TGF-beta inhibits Ang II-induced MAPK p44/42 signaling in vascular smooth muscle cells by Ang II type 1 receptor downregulation.

Vascular changes in diabetes are characterized by reduced vasoconstriction and vascular remodeling. Previously, we demonstrated that TGF-beta1 impairs Ang II-induced contraction through reduced calcium mobilization. However, the effect of TGF-beta1 on Ang II-induced vascular remodeling is unknown. Therefore, we investigated the effect of TGF-beta1 on Ang II-induced activation of the MAPK p44/42 pathway in cultured rat aortic smooth muscle cells (RASMC). Activation of MAPK p44/42 was determined with a phospho-specific antibody. Angiotensin type 1 receptor (AT(1)) and AT(1) mRNA levels were measured by [(3)H]candesartan-binding and real-time PCR, respectively. AT(1) gene transcription activity was assessed using AT(1) promoter-reporter constructs and by a nuclear runoff assay. In TGF-beta1-pretreated cells, Ang II-induced phosphorylation of MAPK p44/42 was inhibited by 29 and 46% for p42 and p44, respectively, and AT(1) density was reduced by 31%. Furthermore, pretreatment with TGF-beta1 resulted in a 64% reduction in AT(1) mRNA levels and decreased AT(1) mRNA transcription rate by 42%. Pretreatment with TGF-beta1 blocked Ang II-induced proliferation of RASMC, while stimulating Ang II-induced upregulation of plasminogen activator inhibitor-1. In conclusion, TGF-beta1 attenuates Ang II-mediated MAPK p44/42 kinase signaling in RASMC through downregulation of AT(1) levels, which is mainly caused by the inhibition of transcription of the AT(1) gene.

The role of angiotensin(1-7) in renal vasculature of the rat.

OBJECTIVE: Angiotensin(1-7) is an active component of the renin-angiotensin-aldosterone system. Its exact role in renal vascular function is unclear. We therefore studied the effects of angiotensin(1-7) on the renal vasculature in vitro and in vivo. METHODS: Isolated small renal arteries were studied in an arteriograph system by constructing concentration-response curves to angiotensin II, without and with angiotensin(1-7). In isolated perfused kidneys, the response of angiotensin II on renal vascular resistance was measured without and with angiotensin(1-7). The influence of angiotensin(1-7) on angiotensin II-induced glomerular afferent and efferent constriction was assessed with intravital microscopy in vivo under anaesthesia. In freely moving rats, we studied the effect of angiotensin(1-7) on angiotensin II-induced reduction of renal blood flow with an electromagnetic flow probe. RESULTS: Angiotensin(1-7) alone had no effect on the renal vasculature in any of the experiments. In vitro, angiotensin(1-7) antagonized angiotensin-II-induced constriction of isolated renal arteries (9.71 +/- 1.21 and 3.20 +/- 0.57%, for control and angiotensin(1-7) pre-treated arteries, respectively; P < 0.0005). In isolated perfused kidneys, angiotensin(1-7) reduced the angiotensin II response (100 +/- 16.6 versus 72.6 +/- 15.6%, P < 0.05) and shifted the angiotensin II dose-response curve rightward (pEC50, 6.69 +/- 0.19 and 6.26 +/- 0.12 for control and angiotensin(1-7) pre-treated kidneys, respectively; P < 0.05). Angiotensin(1-7), however, was devoid of effects on angiotensin-II-induced constriction of glomerular afferent and efferent arterioles and on angiotensin-II-induced renal blood flow reduction in freely moving rats in vivo. CONCLUSION: Angiotensin(1-7) antagonizes angiotensin II in renal vessels in vitro, but does not appear to have a major function in normal physiological regulation of renal vascular function in vivo.

Does angiotensin (1-7) contribute to the anti-proteinuric effect of ACE-inhibitors.

Angiotensin-converting enzyme inhibitors (ACE-I) reduce proteinuria and protect the kidney in proteinuric renal disease. During ACE-I therapy, circulating levels of angiotensin (1-7) [Ang (1-7)] are increased. As cardiac and renal protective effects of Ang (1-7) have been reported, we questioned whether Ang (1-7) contributes to the anti-proteinuric effects of ACE-I treatment. Therefore, we evaluated whether Ang (1-7) infusion reduces proteinuria in a rat model of adriamycin-induced renal disease. In addition, the effect of a selective Ang (1-7) blocker, [D-Ala7]-Ang (1-7) (A779), was investigated in rats treated with the ACE-I, lisinopril (LIS). Six weeks after induction of proteinuria, therapy was started in four different groups: control, Ang (1-7), LIS, and LIS+A779. After two weeks, the rats were sacrificed. Six weeks after injection of adriamycin, the rats had developed proteinuria of 323+/-40 mg/24 hours. The proteinuria remained stable in the control group and in the Ang (1-7) group, but was reduced in both LIS and LIS+A779-treated groups. Similarly, blood pressure (BP) was unchanged in the control and the Ang (1-7) groups, but reduced in both the LIS and the LIS+A779 groups. Plasma levels of Ang (1-7) were increased in the Ang (1-7) and in both LIS-treated groups. We conclude that systemic Ang (1-7) plays no major role in the anti-proteinuric and BPlowering effects of ACE-I in this rat model of adriamycin-induced nephrosis.

