Heleen Rienstra

Spironolactone ameliorates transplant vasculopathy in renal chronic transplant dysfunction in rats.

Chronic transplant dysfunction (CTD) is the leading cause of long-term renal allograft loss and is characterized by specific histological lesions including transplant vasculopathy, interstitial fibrosis, and focal glomerulosclerosis. Increasing evidence indicates that aldosterone is a direct mediator of renal damage via the mineralocorticoid receptor (MR). The MR antagonist spironolactone is renoprotective in native chronic kidney disease, but its effects on CTD are unknown. We studied the effects of spironolactone treatment on CTD development in the Dark Agouti-to-Wistar-Furth renal allograft transplant model, by treatment with 20 mg/kg spironolactone or vehicle daily by oral gavage from 2 days before transplantation (donors and recipients) throughout the experiment (12 wk, recipients). Dark Agouti-to-Dark Agouti isografts served as negative controls. Spironolactone significantly ameliorated the development of transplant vasculopathy in allografts by reducing the number of affected intrarenal arteries. In addition, spironolactone treatment showed a trend toward reduced proteinuria and focal glomerulosclerosis, and significantly reduced glomerular macrophage influx. However, spironolactone treatment did not affect interstitial fibrosis, interstitial macrophage influx, creatinine clearance, and systolic blood pressure. We conclude that spironolactone selectively ameliorates transplant vasculopathy and glomerular lesions in renal CTD in rats. These results suggest that spironolactone may have renoprotective potential as an adjunct treatment in renal transplantation to ameliorate CTD.

Donor and recipient origin of mesenchymal and endothelial cells in chronic renal allograft remodeling.

Chronic transplant dysfunction (CTD) is the leading cause for limited kidney graft survival. Renal CTD is characterized by interstitial and vascular remodeling leading to interstitial fibrosis, tubular atrophy and transplant vasculopathy (TV). The origin of cells and pathogenesis of interstitial and vascular remodeling are still unknown. To study graft‐versus‐recipient origin of interstitial myofibroblasts, vascular smooth muscle cells (SMCs) and endothelial cells (ECs), we here describe a new rat model for renal CTD using Dark Agouti kidney donors and R26 human placental alkaline phosphatase transgenic Fischer344 recipients. This model showed the development of CTD within 12 weeks after transplantation. In interstitial remodeling, both graft‐ and recipient‐derived cells contributed to a similar extent to the accumulation of myofibroblasts. In arteries with TV, we observed graft origin of neointimal SMCs and ECs, whereas in peritubular and glomerular capillaries, we detected recipient EC chimerism. These data indicate that, within the interstitial and vascular compartments of the transplanted kidney, myofibroblasts, SMCs and ECs involved in chronic remodeling are derived from different sources and suggest distinct pathogenetic mechanisms within the renal compartments.

Development of transplant vasculopathy in aortic allografts correlates with neointimal smooth muscle cell proliferative capacity and fibrocyte frequency

OBJECTIVE Transplant vasculopathy consists of neointima formation in graft vasculature resulting from vascular smooth muscle cell recruitment and proliferation. Variation in the severity of vasculopathy has been demonstrated. Genetic predisposition is suggested as a putative cause of this variation, although cellular mechanisms are still unknown. Using a rat aorta transplant model we tested the hypothesis that kinetics of development of transplant vasculopathy are related to neointimal smooth muscle cell proliferative capacity and fibrocyte frequency, the latter being putative neointimal smooth muscle ancestral cells. METHODS Aortic allografts were transplanted in Lewis and Brown Norway, as well as MHC-congenic Lewis.1N and Brown Norway.1L recipients. Severity of transplant vasculopathy was quantified 4, 8, 12 and 24 weeks after transplantation. Host-endothelial chimerism, as a reflection of vascular injury, was determined by specific immunofluorescence. Neointimal smooth muscle cell proliferative capacity was determined in vitro and in situ. Fibrocyte frequency and phenotype were determined after in vitro culture by cell counting, immunofluorescence and in situ zymography. RESULTS Compared to Lewis, Brown Norway recipients developed accelerated transplant vasculopathy which is dependent on the presence of Brown Norway non-MHC-encoded determinants. Accelerated transplant vasculopathy was associated with increased levels of host-endothelial chimerism and increased neointimal smooth muscle cell proliferation, the latter being accompanied by increased endothelial and smooth muscle cell-derived neuropilin-like protein mRNA expression. Moreover, accelerated transplant vasculopathy was associated with increased frequency of circulating gelatinase-expressing CD45(+)vimentin(+) fibrocytes. CONCLUSION Susceptibility for transplant vasculopathy appears to be genetically controlled and correlates with neointimal smooth muscle cell proliferative capacity and circulating fibrocyte frequency.

