Jürgen Wagner

13-cis Retinoic Acid Inhibits Development and Progression of Chronic Allograft Nephropathy

Chronic allograft nephropathy is characterized by chronic inflammation and fibrosis. Because retinoids exhibit anti-proliferative, anti-inflammatory, and anti-fibrotic functions, the effects of low and high doses of 13-cis-retinoic acid (13cRA) were studied in a chronic Fisher344-->Lewis transplantation model. In 13cRA animals, independent of dose (2 or 20 mg/kg body weight/day) and start (0 or 14 days after transplantation) of 13cRA administration, serum creatinine was significantly lower and chronic rejection damage was dramatically reduced, including subendothelial fibrosis of preglomerular vessels and chronic tubulointerstitial damage. The number of infiltrating mononuclear cells and their proliferative activity were significantly diminished. The mRNA expression of chemokines (MCP-1/CCL2, MIP-1alpha/CCL3, IP-10/CXCL10, RANTES/CCL5) and proteins associated with fibrosis (plasminogen activator inhibitor-1, transforming growth factor-beta1, and collagens I and III) were strikingly lower in treated allografts. In vitro, activated peritoneal macrophages of 13cRA-treated rats showed a pronounced decrease in protein secretion of inflammatory cytokines (eg, tumor necrosis factor-alpha, interleukin-6). The suppression of the proinflammatory chemokine RANTES/CCL5 x 13cRA in fibroblasts could be mapped to a promoter module comprising IRF-1 and nuclear factor-kappaB binding elements, but direct binding of retinoid receptors to promoter elements could be excluded. In summary, 13cRA acted as a potent immunosuppressive and anti-fibrotic agent able to prevent and inhibit progression of chronic allograft nephropathy.

Differential regulation of transforming growth factor receptors by angiotensin II and transforming growth factor-β1 in vascular smooth muscle

Abstractu2002Angiotensin II (Ang II) and transforming growth factor (TGF) β1 play a role in vascular remodeling in hypertension. In this process they may interact on various levels, including that of receptor regulation. This consideration prompted the present study on transcriptional regulation of TGF-β receptors by Ang II and TGF-β in vascular smooth muscle cells. Transcriptional expression of the components of the TGF-β system was demonstrated for TGF-β and for TGF-β receptors I, II, and III. As measured by quantitative reverse transcriptase polymerase chain reaction, TGF-β mRNA increased about 2.4-fold in the presence of 40 pM exogenous TGF-β. Ang II at 10–6 M increased TGF-β mRNA 2.5-fold compared to control cells (P<0.05). Ang II also significantly increased TGF-β protein concentration in the supernatant of confluent vascular smooth muscle cells. Ang II caused the induction of TGF-β, but short-term experiments showed TGF-β receptor II mRNA to be differentially regulated by Ang II and TGF-β; while TGF-β caused a 40% decrease in TGF-β receptor II mRNA after 4 h (P<0.05), Ang II caused an increase by about 70%. In contrast, both TGF-β and Ang II increased TGF-β receptor I mRNA to about 260% or 180% of controls (P<0.05). TGF-β effects were abrogated by coincubation with a TGF-β neutralizing antibody, and Ang II effects were abrogated by losartan, an AT-1 receptor antagonist. Coincubation of Ang II with the TGF-β neutralizing antibody did not inhibit the effect of Ang II, indicating that the short-term effects of Ang II on the expression of the TGF-β receptors are not mediated via TGF-β. Furthermore, Ang II stimulated DNA synthesis even in the presence of the TGF-β neutralizing antibody. In conclusion, this study indicates (a) that in vascular smooth muscle TGF-β receptors are regulated on the RNA level by TGF-β and Ang II, and (b) that Ang II dependent regulation of TGF-β receptors is at least partially independent of endogenous TGF-β. Stimulation of the transcriptional expression of TGF-β receptors by Ang II may increase sensitivity of vascular smooth muscle cells to TGF-β.

