ß-Cyclodextrin counteracts obesity in Western diet-fed mice but elicits a nephrotoxic effect.

Obesity has become a worldwide health crisis and is associated with a plethora of comorbidities. The multi-organ effects of obesity have been linked to ectopic lipid accumulation. Thus, there is an urgent need to tackle the obesity crisis by developing effective lipid-lowering therapies. 2-hydroxypropyl-ß-Cyclodextrin (2HP-ß-CD) has been previously shown to reduce lysosomal cholesterol accumulation in a murine model of Niemann Pick Type C (NPC) disease. Using a murine model of Western diet-induced obesity (DIO), we report the effects of 2HP-ß-CD in counteracting weight gain, expansion of adipose tissue mass and ectopic lipid accumulation. Interestingly, DIO caused intracellular storage of neutral lipids in hepatic tissues and of phospholipids in kidneys, both of which were prevented by 2HP-ß-CD. Importantly, this report brings attention to the nephrotoxic effects of 2HP-ß-CD: renal tubular damage, inflammation and fibrosis. These effects may be overlooked, as they are best appreciated upon assessment of renal histology.

Excessive dietary lipid intake provokes an acquired form of lysosomal lipid storage disease in the kidney.

Obesity and dyslipidaemia are features of the metabolic syndrome and risk factors for chronic kidney disease. The cellular mechanisms connecting metabolic syndrome with chronic kidney disease onset and progression remain largely unclear. We show that proximal tubular epithelium is a target site for lipid deposition upon overnutrition with a cholesterol-rich Western-type diet. Affected proximal tubule epithelial cells displayed giant vacuoles of lysosomal or autophagosomal origin, harbouring oxidised lipoproteins and concentric membrane layer structures (multilamellar bodies), reminiscent of lysosomal storage diseases. Additionally, lipidomic analysis revealed renal deposition of cholesterol and phospholipids, including lysosomal phospholipids. Proteomic profiles of renal multilamellar bodies were distinct from those of epidermis or lung multilamellar bodies and of cytoplasmic lipid droplets. Tubular multilamellar bodies were observed in kidney biopsies of obese hypercholesterolaemic patients, and the concentration of the phospholipidosis marker di-docosahexaenoyl (22:6)-bis(monoacylglycerol) phosphate was doubled in urine from individuals with metabolic syndrome and chronic kidney disease. The enrichment of proximal tubule epithelial cells with phospholipids and multilamellar bodies was accompanied by enhanced inflammation, fibrosis, tubular damage markers, and higher urinary electrolyte content. Concomitantly to the intralysosomal lipid storage, a renal transcriptional response was initiated to enhance lysosomal degradation and lipid synthesis. In cultured proximal tubule epithelial cells, inhibition of cholesterol efflux transport or oxysterol treatment induced effects very similar to the in vivo situation, such as multilamellar body and phospholipid amassing, and induction of damage, inflammatory, fibrotic, and lipogenic molecules. The onset of phospholipidosis in proximal tubule epithelial cells is a novel pathological trait in metabolic syndrome-related chronic kidney disease, and emphasises the importance of healthy lysosomes and nutrition for kidney well-being. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

High glucose induces HGF-independent activation of Met receptor in human renal tubular epithelium.

CONTEXT: The role of hepatocyte growth factor (HGF) in diabetic kidney damage remains controversial. OBJECTIVE: To test the hypothesis that high glucose levels activate pathways related to HGF and its receptor Met and that this could participate in glucose-induced renal cell damage. MATERIALS AND METHODS: HK2 cells, a human proximal tubule epithelial cell line, were stimulated with high glucose for 48 hours. Levels of pMet/Met, pEGFR/EGFR, pSTAT3/STAT3, pAkt/Akt and pERK1/2/ERK1/2 were studied by immunoblotting. Absence of HGF was verified by qRT-PCR and ELISA. RESULTS: High glucose level activated Met and its downstream pathways STAT3, Akt and ERK independently of HGF. High glucose induced an integrin ligand fibronectin. HGF-independent Met phosphorylation was prevented by inhibition of integrin α5ß1, Met inhibitor crizotinib, Src inhibitors PP2 and SU5565, but not by EGFR inhibitor AG1478. High glucose increased the expression of TGFß-1, CTGF and the tubular damage marker KIM-1 and increased apoptosis of HK2 cells, effects inhibited by crizotinib. CONCLUSION: High glucose activated Met receptor in HK2 cells independently of HGF, via induction of integrin α5ß1 and downstream signaling. This mode of Met activation was associated with tubular cell damage and apoptosis and it may represent a novel pathogenic mechanism and a treatment target in diabetic nephropathy.

