Renal endothelial function is associated with the anti-proteinuric effect of ACE inhibition in 5/6 nephrectomized rats.

In healthy rats, the physiological variation of baseline endothelial function of intrarenal arteries correlates with the severity of renal damage in response to a subsequent specific renal injury. However, whether such a variation in endothelial function may also condition or predict the variable response to angiotensin-converting enzyme-inhibiting treatment in these individuals has not been addressed before. To study this, 5/6 nephrectomy was performed to induce renal injury and chronic kidney disease in a group of healthy Wistar rats. At the time of nephrectomy, interlobar arteries were obtained from the extirpated right kidney and studied in vitro for endothelium-dependent relaxation to acetylcholine. Six weeks thereafter, treatment with lisinopril was started (n = 11) and continued for 9 wk. Proteinuria (metabolic cages) and systolic blood pressure (SBP; tail cuff) were evaluated weekly, and these were analyzed in relation to renal endothelial function at baseline. 5/6 Nephrectomy induced an increase in SBP and progressive proteinuria. Treatment with lisinopril reduced SBP and slowed proteinuria, albeit to a variable degree among individuals. The acetylcholine-induced renal artery dilation at baseline negatively correlated with lisinopril-induced reduction of proteinuria (r(2) = 0.648, P = 0.003) and with the decrease in SBP (r(2) = 0.592, P = 0.006). Our data suggest that angiotensin-converting enzyme-inhibitor attenuates the progression of renal damage the most in those individuals with decreased basal renal endothelial-mediated vasodilation.

Genetic deletion of growth differentiation factor 15 augments renal damage in both type 1 and type 2 models of diabetes.

Growth differentiation factor 15 (GDF15) is emerging as valuable biomarker in cardiovascular disease and diabetic kidney disease. Also, GDF15 represents an early response gene induced after tissue injury and studies performed in GDF15 knockout (KO) mice suggest that GDF15 plays a protective role after injury. In the current study, we investigated the role of GDF15 in the development of diabetic kidney damage in type 1 and type 2 models of diabetes. Renal damage was assessed in GDF15 KO mice and wild-type (WT) mice in streptozotocin type 1 and db/db type 2 diabetic models. Genetic deletion of GDF15 augmented tubular and interstitial damage in both models of diabetes, despite similar diabetic states in KO and WT mice. Increased tubular damage in KO animals was associated with increased glucosuria and polyuria in both type 1 and type 2 models of diabetes. In both models of diabetes, KO mice showed increased interstitial damage as indicated by increased α-smooth muscle actin staining and collagen type 1 expression. In contrast, glomerular damage was similarly elevated in diabetic KO and WT mice. In type 1 diabetes, GDF15 KO mice demonstrated increased expression of inflammatory markers. In type 2 diabetes, elevated levels of plasma creatinine indicated impaired kidney function in KO mice. GDF15 protects the renal interstitium and tubular compartment in experimental type 1 and 2 diabetes without affecting glomerular damage.

Growth differentiation factor 15 impairs aortic contractile and relaxing function through altered caveolar signaling of the endothelium.

Growth differentiation factor 15 (GDF15) is an independent predictor of cardiovascular disease, and increased GDF15 levels have been associated with endothelial dysfunction in selected patients. We therefore investigated whether GDF15 modulates endothelial function in aortas of wild-type (WT) and GDF15 knockout (KO) mice. Vascular contractions to phenylephrine and relaxation to ACh were assessed in aortas obtained from healthy WT and GDF15 KO mice. The effects of GDF15 pretreatment and the involvement of ROS or caveolae were determined. Phenylephrine-induced contractions and ACh-mediated relaxations were similar in WT and GDF15 KO mice. Pretreatment with GDF15 inhibited contraction and relaxation in both groups. Inhibition of contraction by GDF15 was absent in denuded vessels or after blockade of nitric oxide (NO) synthase. Relaxation in WT mice was mediated mainly through NO and an unidentified endothelium-derived hyperpolarizin factor (EDHF), whereas GDF15 KO mice mainly used prostaglandins and EDHF. Pretreatment with GDF15 impaired relaxation in WT mice by decreasing NO; in GDF15 KO mice, this was mediated by decreased action of prostaglandins. Disruption of caveolae resulted in a similar inhibition of vascular responses as GDF15. ROS inhibition did not affect vascular function. In cultured endothelial cells, GDF15 pretreatment caused a dissociation between caveolin-1 and endothelial NO synthase. In conclusion, GDF15 impairs aortic contractile and relaxing function through an endothelium-dependent mechanism involving altered caveolar endothelial NO synthase signaling.