Donor and Recipient Contribution to Transplant Vasculopathy in Chronic Renal Transplant Dysfunction

Background. Chronic transplant dysfunction is the leading cause of long-term renal allograft loss. One of the histologic hallmarks of chronic transplant dysfunction is transplant vasculopathy characterized by accumulation of smooth muscle cells (SMCs) in the arterial subendothelial space, leading to ischemic graft failure. Currently, no therapy is available for transplant vasculopathy, and knowledge of the origin (donor vs. recipient) of neointimal cells may contribute to develop adequate strategies. Methods. Origin of neointimal SMCs, endothelial, and tubular cells was determined in four nephrectomy samples from male recipients transplanted with a female kidney. Recipient-derived cells were detected using X- and Y-chromosome-specific fluorescent in situ hybridization combined with immunofluorescent staining. Specificity and sensitivity of fluorescent in situ hybridization were determined with corresponding controls. Results. No Y-chromosome-positive cells were detected in the female to female graft, whereas approximately 31% of nucleated cells in male to male grafts had a detectable Y-chromosome. In female to male grafts, a recipient-derived population of neointimal &agr;-smooth muscle actin-positive SMCs were detected (6%, range 3%–11%). Percentages of recipient-derived arterial endothelial cells, glomerular endothelial cells, and tubular epithelial cells were 14% (range 4%–32%), 19% (range 7%–31%) and 3% (range 2%–5%), respectively. Conclusions. Both donor- and recipient-derived cells contribute to vascular remodeling in clinical renal transplantation. The presence of &agr;-smooth muscle actin in donor- and recipient-derived cells supports a constructive role for these cells in neointimal formation. However, the predominance of donor-derived cells in the neointima points to these cells as the likely therapeutic target.

Local Medial Microenvironment Directs Phenotypic Modulation of Smooth Muscle Cells After Experimental Renal Transplantation

Smooth muscle cells (SMCs) play a key role in the pathogenesis of occlusive vascular diseases, including transplant vasculopathy. Neointimal SMCs in experimental renal transplant vasculopathy are graft‐derived. We propose that neointimal SMCs in renal allografts are derived from the vascular media resulting from a transplantation‐induced phenotypic switch. We examined the molecular changes in the medial microenvironment that lead to phenotypic modulation of SMCs in rat renal allograft arteries with neointimal lesions. Dark Agouti donor kidneys were transplanted into Wistar Furth recipients and recovered at day 56. Neointimal and medial layers were isolated using laser microdissection. Gene expression was analyzed using low‐density arrays and confirmed by immunostaining. In allografts, neointimal SMCs expressed increased levels of Tgf β1 and Pdgfb. In medial allograft SMCs, gene expression of Ctgf, Tgf β1 and Pdgfrb was upregulated. Gene expression of Klf4 was upregulated as well, while expression of Sm22α was downregulated. Finally, PDGF‐BB‐stimulated phenotypically modulated SMCs, as evidenced by reduced contractile function in vitro which was accompanied by increased Klf4 and Col1α1, and reduced α‐Sma and Sm22α expression. In transplant vasculopathy, neointimal PDGF‐BB induces phenotypic modulation of medial SMCs, through upregulation of KLF4 in the media to contribute to (further) expansion of the neointima.