Increased Circulating and Urinary Levels of Soluble TAM Receptors in Diabetic Nephropathy.

TAM receptors (Tyro3, Axl, and Mer) have been implicated in innate immunity. Circulating TAM receptor soluble forms (sTyro3, sAxl, sMer) are related to autoimmune disorders. We investigated TAM and their ligand protein S in patients with diabetes. Urinary and plasma levels of protein S, sTyro3, sAxl, and sMer were determined in 126 patients with diabetes assigned to a normoalbuminuric or macroalbuminuric (urinary albumin excretion <30 mg/24 hours and >300 mg/24 hours, respectively) study group and 18 healthy volunteers. TAM and protein S immunostaining was performed on kidney biopsy specimens from patients with diabetic nephropathy (n = 9) and controls (n = 6). TAM expression and shedding by tubular epithelial cells were investigated by PCR and enzyme-linked immunosorbent assay in an in vitro diabetes model. Patients with macroalbuminuria diabetes had higher circulating levels of sMer and more urinary sTyro3 and sMer than normoalbuminuric diabetics. Increased clearance of sTyro3 and sMer was associated with loss of tubular Tyro3 and Mer expression in diabetic nephropathy tissue and glomerular depositions of protein S. During in vitro diabetes, human kidney cells had down-regulation of Tyro3 and Mer mRNA and increased shedding of sTyro3 and sMer. Renal injury in diabetes is associated with elevated systemic and urine levels of sMer and sTyro3. This is the first study reporting excretion of sTAM receptors in urine, identifying the kidney as a source of sTAM.

Metabolic injury-induced NLRP3 inflammasome activation dampens phospholipid degradation.

The collateral effects of obesity/metabolic syndrome include inflammation and renal function decline. As renal disease in obesity can occur independently of hypertension and diabetes, other yet undefined causal pathological pathways must be present. Our study elucidate novel pathological pathways of metabolic renal injury through LDL-induced lipotoxicity and metainflammation. Our in vitro and in vivo analysis revealed a direct lipotoxic effect of metabolic overloading on tubular renal cells through a multifaceted mechanism that includes intralysosomal lipid amassing, lysosomal dysfunction, oxidative stress, and tubular dysfunction. The combination of these endogenous metabolic injuries culminated in the activation of the innate immune NLRP3 inflammasome complex. By inhibiting the sirtuin-1/LKB1/AMPK pathway, NLRP3 inflammasome dampened lipid breakdown, thereby worsening the LDL-induced intratubular phospholipid accumulation. Consequently, the presence of NLRP3 exacerbated tubular oxidative stress, mitochondrial damage and malabsorption during overnutrition. Altogether, our data demonstrate a causal link between LDL and tubular damage and the creation of a vicious cycle of excessive nutrients-NLRP3 activation-catabolism inhibition during metabolic kidney injury. Hence, this study strongly highlights the importance of renal epithelium in lipid handling and recognizes the role of NLRP3 as a central hub in metainflammation and immunometabolism in parenchymal non-immune cells.

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.

Perinatally administered losartan augments renal ACE2 expression but not cardiac or renal Mas receptor in spontaneously hypertensive rats.

Since the identification of the alternative angiotensin converting enzyme (ACE)2/Ang-(1-7)/Mas receptor axis, renin-angiotensin system (RAS) is a new complex target for a pharmacological intervention. We investigated the expression of RAS components in the heart and kidney during the development of hypertension and its perinatal treatment with losartan in young spontaneously hypertensive rats (SHR). Expressions of RAS genes were studied by the RT-PCR in the left ventricle and kidney of rats: normotensive Wistar, untreated SHR, SHR treated with losartan since perinatal period until week 9 of age (20 mg/kg/day) and SHR treated with losartan only until week 4 of age and discontinued until week 9. In the hypertrophied left ventricle of SHR, cardiac expressions of Ace and Mas were decreased while those of AT1 receptor (Agtr1a) and Ace2 were unchanged. Continuous losartan administration reduced LV weight (0.43 ± 0.02; P < 0.05 versus SHR) but did not influence altered cardiac RAS expression. Increased blood pressure in SHR (149 ± 2 in SHR versus 109 ± 2 mmHg in Wistar; P < 0.05) was associated with a lower renal expressions of renin, Agtr1a and Mas and with an increase in ACE2. Continuous losartan administration lowered blood pressure to control levels (105 ± 3 mmHg; P < 0.05 versus SHR), however, only renal renin and ACE2 were significantly up-regulated (for both P < 0.05 versus SHR). Conclusively, prevention of hypertension and LV hypertrophy development by losartan was unrelated to cardiac or renal expression of Mas. Increased renal Ace2, and its further increase by losartan suggests the influence of locally generated Ang-(1-7) in organ response to the developing hypertension in SHRs.