Thiosulfate sulfurtransferase deficiency promotes oxidative distress and aberrant NRF2 function in the brain.

Thiosulfate sulfurtransferase (TST, EC 2.8.1.1) was discovered as an enzyme that detoxifies cyanide by conversion to thiocyanate (rhodanide) using thiosulfate as substrate; this rhodanese activity was subsequently identified to be almost exclusively located in mitochondria. More recently, the emphasis regarding its function has shifted to hydrogen sulfide metabolism, antioxidant defense, and mitochondrial function in the context of protective biological processes against oxidative distress. While TST has been described to play an important role in liver and colon, its function in the brain remains obscure. In the present study, we therefore sought to address its potential involvement in maintaining cerebral redox balance in a murine model of global TST deficiency (Tst-/- mice), primarily focusing on characterizing the biochemical phenotype of TST loss in relation to neuronal activity and sensitivity to oxidative stress under basal conditions. Here, we show that TST deficiency is associated with a perturbation of the reactive species interactome in the brain cortex secondary to altered ROS and RSS (specifically, polysulfide) generation as well as mitochondrial OXPHOS remodeling. These changes were accompanied by aberrant Nrf2-Keap1 expression and thiol-dependent antioxidant function. Upon challenging mice with the redox-active herbicide paraquat (25 mg/kg i.p. for 24 h), Tst-/- mice displayed a lower antioxidant capacity compared to wildtype controls (C57BL/6J mice). These results provide a first glimpse into the molecular and metabolic changes of TST deficiency in the brain and suggest that pathophysiological conditions associated with aberrant TST expression and/or activity renders neurons more susceptible to oxidative stress-related malfunction.

Gene therapy with adenovirus-delivered indoleamine 2,3-dioxygenase improves renal function and morphology following allogeneic kidney transplantation in rat.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in the tryptophan catabolism, has recently emerged as an important immunosuppressive enzyme involved in the regulation of both physiologic (maternal tolerance), as well as pathologic (neoplasia, autoimmune diseases, asthma) processes. Accumulating evidence points to a role for IDO in suppressing T-cell responses, thereby promoting tolerance. In the present study, we investigate the effects of adenovirus-mediated gene therapy with IDO on the acute rejection of the transplanted kidneys. METHODS: The experiments were performed in a rat Fisher to Lewis acute renal rejection model. RGD modified adenovirus carrying IDO gene (RGD-AdTIDO, n = 9) or RGD modified adenovirus carrying green fluorescent protein gene (RGD-AdTL, n = 8) were injected into the renal artery of the donor kidney before transplantation. A group receiving saline (n = 8) served as control. Rats were sacrificed after 7 days. RESULTS: Successful gene delivery was confirmed with real-time polymerase chain reaction and immunohistochemistry. RGD-AdTIDO significantly decreased elevated plasma creatinine (93.7 ± 18.9 µmol/l) compared to the RGD-AdTL (248.2 ± 43.6 µmol/l) and saline (228.3 ± 46.4 µmol/l) treated rats. Moreover, RGD-AdTIDO therapy diminished the infiltration of CD8+ T cells and macrophages into the graft and reduced renal interstitial pre-fibrosis. Also, it limited the up-regulation of kidney injury molecule-1, interleukin (IL)-2, IL-17 and transforming growth factor-ß mRNA expression, and increased foxp3 mRNA expression compared to controls. CONCLUSIONS: RGD-AdTIDO therapy improves renal function and morphology in a clinically relevant model of acute rejection.

Caveolin-1 is enriched in the peroxisomal membrane of rat hepatocytes.

