Bone marrow-derived cells contribute to glomerular cell turnover and repair, but the cell types involved are unknown. Whether rat mesenchymal stem cells (MSC) can accelerate recovery from damage in rat mesangioproliferative anti-Thy1.1 glomerulonephritis was studied. After injection into the left renal artery on day 2 after disease induction, fluorescently labeled MSC were detected in 20 to 50% of glomeruli and rare intrarenal vessels but not in the tubulointerstitium, in contralateral kidneys, or in medium controls. In control experiments, injected mesangial cells were detected less frequently in glomeruli in comparison with injected MSC. In nephritic outbred Wistar rats, MSC injection led to an approximately 50% reduction of mesangiolysis on days 4 and 6 after disease induction, accompanied by three-to four-fold higher intraglomerular cell proliferation on day 4 and more rapid mesangial reconstitution as detected by ␣-smooth muscle actin expression. Injection of MSC into tail veins or intra-arterial injection of mesangial cells instead of MSC failed to reproduce any of these findings. In inbred Lewis rats, anti-Thy1.1 nephritis followed an aggravated course with transient acute renal failure. Acute renal failure was ameliorated by MSC injection into the left renal artery on day 2 after disease induction. Again, MSC led to more rapid recovery from mesangiolysis, increased glomerular cell proliferation, and reduction of proteinuria by 28%. Double immunostaining of 5-bromo-2-deoxyuridine-labeled MSC for endothelial, mesangial, or monocyte/macrophage antigens showed that 85 to 95% of MSC that localized in glomeruli on day 6 failed to express these markers. In vitro, MSC secreted high amounts of vascular endothelial growth factor and TGF-␤1 but not PDGF-BB. In conclusion, even low numbers of MSC can markedly accelerate glomerular recovery from mesangiolytic damage possibly related to paracrine growth factor release and not to differentiation into resident glomerular cell types or monocytes/macrophages.

Podocyte loss contributes to the development of glomerulosclerosis. Although podocyte detachment has been recognized as a new mechanism of podocyte loss in glomerular diseases, its time course and relationship to disease activity are not known. Urinary excretion of viable podocytes was quantified in two models of transient glomerular injury, i.e., rats with puromycin aminonucleoside-induced nephrosis (PAN) and mesangioproliferative nephropathy (anti-Thy 1.1 nephritis model), as well as in a model of continuous glomerular injury, i.e., hypertensive nephropathy (5/6-nephrectomy model), and in aging rats. The number of glomerular Wilm's tumor (WT)-1-positive podocytes and the glomerular expression of cell-cycle proteins in vivo were assessed. Urinary podocyte loss occurred in both primary (PAN) and secondary (anti-Thy 1.1 nephritis) in parallel to the onset of proteinuria. However, subsequently proteinuria persisted despite remission of podocyturia. In continuous glomerular injury, i.e., after 5/6-nephrectomy, podocyturia paralleled the course of proteinuria and of systemic hypertension, whereas no podocyturia became detectable during normal aging (up to 12 mo). Despite podocyte detachment of varying degrees, no decrease in glomerular podocyte counts (i.e., WT-1 positive nuclei) was noted in either disease model. Podocyturia in the PAN and anti-Thy 1.1 nephritis model was preceded by entry of glomerular podocytes into the cell cycle, i.e., cyclin D1, cdc2, and/or proliferating cell nuclear antigen (PCNA) expression. Podocyturia is a widespread phenomenon in glomerular disease and not simply a reflection of proteinuria because it is limited to phases of ongoing glomerular injury. The data suggest that podocyturia may become a more sensitive means to assess the activity of glomerular damage than proteinuria.