Approaches and methods in gene therapy for kidney disease.

Renal gene therapy may offer new strategies to treat diseases of native and transplanted kidneys. Several experimental techniques have been developed and employed using nonviral, viral, and cellular vectors. The most efficient vector for in vivo transfection appears to be adenovirus. Glomeruli, blood vessels, interstitial cells, and pyelum can be transfected with high efficiency. In addition, electroporation and microbubbles with ultrasound, both being enhanced naked plasmid techniques, offer good opportunities. Trapping of mesangial cells into the glomeruli as well as natural targeting of monocytes or macrophages to inflamed kidneys are elegant methods for site-specific delivery of genes. For gene therapy in kidney transplantation, hemagglutinating virus of Japan liposomes are efficient vectors for tubular transfection, whereas enhanced naked plasmid techniques are suitable for glomerular transfection. However, adenovirus offers the best opportunities in a renal transplantation setup because varying parameters of graft perfusion allows targeting of different cell types. In renal grafts, lymphocytes can be used for selective targeting to sites of inflammation. In conclusion, for both in vivo and ex vivo renal transfection, enhanced naked plasmids and adenovirus offer the best perspectives for effective clinical application. Moreover, the development of safer, nonimmunogenic vectors and the large-scale production could make clinical renal gene therapy a realistic possibility for the near future.

Targeting of doxorubicin to the urinary bladder of the rat shows increased cytotoxicity in the bladder urine combined with an absence of renal toxicity.

Targeting of anti-tumor drugs to the urinary bladder for the treatment of bladder carcinoma may be useful, since these agents generally have a low degree of urinary excretion and are highly toxic elsewhere in the body. The anti-tumor drug doxorubicin was coupled to the low-molecular weight protein lysozyme via the acid-sensitive cis-aconityl linker. All free amino groups of the lysozyme were used for drug attachment to achieve intact excretion of the doxorubicin-aconityl-lysozyme conjugate into the bladder. In the bladder, the cytotoxic drug should be regenerated through acidification of the urine. First, the doxorubicin-aconityl-lysozyme conjugate was tested in rats for its target specificity and general toxicity. Wistar rats were injected intravenously with 2 mg/kg free doxorubicin or 10 mg/kg lysozyme-conjugated doxorubicin. Total urinary excretion of doxorubicin was about 10 times higher if the drug was coupled to lysozyme (39 +/- 3% versus 4.4 +/- 0.4%). Free doxorubicin had no detectable toxic effects on heart, liver and lung but caused severe renal damage (proteinuria, N-acetylglucosaminidase excretion and glomerulosclerosis). None of the rats injected with doxorubicin-lysozyme conjugate showed such renal toxicity. Second, we tested whether doxorubicin could be released from the conjugate in the bladder through acidification of the urine and if the released doxorubicin could still exert a cytotoxic effect. Doxorubicin-aconityl-lysozyme (2 mg/kg conjugated doxorubicin, i.v.) was administered in rats with acidified urine (pH 6.1 +/- 0.1) and in rats with a high urinary pH (8.2 +/- 0.4). Ten times more doxorubicin was released from the conjugate in the group with acidified urine (15 +/- 7% versus 1.7 +/- 0.1%). In agreement with this, cytotoxicity was also higher in the low pH group (IC50 of 255 +/- 47 nM versus 684 +/- 84 nM doxorubicin). In conclusion, a specific delivery of doxorubicin to the urinary bladder combined with a reduced toxicity of doxorubicin in the kidneys can be achieved by coupling this anti-tumor drug to the low-molecular weight protein lysozyme via an acid-labile linker. A release of cytotoxic doxorubicin in the urinary bladder can be achieved by acidification of the urine. This technology, after further optimization, may provide an interesting tool for the treatment of bladder carcinoma.

Extracorporeal elimination in acute valproate intoxication.

Severe poisoning with valproate may result in coma and death. The management of valproate intoxication is principally supportive. Valproate is scarcely excreted renally and is mainly protein bound and, therefore, not considered to be amenable for extracorporeal elimination. Despite these unfavourable pharmacokinetic properties, several case reports showed successful treatment of valproate intoxication with haemodialysis and/or haemoperfusion. We describe a male patient (57 years) after ingestion of 64 g of valproate. The patient was successfully treated with haemodialysis for 6 h. Haemodialysis was followed by continuous venovenous haemodiafiltration (CVVH-D) for 18 h to prevent a rebound phenomenon. This report confirms the benefit of haemodialysis in serious valproate overdose. A review of the literature shows that haemodialysis followed by CVVH-D is the treatment of choice in severe valproate intoxication.