Rosiglitazone attenuates transplant arteriosclerosis after allogeneic aorta transplantation in rats.

Background. Transplant arteriosclerosis is a leading cause of chronic transplant dysfunction and is characterized by occlusive neointima formation in intragraft arteries. Development of transplant arteriosclerosis is refractory to conventional immunosuppressive drugs and adequate therapy is not available. In this study, we determined the efficacy of the synthetic peroxisome proliferator-activated receptor (PPAR)-&ggr; agonist rosiglitazone to attenuate the development of transplant arteriosclerosis in rat aortic allografts. Methods. Lewis aortic allografts were transplanted into Brown Norway recipient rats. Recipient rats received either ∼5 mg rosiglitazone/day (starting 1 week before transplantation until the end of the experiment) or were left untreated. Transplant arteriosclerosis was quantified using morphometric analysis. Alloreactivity was measured in vitro using mixed lymphocyte reactions. Regulatory T cell frequency and function were analyzed using flow cytometry and in vitro suppression assays, respectively. Intragraft gene expression was analyzed using real-time polymerase chain reaction. Finally, medial and neointimal vascular smooth muscle cell proliferation was analyzed in vitro. Results. Rosiglitazone significantly reduced transplant arteriosclerosis development 8 weeks after transplantation (P<0.01 vs. nontreated). Rosiglitazone reduced T cell alloreactivity which was not mediated through modulation of CD4+CD25+FoxP3+ regulatory T cells. Reduced development of transplant arteriosclerosis coincided with reduced intragraft expression of stromal-derived factor-1&agr; and platelet-derived growth factor receptor-&bgr;. Finally, rosiglitazone reduced growth-factor-driven proliferation of both medial and neointimal vascular smooth muscle cells in vitro, which was not mediated through PPAR&ggr;. Conclusion. PPAR&ggr; agonists may offer a new therapeutic strategy in clinical transplantation to attenuate the development of transplant arteriosclerosis and thereby chronic transplant dysfunction.

Renal heparan sulfate proteoglycans modulate fibroblast growth factor 2 signaling in experimental chronic transplant dysfunction.

Depending on the glycan structure, proteoglycans can act as coreceptors for growth factors. We hypothesized that proteoglycans and their growth factor ligands orchestrate tissue remodeling in chronic transplant dysfunction. We have previously shown perlecan to be selectively up-regulated in the glomeruli and arteries in a rat renal transplantation model. Using the same model, here we present quantitative RT-PCR profiling data on proteoglycans and growth factors from laser-microdissected glomeruli, arterial tunicae mediae, and neointimae at 12 weeks after transplantation. In glomeruli and neointimae of allografts, selective induction of the matrix heparan sulfate proteoglycan perlecan was observed, along with massive accumulation of fibroblast growth factor 2 (FGF2). Profiling the heparan sulfate polysaccharide side chains revealed conversion from a non-FGF2-binding heparan sulfate phenotype in control and isografted kidneys toward a FGF2-binding phenotype in allografts. In vitro experiments with perlecan-positive rat mesangial cells showed that FGF2-induced proliferation is dependent on sulfation and can be inhibited by exogenously added heparan sulfate. These findings indicate that matrix proteoglycans such as perlecan serve as functional docking platforms for FGF2 in chronic transplant dysfunction. We speculate that heparin-like glycomimetics could be a promising intervention to retard development of glomerulosclerosis and neointima formation in chronic transplant dysfunction.

Dichotomous effects of rosiglitazone in transplantation-induced systemic vasodilator dysfunction in rats