Pioglitazone, a PPARγ agonist, provides comparable protection to angiotensin converting enzyme inhibitor ramipril against adriamycin nephropathy in rat.

Peroxisome proliferator-activated receptor γ (PPARγ) agonists have been shown to ameliorate diabetic nephropathy, but much less are known about their effects in non-diabetic nephropathies. In the present study, metabolic parameters, blood pressure, aortic endothelial function along with molecular and structural markers of glomerular and tubulointerstitial renal damage, were studied in a rat model of normotensive nephropathy induced by adriamycin and treated with PPARγ agonist pioglitazone (12mg/kg, po), angiotensin converting enzyme (ACE) inhibitor ramipril (1mg/kg, po) or their combination. Pioglitazone had no effect on systolic blood pressure, marginally reduced glycemia and improved aortic endothelium-dependent relaxation. In the kidney, pioglitazone prevented the development of proteinuria and focal glomerulosclerosis to the similar extent as blood-pressure lowering ramipril. Renoprotection provided by either treatment was associated with a reduction in the cortical expression of profibrotic plasminogen activator inhibitor-1 and microvascular damage-inducing endothelin-1, and a limitation of interstitial macrophage influx. Treatment with PPARγ agonist, as well as ACE inhibitor comparably affected renal expression of the renin-angiotensin system (RAS) components, normalizing increased renal expression of ACE and enhancing the expression of Mas receptor. Interestingly, combined pioglitazone and ramipril treatment did not provide any additional renoprotection. These results demonstrate that in a nondiabetic renal disease, such as adriamycin-induced nephropathy, PPARγ agonist pioglitazone provides renoprotection to a similar extent as an ACE inhibitor by interfering with the expression of local RAS components and attenuating related profibrotic and inflammatory mechanisms. The combination of the both agents, however, does not lead to any additional renal benefit.

Enalaprilat increases PPARß/δ expression, without influence on PPARα and PPARγ, and modulate cardiac function in sub-acute model of daunorubicin-induced cardiomyopathy.

Anthracycline therapy is limited by a cardiotoxicity that may eventually lead to chronic heart failure which is thought to be prevented by ACE inhibitors (ACEi). However, the protective effect of ACEi in early stages of this specific injury remains elusive. Activated nuclear transcription factors peroxisome proliferator-activated receptors (PPAR) regulate cellular metabolism, but their involvement in anthracycline cardiomyopathy has not been investigated yet. For this purpose, Wistar rats were administered with daunorubicin (i.p., 3 mg/kg, in 48 h intervals) or co-administered with daunorubicine and enalaprilat (i.p., 5 mg/kg in 12 h intervals). Control animals received vehicle. Left ventricular function was measured invasively under anesthesia. Cell-shortening was measured by videomicroscopy in isolated cardiomyocytes. Expression of PPARs mRNA in cardiac tissue was measured by Real-Time PCR. Although the hemodynamic parameters of daunorubicin-treated rats remained altered upon ACEi co-administration, ACEi normalized daunorubicin-induced QT prolongation. On cellular level, ACEi normalized altered basal and isoproterenol-stimulated cardiac cell shortening in daunorubicine-treated group. Moreover, anthracycline administration significantly up-regulated heart PPARα mRNA and its expression remained increased after ACEi co-administration. On the other hand, the expression of cardiac PPARß/δ was not altered in anthracycline-treated animals, whereas co-administration of ACEi increased its expression. Conclusively, effect of ACEi can be already detected in sub-acute phase of anthracycline-induced cardiotoxicity. Altered expression of heart PPARs may suggest these nuclear receptors as a novel target in anthracycline cardiomyopathy.

