Recent data suggest that the phosphatidylinositol 3-kinase (PI3-K)/Akt/mammalian target of rapamycin (mTOR) pathway is important in diabetic nephropathy. The effect of mTOR blockade by sirolimus (SRL) in diabetic kidney disease in rats was investigated. Diabetes was induced by streptozotocin in male Sprague-Dawley rats. Sixteen weeks later, diabetic animals were divided into the following groups: diabetes (D; n ‫؍‬ 8), diabetes ؉ SRL at 1 mg/kg per d, SRL trough level 2.3 ؎ 0.25 ng/ml (D؉SRL; n ‫؍‬ 7); and diabetes ؉ normoglycemia maintained by insulin implants (D؉NG; n ‫؍‬ 5). There was an age-matched nondiabetic group (ND; n ‫؍‬ 6). All animals were followed for 4 wk. The D group showed glomerular hypertrophy (mean glomerular volume 5.0 ؎ 0.4 in D versus 3.3 ؎ 0.2 10 6 3 in ND; P < 0.05) without renal hyperplasia (calculated by reverse transcription-PCR of proliferative cell nuclear antigen) and albuminuria (29 ؎ 4 in D versus 1.4 ؎ 1.5 mg/24 h in ND; P < 0.05). Both D؉NG and D؉SRL groups had a significant reduction of albuminuria, although glomerular hypertrophy was still present. SRL treatment did not modify the number of infiltrating renal ED1 ؉ cells. Diabetic animals had greater expression of p-Akt and mTOR, unlike ND rats. NG and SRL treatment reduced p-Akt and normalized mTOR. It is interesting that D؉SRL was associated with a significant reduction of renal TGF-␤1 and glomerular connective tissue growth factor. SRL treatment reduced glomerular ␣-smooth muscle actin overexpression and reduced significantly the mesangial matrix accumulation that is characteristic of diabetic nephropathy. In conclusion, mTOR blockade by low-dose SRL has a beneficial effect in diabetic kidney disease, suggesting that the mTOR pathway has an important pathogenic role in diabetic nephropathy.

In solid organ transplantation, mesenchymal stem cell (MSC) therapy is strongly emerging among other cell therapies due to the positive results obtained in vitro and in vivo as an immunomodulatory agent and their potential regenerative role. We aimed at testing whether a single dose of MSCs, injected at 11 weeks after kidney transplantation for the prevention of chronic mechanisms, enhanced regeneration and provided protection against the inflammatory and fibrotic processes that finally lead to the characteristic features of chronic allograft nephropathy (CAN). Either bone marrow mononuclear cells (BMCs) injection or no-therapy (NT) were used as control treatments. A rat kidney transplantation model of CAN with 2.5 h of cold ischemia was used, and functional, histological, and molecular parameters were assessed at 12 and 24 weeks after transplantation. MSC and BMC cell therapy preserves renal function at 24 weeks and abrogates proteinuria, which is typical of this model (NT24w: 68.9 -26.5 mg/24 h, MSC24w: 16.6 -2.3 mg/24 h, BMC24w: 24.1 -5.3 mg/24 h, P < 0.03). Only MSC-treated animals showed a reduction in interstitial fibrosis and tubular atrophy (NT24w: 2.3 -0.29, MSC24w: 0.4 -0.2, P < 0.03), less T cells (NT: 39.6 -9.5, MSC: 8.1 -0.9, P < 0.03) and macrophages (NT: 20.9 -4.7, MSC: 5.9 -1.7, P < 0.05) infiltrating the parenchyma and lowered expression of inflammatory cytokines while increasing the expression of anti-inflammatory factors. MSCs appear to serve as a protection from injury development rather than regenerate the damaged tissue, as no differences were observed in Ki67 expression, and kidney injury molecule-1, Clusterin, NGAL, and hepatocyte growth factor expression were only up-regulated in nontreated animals. Considering the results, a single delayed MSC injection is effective for the long-term protection of kidney allografts.

Macrophage infiltration is a common feature of the early phase of renal ischaemia/reperfusion injury. Indeed, it is generally regarded as the cause of tissue injury in this phase, although it is also clear that it can lead to tissue repair in other phases. In order to ascertain whether macrophages are directly involved in the repair/late phase, which follows the proinflammatory and injury process of renal ischaemia/reperfusion, we used two different approaches based on macrophage depletion. Firstly, we produced renal ischaemia in mice that were previously treated with clodronate liposome. Secondly, during reperfusion we re-injected RAW 264.7 to macrophage-depleted mice 24 h prior to sacrifice. The results showed that regeneration, as evaluated by stathmin and PCNA markers, was macrophagedependent: it was blocked when macrophage depletion was provoked and recovered with macrophage re-injection. The cytokine profile revealed the influence of the inflammatory environment on kidney repair: pro-inflammatory cytokines (MCP-1, MIP-1α) increased during the early stages of reperfusion, coinciding with low regeneration, and the antiinflammatory cytokine IL-10 increased during the longer periods of reperfusion when regeneration was more evident. We conclude that macrophages induce renal regeneration after ischaemia/reperfusion, depending on the inflammatory milieu.

Fifteen minutes of warm ischemia and 10 minutes of reperfusion in the kidney is the most suitable one-cycle schedule for preconditioning since it protects from both warm and cold ischemia. The beneficial effect of preconditioning is related to the local production of NO, and we believe it has promising therapeutic value in clinical renal transplantation.