Isotretinoin ameliorates renal damage in experimental acute renal allograft rejection

Background. Retinoic acids, derivatives of vitamin A, act through retinoid receptors that are expressed in renal and immunocompetent cells (B and T cells; monocytes and macrophages). In experimental models of glomerulonephritis and renal interstitial disease, retinoids were shown to reduce both glomerular and tubular damage and inflammation. We therefore examined whether retinoids reduce cellular rejection and renal damage in a model of acute renal allograft rejection. Methods. Kidneys of Fisher rats (F344, RT11v1) were orthotopically grafted to Lewis rats (RT11). Animals were killed 7 or 14 days after transplantation. Rats undergoing transplantation were treated with isotretinoin (13 cis-retinoic acid) at a low dose of 2 mg/kg body weight per day (LD isotretinoin) or at a high dose of 20 mg/kg body weight per day (HD isotretinoin) or with vehicle. Results. At day 14, albuminuria was reduced by approximately 70% (vehicle: 1.1±0.2 mg/24 hr vs. LD isotretinoin: 0.32±0.1 mg/24 hr; P <0.001). At days 7 and 14 serum creatinine levels were significantly higher in the vehicle-treated group than in the LD and HD isotretinoin-treated rats (P <0.05). Both LD and HD isotretinoin significantly reduced acute vascular injury compared with vehicle-treated rats (score at day 14: LD isotretinoin 20.1±5.1 vs. vehicle 57.8±9.9, P <0.01), acute glomerular injury (score: LD isotretinoin 6.8±1.0 vs. vehicle 10.6±0.9 P <0.05), and the number of glomerular monocytes and macrophages and cytotoxic T cells. Isotretinoin also significantly lessened tubulointerstitial damage, tubulointerstitial cell proliferation, and the number of cells infiltrating the tubulointerstitium. Conclusions. Isotretinoin significantly ameliorated functional, vascular, glomerular, and tubulointerstitial lesions in acute graft rejection. Although the current study did not definitely eliminate the possibility that isotretinoin only delayed the rejection process, retinoic acid derivatives may provide a new approach in the treatment of acute rejection injury.

Efficacy of cationic liposome-mediated gene transfer to mesangial cells in vitro and in vivo

Mesangial cells represent a major target for gene transfer approaches to the kidney. To establish a liposome-based system for transfection of mesangial cells we analyzed the efficacy and toxicity of different cationic liposomes and other nonviral transfection methods in primary cultures of rat and human mesangial cells using the Escherichia coli β-galactosidase (lacZ) gene as a marker. In addition, an expression vector containing a human renin cDNA under the control of the cytomegalovirus immediate-early promoter/enhancer was generated, introduced into mesangial cells, and assayed in a system of transient gene expression. In vivo, gene transfer was studied after infusion of liposome/DNA complexes in the kidney of rats via the renal artery. Transfection efficiency ranged from 5.5% with DMRIE Liposomes in rat mesangial cells to 1.1% with LipofectAmine liposomes in human mesangial cells. Cytotoxicity following transfection was dependent on the transfection method. Transfection with the human renin expression vector led to the secretion of 11xa0pg/104 cells/48xa0h human renin in rat mesangial cells, 3600xa0pg/104 cells/48xa0h in 293 cells, and 113xa0pg/104 cells/48xa0h human renin in opossum kidney cells. In vivo, infusion of liposomes was accompanied by nephrotoxicity and did not result in marker gene expression. Together the data demonstrate that cationic liposomes are useful tools for transferring genes into mesangial cells, including human mesangial cells. Cationic liposomes provide a functional system for the synthesis and secretion of human renin in mesangial cells and other mammalian kidney cells. The current limitation of the evaluated liposomes for an efficient in vivo gene transfer to mesangial cells is the toxicity upon intrarenal arterial administration.

Repression of c-fos and c-jun gene expression is not part of AT2 receptor coupled signal transduction

Abstractu2002The signal transduction mechanism coupled to angiotensin AT2 receptors is still a matter of debate. Based on the findings that AT2 receptor stimulation causes inhibition of proliferation, and that other antiproliferative agents such as transforming growth factor-β, retinoic acid, and MyoD act via repression of immediate early gene (IEG) expression, this study was aimed at elucidating whether downregulation of IEG expression is also part of the AT2 receptor coupled signaling mechanism. Stimulation of angiotensin AT2 receptors in the rat pheochromocytoma cell line PC12xa0W following pretreatment with growth factors was able to counteract growth factor induced proliferation but not to repress growth factor induced c-fos and c-jun expression; neither did AT2 receptor stimulation cause an induction of c-fos expression. We conclude that, in contrast to other growth-inhibiting agents, the antiproliferative effect of angiotensin II via the AT2 receptor is not mediated by repression of the immediate early genes c-fos and c-jun.

