisch und pro-fibrotisch und fördern den Prozess der Transplantatnephropathie. An der Entstehung einer Transplantatnephropathie sind sowohl nicht-immunologische als auch immunologische Faktoren beteiligt. Um das Transplantatüberleben zu verbessern, ist es dringend notwendig andere Medikamente auf ihre Wirksamkeit bezüglich einer effektiven Therapie zur Verhinderung einer chronischen Transplantatnephropathie zu testen. In der vorliegenden Arbeit wurde zum einen der Einfluss von Everolimus, einem mTOR-Inhibitor, auf die Vernarbung des Nierengewebes in einem nicht-immunologisch bedingten chronisch progressiven Nephronenverlust-Modell, der 5/6 Nephrektomie der Ratte, untersucht. Everolimus wirkt anti-fibrotisch, anti-proliferativ und anti-angiogenetisch. Im 5/6 Nephrektomiemodell der Ratte kommt es als Folge eines glomerulären Schadens zu einer Vernarbungsreaktion, der Endothel, Mesangium und Podozyten gleichermaßen betrifft. Eine mTOR-Inhibition in der Anfangsphase (ab Tag 3) nach Modellinduktion führt zu einer verzögerten Reparatur des glomerulären Schadens und zu vermehrter Defektheilung des Gewebes mit Glomerulosklerose und interstitieller Fibrose. Es kam außerdem zu einer Steigerung der Proteinurie und einer Verschlechterung der Nierenfunktion. Im zweiten Teil der Arbeit wurde der Einfluss des mTOR-Inhibitors Sirolimus und des Proteasomen-Inhibitors Bortezomib, allein und in Kombination, auf die immunologische Abstoßungsreaktion nach Nierentransplantation im Fischer-Lewis-Modell der Ratte untersucht. Hierbei konzentrierten wir uns auf die Beeinflussung der chronisch-humoralen Abstossungsreaktion, die durch die Entstehung von Antikörpern gegen Epitope der Spenderniere gekennzeichnet ist. Durch Depletion der Antikörper-produzierenden Plasmazellen in der Milz und im Knochenmark konnten Sirolimus und Bortezomib die weitere Abstoßungsreaktion vermindern. Die behandelten Tiere zeigten zusätzlich histologische Verbesserungen bezüglich der glomerulären, tubulointerstitiellen und vaskulären Strukturen der Niere. Neben den Plasmazellen wurden auch andere Zellen des Immunsystems wie zytotoxische T-Zellen, T-Helferzellen, Makrophagen, Monozyten und B-Zellen durch Sirolimus und Bortezomib moduliert und die Immunantwort abgeschwächt. In dieser Arbeit konnten wir somit zeigen, dass sich der Einsatz des mTOR-Inhibitors Everolimus in einem komplexen glomerulären Schaden negativ auf den Heilungsprozess auswirken und zu einer Verschlechterung der Funktion führen kann. Die Wirkungsweise von Everolimus scheint ganz entscheidend von der Schwere der Schädigung, der Medikamentendosis und dem Behandlungszeitpunkt abhängig zu sein. In der Situation der Antikörper-vermittelten Abstoßungsreaktion können der mTOR-Inhibitor Sirolimus oder der Proteasomen-Inhibitor Bortezomib, sowohl in Kombination als auch einzeln, immunmodulierend wirken und durch Depletion der Plasmazellen die chronisch-humorale und zelluläre Abstoßungsreaktion vermindern

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

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

The mTOR Inhibitor Everolimus Attenuates the Time Course of Chronic Anti-Thy1 Nephritis in the Rat

Background: The antiproliferative immunosuppressant everolimus adversely affects the acute reversible anti-Thy1 nephritis model. We hypothesized that everolimus treatment started after the acute proliferative phase could even be beneficial in the chronic anti-Thy1 nephritis model in the rat. Methods: Chronic anti-Thy1 nephritis was induced by injection of the monoclonal antibody 1-22-3 in 20 male Sprague-Dawley rats 7 days after uninephrectomy. Two weeks after disease induction, rats were randomly treated with either everolimus or vehicle for 14 weeks. Changes in progression of renal disease were investigated by immunohistochemistry and real-time PCR 16 weeks after disease induction. Results: During chronic anti-Thy1 nephritis, the formation of focal segmental glomerulosclerosis lesions, the degree of interstitial fibrosis as well as the increase in proteinuria over 14 weeks was ameliorated by everolimus treatment. Increased glomerular hypertrophy observed in the vehicle-treated rats was completely prevented in the everolimus- treated nephritic rats. Increased glomerular fibronectin mRNA and protein as well as the renal influx of monocytes/ macrophages was significantly reduced in the evero- limus group. Everolimus reduced the pro-angiogenic factor vascular endothelial growth factor (VEGF) and VEGF mRNA in glomeruli, while the transforming growth factor- _ signaling pathway was not affected. Conclusion: ‘Late’ start of everolimus treatment demonstrates beneficial effects on the time course of chronic anti-Thy1 nephritis

Influence of TSP-2 gene therapy on VEGF/VEGF receptor expression and MMP-2 activity.

<p>VEGF and VEGF receptor 1 expression was assessed by semiquantitative scoring of immunohistochemistry. VEGF was reduced in glomeruli (A) as well as in the tubulointerstitial compartment (B) from TSP-2 treated rats. Representative pictures from VEGF stained glomeruli showing pronounced VEGF expression in podocyted as well as mesangial cells in kidneys from control plasmid treated animals (C, brown staining). In contrast, VEGF was only rarely detected in glomeruli from TSP-2 treated rats (D, brown staining). Expression of VEGF receptor 1 was similar in glomeruli (E) as well as cortex (F) of both groups. Glomerular VEGF mRNA expression was comparable in both groups (G). Serum MMP-2 activity at endpoint of the study was detected by zymography (I) and evaluated by densitometry (H). Control (n = 8) vs. TSP-2 treated (A–F:n = 8; G–I:n = 6); *p<0,05.</p

TSP-2 inhibits renal endothelial cell proliferation.

<p>Since total renal proliferation, as assessed by PCNA staining, was similar in both groups (A), TSP-2 gene therapy specifically reduced endothelial proliferative activity (B), as assessed by CD31/PCNA double positive cells (C, endothelial cells stained in brown and PCNA positive nuclei in blue). Control (n = 8) vs. TSP-2 treated (n = 8); *p<0,007. The effect of TSP-2 on proliferation of isolated rat endothelial cells was investigated by a BrdU-incorporation assay (D, n = 6; *p<0,05).</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