Background. Transplantation-induced systemic endothelial dysfunction causes severe cardiovascular morbidity and mortality after transplantation. Interventions that improve systemic endothelial function after transplantation and furthermore reduce intragraft vascular dysfunction might improve graft and patient survival. Treatment with the PPAR&ggr; agonist rosiglitazone is an intervention that potentially fulfills these criteria. In this study, we determined the effect of rosiglitazone treatment on transplantation-induced endothelial dysfunction and vasomotor activity in an experimental model for chronic transplant dysfunction in rats. Methods. Lewis abdominal aortic allografts were orthotopically transplanted into Brown Norway recipients that received either regular chow or chow containing rosiglitazone (∼4.2 mg/day). Endothelium-dependent (response to metacholine) and total (response to sodium nitrite) vasodilatory responses were determined in autologous thoracic aortic rings using an ex vivo organ bath setup. Measurements were performed 8 weeks after transplantation. Results. Aortic allografting induced systemic endothelial dysfunction as measured by reduced endothelium-dependent vasodilation in the recipient’s vascular system. Rosiglitazone treatment restored endothelium-dependent vasodilatory responses to pretransplantation levels. However, rosiglitazone treatment reduced the total dilatory response despite normalized endothelial function, indicating impairment of vascular smooth muscle cell vasomotor activity. Conclusions. Rosiglitazone treatment after allogeneic transplantation restores endothelial function but impairs vascular smooth muscle cell vasomotor activity. This dichotomous effect of rosiglitazone might impede use of rosiglitazone after organ transplantation since this potentially increases cardiovascular risk despite improved endothelial cell function.

The Source of Neointimal Cells in Vein Grafts: Does the Origin Matter?

The focus of this Commentary is the existing plasticity in the origin of neointimal vascular smooth muscle cells and endothelial cells after vein grafting.

SPIRONOLACTONE AMELIORATES FOCAL GLOMERULOSCLEROSIS AND TRANSPLANT ARTERIOPATHY IN EXPERIMENTAL CHRONIC ALLOGRAFT NEPHROPATHY IN RATS: 1528

Background. Chronic allograft nephropathy (CAN) is the leading cause of long-term renal allograft loss, for which no effective therapy is available. Ischemia/reperfusion injury at transplantation predisposes to development of CAN. Increasing evidence indicates that aldosterone is a direct mediator of renal damage via the mineralocorticoid receptor (MR). The aldosterone antagonist spironolactone attenuates renal ischemia/reperfusion injury in rats. Thus, aldosterone blockade might protect against CAN. In this study we determined the efficacy of spironolactone in attenuating CAN after experimental renal transplantation in rats. Methods. Renal transplantation was performed in the allogeneic Dark Agouti-to-Wistar Furth rat strain combination. Dark Agouti-to-Dark Agouti isografts served as controls. Recipients received either spironolactone (20 mg/kg BW) (+) or vehicle (-) daily by oral gavage until the end of the experiment. Treatment started 2 days prior to transplantation. 4 experimental groups were included: 1) allograft(n=9), 2) allograft+ (n=9), 3) isograft(n=8) and 4) isograft+ (n=8). Before and every 2 weeks after transplantation systolic blood pressure and urinary protein excretion were determined. Grafts were retrieved 12 weeks after transplantation and processed for histological analyses (focal glomerulosclerosis (FGS), interstitial fibrosis (IF), transplant arteriopathy (TA) and macrophage influx). Results. Isograft recipients did not develop proteinuria. In contrast, allograft recipients developed clear proteinuria which was attenuated by spironolactone (77±28 mg/day [group 1] vs. 41±10 mg/day [group 2]; p=0.06). In time, spironolactone did not affect systolic blood pressure. In contrast to isografts, allografts presented with prominent FGS and IF. Spironolactone significantly attenuated the severity of FGS in allografts (100±37 A.U. [group 1] vs 47±17 A.U. [group 2], p<0.05) without affecting IF. Reduced FGS in the allografts treated with spironolactone was accompanied by reduced glomerular macrophage influx (p<0.05, group 1 vs. group 2). Allografts developed TA which was significantly ameliorated (p<0.05) by spironolactone. Conclusion. MR blockade by the aldosterone antagonist spironolactone ameliorates the development of proteinuria, FGS and TA in an experimental rat model for CAN, suggesting a role for aldosterone in the pathogenesis of CAN. We propose that the observed glomeruloand vasoprotective effects of spironolactone might result in improved long-term graft survival. PF Hfdst 4:Proefschrift 18-08-2008 13:01 Pagina 98