Sotrastaurin (AEB071) alone and in combination with cyclosporine A prolongs survival times of non-human primate recipients of life-supporting kidney allografts.

Background. Sotrastaurin (STN), a novel oral protein kinase C inhibitor that inhibits early T-cell activation, was assessed in non-human primate recipients of life-supporting kidney allografts. Methods. Cynomolgus monkey recipients of life-supporting kidney allografts were treated orally with STN alone or in combination with cyclosporine A (CsA). Results. STN monotherapy at 50 mg/kg once daily prolonged recipient survival times to the predefined endpoint of 29 days (n=2); when given at 25 mg/kg twice daily, the median survival time (MST) was 27 days (n=4). Neither once-daily monotherapy of STN 20 mg/kg nor CsA 20 mg/kg was effective (MST 6 days [n=2] and 7 days [n=5], respectively). In combination, however, STN 20 mg/kg and CsA 20 mg/kg prolonged MST to more than 100 days (n=5). By combining lower once-daily doses of STN (7 or 2 mg/kg) with CsA (20 mg/kg), MST was more than 100 (n=3) and 22 days (n=2), respectively. Neither in single-dose pharmacokinetic studies nor the transplant recipients were STN or CsA blood levels for combined treatment greater than when either drug was administered alone. STN blood levels in transplant recipients during combination therapy were dose related (20 mg/kg, 30–182 ng/mL; 7 mg/kg, 7–41 ng/mL; and 2 mg/kg, 3–5 ng/mL). STN at a daily dose of up to 20 mg/kg was relatively well tolerated. Conclusions. STN prolonged survival times of non-human primate kidney allograft recipients both as monotherapy and most effectively in combination with CsA. Pharmacokinetic interactions were not responsible for the potentiation of immunosuppressive efficacy by coadministering STN and CsA.

Chronic low-dose isotretinoin treatment limits renal damage in subtotally nephrectomized rats

Retinoids are anti-proliferative and anti-inflammatory compounds. We had previously shown that retinoids alleviate kidney damage in acute models of renal disease. We now examined whether retinoids are also effective in a chronic renal ablation model. Subtotally nephrectomized rats (SNx; two-third ablation) were compared to sham-operated controls (sham). SNx rats were administered either 10xa0mg/kg b.w. (low dose, LD) or 40xa0mg/kg b.w. (high dose, HD) isotretinoin or vehicle (nu2009=u200910 per group). The experiment was terminated after 16xa0weeks. Systolic blood pressure was significantly higher after SNx compared to sham but lower in SNx with LD isotretinoin (vs. SNx + vehicle). Compared to SNx + vehicle, SNx + LD isotretinoin had lower glomerular cell numbers, less glomerular hypertrophy and sclerosis, and less interstitial expansion. Morphological improvement in SNx + LD isotretinoin was accompanied by improvement in creatinine clearance and reduced urinary albumin excretion. In contrast, HD isotretinoin caused aggravation of renal damage with fibrinoid necroses of vessels and elevated urinary albumin excretion despite lower blood pressure. The dichotomous effects of isotretinoin are at least in part due to time- and dose-dependent alterations of transforming growth factor β1 and collagen IV gene expression as also suggested by cell-culture studies in vascular smooth muscle cells. In addition, isotretinoin affected the systemic and the renal renin–angiotensin system (which was further analyzed in a model of angiotensin II infusion of the rat). Isotretinoin failed to cumulate at LD but cumulated at HD in SNx. We conclude that LD isotretinoin attenuates progressive renal damage, whereas HD isotretinoin cumulates and aggravates renal damage independent of blood pressure reduction.

Blood pressure in diabetic nephropathy--current controversies.

Abstract. Ritz E, Rychlík I, Schömig M, Wagner J (Department of Internal Medicine, Ruperto Carola University, Heidelberg, Germany). Blood pressure in diabetic nephropathy – current controversies (Review). J Intern Med 2001; 249: 215–223.