Bradykinin modulates spontaneous nerve growth factor production and stretch-induced ATP release in human urothelium.

The urothelium plays a crucial role in integrating urinary bladder sensory outputs, responding to mechanical stress and chemical stimulation by producing several diffusible mediators, including ATP and, possibly, neurotrophin nerve growth factor (NGF). Such urothelial mediators activate underlying afferents and thus may contribute to normal bladder sensation and possibly to the development of bladder overactivity. The muscle-contracting and pain-inducing peptide bradykinin is produced in various inflammatory and non-inflammatory pathologies associated with bladder overactivity, but the effect of bradykinin on human urothelial function has not yet been characterized. The human urothelial cell line UROtsa expresses mRNA for both B1 and B2 subtypes of bradykinin receptors, as determined by real-time PCR. Bradykinin concentration-dependently (pEC50=8.3, Emax 4434±277nM) increased urothelial intracellular calcium levels and induced phosphorylation of the mitogen-activated protein kinase (MAPK) ERK1/2. Activation of both bradykinin-induced signaling pathways was completely abolished by the B2 antagonist icatibant (1µM), but not the B1 antagonist R715 (1µM). Bradykinin-induced (100nM) B2 receptor activation markedly increased (192±13% of control levels) stretch-induced ATP release from UROtsa in hypotonic medium, the effect being dependent on intracellular calcium elevations. UROtsa cells also expressed mRNA and protein for NGF and spontaneously released NGF to the medium in the course of hours (11.5±1.4pgNGF/mgprotein/h). Bradykinin increased NGF mRNA expression and accelerated urothelial NGF release to 127±5% in a protein kinase C- and ERK1/2-dependent manner. Finally, bradykinin up-regulated mRNA for transient-receptor potential vanilloid (TRPV1) sensory ion channel in UROtsa. In conclusion, we show that bradykinin represents a versatile modulator of human urothelial phenotype, accelerating stretch-induced ATP release, spontaneous release of NGF, as well as expression of sensory ion channel TRPV1. Bradykinin-induced changes in urothelial sensory function might contribute to the development of bladder dysfunction.

Neurotrophins as regulators of urinary bladder function.

Increased voiding frequency and urgency are among the most prevalent storage lower urinary tract symptoms (LUTS), often diagnosed as part of overactive bladder syndrome (OAB). It has been suggested that these symptoms are caused by excessive sensory activation of the neural micturition circuit. It seems likely that sensory pathway remodelling is also responsible for pain perception upon bladder filling in patients with bladder pain syndrome (BPS). Neurotrophins-including nerve growth factor (NGF), brain-derived nerve factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4)-represent master modulators of neural plasticity, both in peripheral and central nervous systems. Accumulating evidence points towards a role for neurotrophins in the control of neural sensory function during micturition and indicates their involvement in the emergence of OAB-related and BPS-related LUTS. Neurotrophins could potentially be used as urinary biomarkers to improve diagnostic accuracy for OAB and BPS and monitor therapy effectiveness. Proof-of-principle clinical evidence has confirmed that NGF is a potential target for treating human bladder overactivity.

Specificity evaluation of antibodies against human ß3-adrenoceptors.

ß(3)-Adrenoceptors are a promising drug target for the treatment of urinary bladder dysfunction, but knowledge about their expression at the protein level and their functional role is limited, partly due to a lack of well validated tools. As many antibodies against G-protein-coupled receptors, including those against ß(3)- and other ß-adrenoceptor subtypes, lack selectivity for their target, we have evaluated the specificity of five antibodies raised against the full-length protein of the human ß(3)-adrenoceptor (H155-B01), its N-terminus (LSA4198 and TA303277) and its C-terminus (AB5122, Sc1472) in immunoblotting and immunocytochemistry. Our primary test system were Chinese hamster ovary cells stably transfected to express each of the three human ß-adrenoceptor subtypes at near physiological levels (100-200 fmol/mg protein). None of the five antibodies exhibited convincing target specificity in immunoblotting with Sc1472 apparently being least unsuitable. In immunocytochemistry, LSA4198 and Sc1472 appeared most promising, exhibiting at least some degree of specificity. As these two antibodies have been raised against different epitopes (N- and C-terminus of the receptor, respectively), we propose that concordant staining by both antibodies provides the most convincing evidence for ß(3)-adrenoceptor labelling in cyto- or histochemistry studies.