Glomerulonephritis (GN) isM esenchymal stem cells (MSC) hold special promise for renal repair, because nephrons are largely of mesenchymal origin (1). The potential of MSC for renal repair has been shown in rodent models of acute renal failure (ARF), where the course of glycerol, cisplatin, or ischemia-reperfusion induced ARF was improved by MSC injection shortly after disease induction (2-5). In addition, we recently reported that injection of rat MSC into a renal artery can accelerate recovery from mesangiolytic damage and prevent transient ARF in rat anti-Thy1.1 glomerulonephritis (GN) (6). AntiThy1.1 nephritis is a model of acute mesangioproliferative glomerulonephritis and is characterized by initial mesangiolysis followed within a few days by glomerular repair via endothelial and mesangial cell proliferation and accumulation of mesangial matrix. We have also provided evidence that MSC likely exerted these effects in glomeruli by paracrine effects, such as the release of high amounts of vascular endothelial growth factor (VEGF) and TGF-␤1 rather than by differentiation into resident glomerular cell types or monocytes/macrophages (6).In this study, we investigated the long-term effects of MSC administration in early anti-Thy1.1 nephritis. Normally, antiThy1.1 nephritis in rats follows a self-limited course, and spontaneous restitution of the glomerular architecture can be observed within approximately 4 wk. For enhancement of the relevance of the model for progressive renal disease in humans, the model in this study was aggravated and transformed into a course of progressive renal failure by previous uninephrectomy of the rats (7,8). Materials and MethodsRats were housed under standard conditions in a light-, temperature-, and humidity-controlled environment with free access to tap water and standard rat diet. All animal protocols were approved by the local government authorities. Harvest and Culture of MSCInbred male Lewis rats that weighed 180 to 210 g (Harlan, Horst, Netherlands) served as bone marrow donors; MSC were prepared as described previously (6). Cells were seeded onto six-well plates (nine

Background-Aldosterone and angiotensin (Ang) II both may cause organ damage. Circulating aldosterone is produced in the adrenals; however, local cardiac synthesis has been reported. Aldosterone concentrations depend on the activity of aldosterone synthase (CYP11B2). We tested the hypothesis that reducing aldosterone by inhibiting CYP11B2 or by adrenalectomy (ADX) may ameliorate organ damage. Furthermore, we investigated how much local cardiac aldosterone originates from the adrenal gland. Methods and Results-We investigated the effect of the CYP11B2 inhibitor FAD286, losartan, and the consequences of ADX in transgenic rats overexpressing both the human renin and angiotensinogen genes (dTGR). dTGR-ADX received dexamethasone and 1% salt. Dexamethasone-treated dTGR-salt served as a control group in the ADX protocol. Untreated dTGR developed hypertension and cardiac and renal damage and had a 40% mortality rate (5/13) at 7 weeks. FAD286 reduced mortality to 10% (1/10) and ameliorated cardiac hypertrophy, albuminuria, cell infiltration, and matrix deposition in the heart and kidney. FAD286 had no effect on blood pressure at weeks 5 and 6 but slightly reduced blood pressure at week7 (177Ϯ6 mm Hg in dTGRϩFAD286 and 200Ϯ5 mm Hg in dTGR). Losartan normalized blood pressure during the entire study. Circulating and cardiac aldosterone levels were reduced in FAD286 or losartan-treated dTGR. ADX combined with dexamethasone and salt treatment decreased circulating and cardiac aldosterone to barely detectable levels. At week 7, ADX-dTGR-dexamethasone-salt had a 22% mortality rate compared with 73% in dTGR-dexamethasone-salt. Both groups were similarly hypertensive (190Ϯ9 and 187Ϯ4 mm Hg). In contrast, cardiac hypertrophy index, albuminuria, cell infiltration, and matrix deposition were significantly reduced after ADX (PϽ0.05). Conclusions-Aldosterone