UNLABELLED: Caveolae are a subtype of cholesterol-enriched lipid microdomains/rafts that are routinely detected as vesicles pinching off from the plasma membrane. Caveolin-1 is an essential component of caveolae. Hepatic caveolin-1 plays an important role in liver regeneration and lipid metabolism. Expression of caveolin-1 in hepatocytes is relatively low, and it has been suggested to also reside at other subcellular locations than the plasma membrane. Recently, we found that the peroxisomal membrane contains lipid microdomains. Like caveolin-1, hepatic peroxisomes are involved in lipid metabolism. Here, we analyzed the subcellular location of caveolin-1 in rat hepatocytes. The subcellular location of rat hepatocyte caveolin-1 was analyzed by cell fractionation procedures, immunofluorescence, and immuno-electron microscopy. Green fluorescent protein (GFP)-tagged caveolin-1 was expressed in rat hepatocytes. Lipid rafts were characterized after Triton X-100 or Lubrol WX extraction of purified peroxisomes. Fenofibric acid-dependent regulation of caveolin-1 was analyzed. Peroxisome biogenesis was studied in rat hepatocytes after RNA interference-mediated silencing of caveolin-1 and caveolin-1 knockout mice. Cell fractionation and microscopic analyses reveal that caveolin-1 colocalizes with peroxisomal marker proteins (catalase, the 70 kDa peroxisomal membrane protein PMP70, the adrenoleukodystrophy protein ALDP, Pex14p, and the bile acid-coenzyme A:amino acid N-acyltransferase BAAT) in rat hepatocytes. Artificially expressed GFP-caveolin-1 accumulated in catalase-positive organelles. Peroxisomal caveolin-1 is associated with detergent-resistant microdomains. Caveolin-1 expression is strongly repressed by the peroxisome proliferator-activated receptor-alpha agonist fenofibric acid. Targeting of peroxisomal matrix proteins and peroxisome number and shape were not altered in rat hepatocytes with 70%-80% reduced caveolin-1 levels and in livers of caveolin-1 knockout mice. CONCLUSION: Caveolin-1 is enriched in peroxisomes of hepatocytes. Caveolin-1 is not required for peroxisome biogenesis, but this unique subcellular location may determine its important role in hepatocyte proliferation and lipid metabolism.

Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation.

Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution of 4.4- and 70-kDa dextrans through the cytosol, and localization of 155- and 500-kDa dextrans in distinct vesicles after UMTD. After ATP depletion, reduced uptake of 4.4-kDa dextran and no uptake of 500-kDa dextran was observed after UMTD. Independently inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis significantly decreased intracellular delivery of 4.4- to 500-kDa dextrans. Furthermore, 3D fluorescence microscopy demonstrated dextran vesicles (500 kDa) to colocalize with caveolin-1 and especially clathrin. Finally, after UMTD of dextran (500 kDa) into rat femoral artery endothelium in vivo, dextran molecules were again localized in vesicles that partially colocalized with caveolin-1 and clathrin. Together, these data indicated uptake of molecules via endocytosis after UMTD. In addition to triggering endocytosis, UMTD also evoked transient pore formation, as demonstrated by the influx of calcium ions and cellular release of preloaded dextrans after US and microbubble exposure. In conclusion, these data demonstrate that endocytosis is a key mechanism in UMTD besides transient pore formation, with the contribution of endocytosis being dependent on molecular size.

The protective effect of 1-methyltryptophan isomers in renal ischemia-reperfusion injury is not exclusively dependent on indolamine 2,3-dioxygenase inhibition.

BACKGROUND AND PURPOSE: Indolamine 2,3-dioxygenase (IDO), an enzyme that catalyses the metabolism of tryptophan, may play a detrimental role in ischemia-reperfusion injury (IRI). IDO can be inhibited by 1-methyl-tryptophan, which exists in a D (D-MT) or L (L-MT) isomer. These forms show different pharmacological effects besides IDO inhibition. Therefore, we sought to investigate whether these isomers can play a protective role in renal IRI, either IDO-dependent or independent. EXPERIMENTAL APPROACH: We studied the effect of both isomers in a rat renal IRI model with a focus on IDO-dependent and independent effects. KEY RESULTS: Both MT isomers reduced creatinine and BUN levels, with D-MT having a faster onset of action but shorter duration and L-MT a slower onset but longer duration (24 h and 48 h vs 48 h and 96 h reperfusion time). Interestingly, this effect was not exclusively dependent on IDO inhibition, but rather from decreased TLR4 signalling, mimicking changes in renal function. Additionally, L-MT increased the overall survival of rats. Moreover, both MT isomers interfered with TGF-ß signalling and epithelial-mesenchymal transition. In order to study the effect of isomers in all mechanisms involved in IRI, a series of in vitro experiments was performed. The isomers affected signalling pathways in NK cells and tubular epithelial cells, as well as in dendritic cells and T cells. CONCLUSION AND IMPLICATIONS: This study shows that both MT isomers have a renoprotective effect after ischemia-reperfusion injury, mostly independent of IDO inhibition, involving mutually different mechanisms. We bring novel findings in the pharmacological properties and mechanism of action of MT isomers, which could become a novel therapeutic target of renal IRI.