The 'injury hypothesis' in organ transplantation suggests that ischemia-reperfusion injury is involved in the adaptative alloimmune response. We previously found that a strong immune/inflammatory response was induced by ischemia during kidney transplantation in rats. We show here that immature dendritic cells (DCs) undergo hypoxia-mediated differentiation comparable to allogeneic stimulation. Hypoxia-differentiated DCs overexpress hypoxia inducible factor-1alpha (HIF-1alpha) and its downstream target genes, such as vascular endothelial growth factor or glucose transporter-1. Rapamycin attenuated DC differentiation, HIF-1alpha expression, and its target gene expression in a dose-dependent manner along with downregulated interleukin-10 secretion. Coculture of hypoxia-differentiated DCs with CD3 lymphocytes induced proliferation of lymphocytes, a process also neutralized by rapamycin. Furthermore, in vivo examination of ischemia-reperfusion-injured mouse kidneys showed a clear maturation of resident DCs that was blunted by rapamycin pretreatment. Our results suggest that hypoxia is a central part of the 'injury hypothesis' triggering DC differentiation under hypoxic conditions. Rapamycin attenuates the hypoxic immune-inflammatory response through inhibition of the HIF-1alpha pathway.

This study assesses the individual contributions of the nonalloreactive factor, cold ischemia (CI), and alloreactivity to late functional and structural renal graft changes, and examines the effect of the association of both factors on the progression of chronic allograft nephropathy. Lewis rats acted as receptors of kidneys from either Lewis or Fischer rats. For CI, kidneys were preserved for 5 hours. The rats were divided into four groups: Syn, syngeneic graft; SynI, syngeneic graft and CI; Allo, allogeneic graft; AlloI, allogeneic graft and CI. Renal function was assessed every 4 weeks for 24 weeks. Grafts were evaluated for acute inflammatory response at 1 week and for chronic histological damage at 24 weeks. Only when CI and allogenicity were combined did immediate posttransplant mortality occur, while survivors showed accelerated renal insufficiency that induced further mortality at 12 weeks after transplant. Solely ischemic rats developed renal insufficiency. Renal structural damage in ischemic rats was clearly tubulointerstitial, while significant vasculopathy and glomerulosclerosis appeared only in the allogeneic groups. There was increased infiltration of macrophages and expression of mRNA-transforming growth factor-␤1 in the ischemic groups, irrespective of the allogeneic background. The joint association of CI plus allogenicity significantly increased cellular infiltration at both early and late stages, aggravating tubulointerstitial and vascular damage considerably. In summary, CI is mainly responsible for tubulointerstitial damage, whereas allogenicity leads to vascular lesion. Chronic allograft nephropathy (CAN) is a multifactorial process that leads to late allograft loss in renal transplantation and is caused by both alloreactive and nonalloreactive factors.

Diabetic nephropathy is the main cause of end-stage renal disease requiring dialysis in developed countries.In this study, we demonstrated the therapeutic effect of hepatocyte growth factor (HGF) on advanced rather than early diabetic nephropathy using a rat model of streptozotocin-induced diabetes. Early diabetic nephropathy (16 weeks after induction of diabetes) was characterized by albuminuria, hyperfiltration, and glomerular hypertrophy, whereas advanced diabetic nephropathy showed prominent transforming growth factor (TGF)-␤1 upregulation, mesangial expansion, and glomerulosclerosis. An SP1017-formulated human HGF (hHGF) plasmid was administered by intramuscular injection combined with electroporation over a 30-day follow-up in rats with early and advanced diabetic nephropathy. hHGF gene therapy upregulated endogenous rat HGF in the diabetic kidney (rat HGF by RT-PCR was threefold higher than in diabetic rats without therapy). hHGF gene therapy did not improve functional or morphologic abnormalities in early diabetic nephropathy. hHGF gene therapy reduced albuminuria and induced strong regression of mesangial expansion and glomerulosclerosis in advanced diabetic nephropathy. These findings were associated with suppression of renal TGF-␤1 and mesangial connective tissue growth factor (CTGF) upregulation, inhibition of renal tissue inhibitor of metalloproteinase (TIMP)-1 expression, and reduction of renal interstitial myofibroblasts. In conclusion, our results suggest that hHGF gene therapy may be considered as an innovative therapeutic strategy to treat advanced diabetic nephropathy.

Inflammatory processes and tissue scarring are characteristic features of chronic allograft nephropathy. Hepatocyte growth factor (HGF) has beneficial effects on renal fibrosis and it also ameliorates renal interstitial inflammation as it has been recently described. Contrarily to protein administration, intramuscular gene electrotransfer allows sustained release of HGF. So, here we hypothesized that gene therapy with human HGF would diminish the characteristic scarring of chronic allograft nephropathy either by antagonizing tissue fibrosis mechanisms or by reducing inflammation. Lewis rats transplanted with cold preserved Fischer kidneys received vehicle (NoHGF) or intramuscular plasmid DNA encoding HGF plus electroporation either before transplantation (IniHGF, early post-transplant cytoprotection of tubular cells) or 8/10 weeks after transplantation (DelHGF, delayed prevention of chronic mechanisms). Serum creatinine and proteinuria were measured every 4 weeks for 24 weeks. Grafts at 12 or 24 weeks were evaluated for glomerulosclerosis, fibrosis inflammatory cells and mediators, cell regeneration and tubulo-interstitial damage. Nontreated animals developed renal insufficiency, progressive proteinuria and fibrosis among other characteristic histological features of chronic allograft nephropathy. Treatment with human HGF, especially when delayed until the onset of fibrogenic mechanisms, reduced renal failure and mortality, diminished tubule-interstitial damage, induced cell regeneration, decreased inflammation, NF-kappaB activation, and profibrotic markers at 12 weeks and prevented late interstitial fibrosis and glomerulosclerosis. The effectiveness of HGF-gene therapy in the prevention of renal allograft scarring is related with the halt of profibrotic inflammatory-induced mechanisms.