IntroductionNaive B cells are poor antigen-presenting cells (APC) for naive T cells. In many systems, they have been dispensable for CD4 ϩ T-cell priming. 1,2 Yet antigen presentation by naive B cells is not an immunologic null event. Animals can be rendered tolerant toward antigens presented by naive B cells. 3 Indeed, evidence suggests that B cells can take part in antigen presentation via major histocompatibility complex (MHC) II molecules 4-7 and might even be required to reach full T-cell effector potential. 8 We have previously shown that naive B cells, despite low efficiency on a per cell basis, when loaded with specific peptide antigen can induce antigen-specific proliferation in naive T cells. 9 However, the biophysics of the underlying T-B contact was very different from bona fide T-cell activation by dendritic cells (DCs). 10 While T-cell contacts to DCs were dynamic and sequential, contacts to B cells were mostly very stable in vitro and in vivo. 9,11 The functional significance of this discrepancy is not clear.Information transfer between T cell and APC during cell-cell interaction is characterized by the formation of a supramolecular assembly of signaling and adhesion molecules termed immunologic synapse (IS). 12 Its exact function is still debated yet it is generally accepted as a T-cell activating structure. 13,14,14 A mature IS presents with a distinguished central part of the supramolecular activation cluster (c-SMAC) enriched in signaling molecules like T-cell receptor (TCR) and MHC, and a peripheral part (p-SMAC) enriched in adhesion molecules. 15 The formation of a mature IS takes between 30 to 60 minutes. 12 Encounters between DCs and T cells in early and late phases of immune responses, however, last only for several minutes. 16,17 This is too short to form a mature IS. In contrast, antigen-specific contacts to naive B cells invariably last several hours. 9 It is unclear whether a mature synapse can form under these circumstances.Based on these arguments, we reasoned that the molecular organization of the IS between T cells and naive B cells might be different from the one found in T-DC pairs. We also wanted to test the potential consequences for the resulting activated CD4 ϩ T cells. To further evaluate the outcome of antigen-specific CD4 ϩ T-cell stimulation by naive B cells, we investigated the molecular structure of the underlying IS and the phenotype of CD4 ϩ T cells after in vitro contact with specific-antigen loaded naive B cells or DCs.We present evidence that stimulation of naive T cells by naive B cells results in formation of a mature IS which is absent in DC-T-cell pairs. In addition, while DC stimulation generates classical effector T cells, naive B-cell-activated T cells show regulatory capacity in vitro and in vivo. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 10, 2018. by gue...

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Complement activation plays a key role in mediating apoptosis, inflammation, and transplant rejection. In this study, the role of the complement 5a receptor (C5aR) was examined in human renal allografts and in an allogenic mouse model of renal transplant rejection. In human kidney transplants with acute rejection, C5aR expression was increased in renal tissue and in cells infiltrating the tubulointerstitium. Similar findings were observed in mice. When recipient mice were treated once daily with a C5aR antagonist before transplantation, long-term renal allograft survival was markedly improved compared with vehicle-treatment (75 versus 0%), and apoptosis was reduced. Furthermore, treatment with a C5aR antagonist significantly attenuated monocyte/macrophage infiltration, perhaps a result of reduced levels of monocyte chemoattractant protein 1 and the intercellular adhesion molecule 1. In vitro, C5aR antagonism inhibited intercellular adhesion molecule 1 upregulation in primary mouse aortic endothelial cells and reduced adhesion of peripheral blood mononuclear cells. Furthermore, C5aR blockade markedly reduced alloreactive T cell priming. These results demonstrate that C5aR plays an important role in mediating acute kidney allograft rejection, suggesting that pharmaceutical targeting of C5aR may have potential in transplantation medicine.

Dysregulation of the actin cytoskeleton in podocytes represents a common pathway in the pathogenesis of proteinuria across a spectrum of chronic kidney diseases (CKD). The GTPase dynamin has been implicated in the maintenance of cellular architecture in podocytes through its direct interaction with actin. Furthermore, the propensity of dynamin to oligomerize into higher-order structures in an actin-dependent manner and to crosslink actin microfilaments into higher order structures have been correlated with increased actin polymerization and global organization of the actin cytoskeleton in the cell. We found that use of the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus increased actin polymerization in injured podocytes, was sufficient to improve renal health in diverse models of both transient kidney disease and of CKD. In particular, administration of Bis-T-23 in these renal disease models restored the normal ultrastructure of podocyte foot processes, lowered proteinuria, lowered collagen IV deposits in the mesangial matrix, diminished mesangial matrix expansion and extended lifespan. These results further establish that alterations in the actin cytoskeleton of kidney podocytes is a common hallmark of CKD, while also underscoring the significant regenerative potential of injured glomeruli and that targeting the oligomerization cycle of dynamin represents an attractive potential therapeutic target to treat CKD.