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.

Immune modulation and graft protection by gene therapy in kidney transplantation.

Kidney transplantation represents the therapy of choice for many patients with end-stage renal disease. However, the success of renal engraftment is hindered by a number of factors, the most important of which being adverse effects of systemic immunosuppressive therapy, chronic transplant dysfunction and a severe shortage of donor kidneys. Gene therapy approaches may provide valuable strategies in each of these areas. First, gene therapy holds the potential of local therapy, thus circumventing systemic side effects of chronic immunosuppression. Second, chronic transplant dysfunction may be addressed by innovative strategies to induce local immune tolerance, immune suppression and additional graft protecting mechanisms. Third, gene therapy may be instrumental in increasing the quality of the grafts by limiting ischemia-reperfusion injury, especially in non-heart beating donors, thereby expanding the donor pool. In this article, we give an overview of the current state of gene therapy in experimental models of kidney transplantation.

Modulation of glutathione peroxidase activity by age-dependent carbonylation in glomeruli of diabetic mice.

AIMS: Low levels of reactive oxygen species and resulting oxidative protein modifications may play a beneficial role in cellular function under stress conditions. Here we studied the influence of age-dependent protein carbonylation on expression and activity of the anti-oxidative selenoenzyme glutathione peroxidase (GPx) in insulin-deficient Ins2Akita mice and type 2 diabetic obese db/db mice in context of diabetic nephropathy. METHODS: Protein carbonylation, GPx expression and activity were examined in kidney tissue and lysates by common histological and protein biochemical methods. RESULTS: In kidneys of Ins2Akita mice, carbonylated proteins, GPx-1 and GPx-4 expression were mainly detected in podocytes and mesangial cells. GPx activity was increased in kidney cortex homogenates of these mice. Remarkably, young animals did not show a concomitant increase in GPx expression but enhanced GPx carbonylation. No carbonylation-dependent modification of GPx activity was detected in db/db mice. In cultured podocytes hyperglycemia induced an increase in GPx expression but had no effect on activity or carbonylation. In kidney tissue sections of type 1 or type 2 diabetes patients, GPx-1 and GPx-4 expression but not overall protein carbonylation was significantly decreased. CONCLUSIONS: These results indicate the existence of a threshold for beneficial carbonylation-dependent redox signaling during the progression of diabetic nephropathy.

Optimization of ultrasound and microbubbles targeted gene delivery to cultured primary endothelial cells.

Ultrasound and microbubbles targeted gene delivery (UMTGD) is a promising technique for local gene delivery. As the endothelium is a primary target for systemic UMTGD, this study aimed at establishing the optimal parameters of UMTGD to primary endothelial cells. For this, an in vitro ultrasound (US) setup was employed in which individual UMTGD parameters were systematically optimized. The criteria for the final optimized protocol were: (1) relative high reporter gene expression levels, restricted to the US exposed area and (2) induction of not more than 5% cell death. US frequency and timing of medium replacement had a strong effect on UMTGD efficiency. Furthermore, US intensity, DNA concentration and total duration of US all affected UMTGD efficiency. Optimal targeted gene delivery to primary endothelial cells can be accomplished with Sonovue microbubbles, using 20 microg/ml plasmid DNA, a 1 MHz US exposure of Ispta 0.10 W/cm(2) for 30 s with immediate medium change after UMTGD. This optimized protocol resulted in both an increase in the number of transfected cells (more than three fold) and increased levels of transgene expression per cell (170%).

Differences in mitochondrial function and morphology during cooling and rewarming between hibernator and non-hibernator derived kidney epithelial cells.

Hibernators show superior resistance to ischemia and hypothermia, also outside the hibernation season. Therefore, hibernation is a promising strategy to decrease cellular damage in a variety of fields, such as organ transplantation. Here, we explored the role of mitochondria herein, by comparing epithelial cell lines from a hibernator (hamster kidney cells, HaK) and a non-hibernator (human embryonic kidney cells, HEK293) during cold preservation at 4 °C and rewarming. Cell survival (Neutral Red), ATP and MDA levels, mitochondrial membrane potential (MMP), mitochondrial morphology (using fluorescent probes) and metabolism (seahorse XF) were assessed. Hypothermia induced dispersion of the tubular mitochondrial network, a loss of MMP, increased oxygen radical (MDA) and decreased ATP production in HEK293. In contrast, HaK maintained MMP and ATP production without an increase in oxygen radicals during cooling and rewarming, resulting in superior cell survival compared to HEK293. Further, normothermic HaK showed a dispersed mitochondrial network and higher respiratory and glycolysis capacity compared to HEK293. Disclosing the mechanisms that hibernators use to counteract cell death in hypothermic and ischemic circumstances may help to eventually improve organ preservation in a variety of fields, including organ transplantation.

Metformin Improves Endothelial Function and Reduces Blood Pressure in Diabetic Spontaneously Hypertensive Rats Independent from Glycemia Control: Comparison to Vildagliptin.

Metformin confers vascular benefits beyond glycemia control, possibly via pleiotropic effects on endothelial function. In type-1-diabetes-mellitus (T1DM-)patients metformin improved flow-mediated dilation but also increased prostaglandin(PG)-F2α, a known endothelial-contracting factor. To explain this paradoxical finding we hypothesized that metformin increased endothelial-vasodilator mediators (e.g. NO and EDHF) to an even larger extent. Spontaneously-hypertensive-rats (SHR) display impaired endothelium-dependent relaxation (EDR) involving contractile PGs. EDR was studied in isolated SHR aortas and the involvement of PGs, NO and EDHF assessed. 12-week metformin 300 mg/kg/day improved EDR by up-regulation of NO and particularly EDHF; it also reduced blood pressure and increased plasma sulphide levels (a proxy for H2S, a possible mediator of EDHF). These effects persisted in SHR with streptozotocin (STZ)-induced T1DM. Vildagliptin (10 mg/kg/day), targeting the incretin axis by increasing GLP-1, also reduced blood pressure and improved EDR in SHR aortas, mainly via the inhibition of contractile PGs, but not in STZ-SHR. Neither metformin nor vildagliptin altered blood glucose or HbA1c. In conclusion, metformin reduced blood pressure and improved EDR in SHR aorta via up-regulation of NO and particularly EDHF, an effect that was independent from glycemia control and maintained during T1DM. A comparison to vildagliptin did not support effects of metformin mediated by GLP-1.

Predictive Properties of Biomarkers GDF-15, NTproBNP, and hs-TnT for Morbidity and Mortality in Patients With Type 2 Diabetes With Nephropathy.

OBJECTIVE: Although patients with type 2 diabetes (T2D) with nephropathy are at high risk for renal and cardiovascular complications, relevant biomarkers have been poorly identified. Because renal impairment may increase biomarker levels, this potentially confounds associations between biomarker levels and risk. To investigate the predictive value of a biomarker in such a setting, we examined baseline levels of growth differentiation factor-15 (GDF-15), N-terminal prohormone of B-type natriuretic peptide (NTproBNP), and high-sensitivity troponin T (hs-TnT) in relation to renal and cardiovascular risk in T2D patients with nephropathy. RESEARCH DESIGN AND METHODS: Eight hundred sixty-one T2D patients from the sulodexide macroalbuminuria (Sun-MACRO) trial were included in our post hoc analysis. Prospective associations of baseline serum GDF-15, NTproBNP, and hs-TnT with renal and cardiovascular events were determined by Cox multiple regression and C-statistic analysis. Renal base models included albumin-to-creatinine ratio (ACR), serum creatinine, hemoglobin, age, and sex. Cardiovascular base models included diastolic blood pressure, ACR, cholesterol, age, and sex. RESULTS: The mean (±SD) estimated glomerular filtration rate was 33 ± 9 mL/min/1.73 m2, and the median serum concentration for GDF-15 was 3,228 pg/mL (interquartile range 2,345-4,310 pg/mL), for NTproBNP was 380 ng/L (155-989 ng/L), and for hs-TnT was 30 ng/L (20-47 ng/L). In multiple regression analysis, GDF-15 (hazard ratio [HR] 1.83, P = 0.04), NTproBNP (HR 2.34, P = 0.004), and hs-TnT (HR 2.09, P = 0.014) were associated with renal events, whereas NTproBNP (HR 3.45, P < 0.001) was associated with cardiovascular events. The C-statistic was improved by adding NTproBNP and hs-TNT to the renal model (0.793 vs. 0.741, P = 0.04). For cardiovascular events, the C-statistic was improved by adding NTproBNP alone (0.722 vs. 0.658, P = 0.018). CONCLUSIONS: Biomarkers GDF-15, NTproBNP, and hs-TnT associate independently with renal risk, whereas NTproBNP independently predicts cardiovascular risk.

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.

Differential Effects of Long Term FTY720 Treatment on Endothelial versus Smooth Muscle Cell Signaling to S1P in Rat Mesenteric Arteries.

The sphingosine-1-phosphate (S1P) analog FTY720 exerts pleiotropic effects on the cardiovascular system and causes down-regulation of S1P receptors. Myogenic constriction is an important mechanism regulating resistance vessel function and is known to be modulated by S1P. Here we investigated myogenic constriction and vascular function of mesenteric arteries of rats chronically treated with FTY720. Wistar rats received FTY720 1mg/kg/daily for six weeks. At termination, blood pressure was recorded and small mesenteric arteries collected for vascular studies in a perfusion set up. Myogenic constriction to increased intraluminal pressure was low, but a sub-threshold dose of S1P profoundly augmented myogenic constriction in arteries of both controls and animals chronically treated with FTY720. Interestingly, endothelial denudation blocked the response to S1P in arteries of FTY720-treated animals, but not in control rats. In acute experiments, presence of FTY720 significantly augmented the contractile response to S1P, an effect that was partially abolished after the inhibition of cyclooxygenase (COX-)-derived prostaglandins. FTY720 down regulated S1P1 but not S1P2 in renal resistance arteries and in cultured human endothelial cells. This study therefore demonstrates the endothelium is able to compensate for the complete loss of responsiveness of the smooth muscle layer to S1P after long term FTY720 treatment through a mechanism that most likely involves enhanced production of contractile prostaglandins by the endothelium.

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.

Early Posttransplant Tryptophan Metabolism Predicts Long-term Outcome of Human Kidney Transplantation.

BACKGROUND: Chronic transplant dysfunction (CTD) is the leading cause of long-term loss of the renal allograft. So far, no single test is available to reliably predict the risk for CTD. Monitoring of tryptophan (trp) metabolism through indoleamine 2.3-dioxygenase (IDO) has been previously proposed to predict acute rejection of human kidney transplants. Here, we investigate the potential of IDO/trp degradation along the kynurenine (kyn) pathway to predict the long-term outcome of human kidney transplantation. METHODS: During the 2-year follow-up blood, urine, and kidney biopsies were collected from 48 renal transplant patients. Concentrations of kyn and trp in serum and urine were measured at 2 weeks, 6 months, and 2 years after transplantation. Kynurenine to tryptophan ratio was calculated as an estimate of trp degradation. To evaluate the histological changes and IDO expression, respectively, periodic acid schiff staining and immunohistochemistry for IDO were performed on biopsies taken at 6 months and 2 years. RESULTS: Two years after transplantation, kyn/trp was increased in urine and decreased in serum as compared to 2-week values. In 2-year biopsies, IDO expression was mainly found in infiltrating inflammatory cells and in the glomeruli. The urine level of trp 2 weeks after transplantation predicted the serum creatinine 6 months and the estimated creatinine clearance 2 years after transplantation. Additionally, serum level of kyn 6 months after transplantation predicted the serum creatinine 2 years after transplantation. CONCLUSIONS: Early serum and urine levels of trp and kyn may offer a novel route for early detection of patients at risk for developing CTD.

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.