Expression profiling of G-protein-coupled receptors in human urothelium and related cell lines.

UNLABELLED: What's known on the subject? and What does the study add? Urothelium emerged as a crucial integrator of sensory inputs and outputs in the bladder wall, and urothelial G-protein-coupled receptors (GPCRs) may represent plausible targets for treatment of various bladder pathologies. Urothelial cell lines provide a useful tool to study urothelial receptor function, but their validity as models for native human urothelium remains unclear. We characterize the mRNA expression of genes coding for GPCRs in human freshly isolated urothelium and compare the expression pattern with those in human urothelial cell lines. OBJECTIVES: To characterize the mRNA expression pattern of genes coding for G-protein-coupled receptors (GPCRs) in human freshly isolated urothelium. To compare GPCR expression in human urothelium-derived cell lines to explore the suitability of these cell lines as model systems to study urothelial function. MATERIALS AND METHODS: Native human urothelium (commercially sourced) and human urothelium-derived non-cancer (UROtsa and TERT-NHUC) and cancer (J82) cell lines were used. For mRNA expression profiling we used custom-designed real-time polymerase chain reaction array for 40 receptors and several related genes. RESULTS: Native urothelium expressed a wide variety of GPCRs, including α(1A), α(1D) and all subtypes of α(2) and ß adrenoceptors. In addition, M(2) and M(3) cholinergic muscarinic receptors, angiotensin II AT(1) receptor, serotonin 5-HT(2A) receptor and all subtypes of bradykinin, endothelin, cannabinoid, tachykinin and sphingosine-1-phosphate receptors were detected. Nerve growth factor and both its low- and high-affinity receptors were also expressed in urothelium. In all cell lines expression of most GPCRs was markedly downregulated, with few exceptions. In UROtsa cells, but much less in other cell lines, the expression of ß(2) adrenoceptors, M(3) muscarinic receptors, B(1) and B(2) bradykinin receptors, ET(B) endothelin receptors and several subtypes of sphingosine-1-phosphate receptors was largely retained. CONCLUSIONS: Human urothelium expresses a wide range of receptors which enables sensing and integration of various extracellular signals. Human urothelium-derived cell lines, especially UROtsa cells, show comparable mRNA expression to native tissue for several physiologically relevant GPCRs, but lose expression of many other receptors. The use of cell lines as model systems of human urothelium requires careful validation of suitability for the genes of interest.

Nerve growth factor in bladder dysfunction: contributing factor, biomarker, and therapeutic target.

In the last two decades, nerve growth factor (NGF), initially described as a prototypical trophic factor in the development of sensory and sympathetic innervation, has emerged as a complex regulator of neural plasticity along the micturition pathways. This review aims to summarize the current experimental and clinical evidence for a role of NGF in urinary bladder. Experimental administration of NGF elicits the states of increased sensation, urgency, and bladder hyperreflexia, resembling pathologies associated with bladder overactivity and inflammatory pain, such as overactive bladder syndrome (OAB) and interstitial cystitis/painful bladder syndrome (IC/PBS). There is strong experimental evidence, including the effective therapeutic targeting, on the direct causal role of NGF in rodent models of bladder outlet obstruction, spinal cord injury, diabetic bladder dysfunction, and interstitial inflammation. In humans, there are attempts to employ urinary NGF levels as a diagnostic marker in various forms of OAB and IC/PBS. In near future, use of novel experimental tools, such as urothelium-specific NGF transgenic mice or more specific low-molecular weight NGF receptor modulators, may provide better understanding of several unresolved issues in NGF-related bladder dysfunction. Moreover, successful experimental therapeutic approaches, such as NGF sequestering proteins or modified NGF antibodies, await the translation to the clinical treatment of bladder disorders.

ß-adrenoceptor agonist effects in experimental models of bladder dysfunction.

ß-adrenoceptor stimulation can enhance the storage function of the urinary bladder by acting on detrusor smooth muscle tone, mediator release from the urothelium and/or afferent nerve activity. In humans this may occur predominantly if not exclusively via the ß3-subtype. The effects of ß-adrenoceptor agonists including several ß3-selective agonists have been studied in vitro and in vivo, in healthy animals of both genders and various age groups and in a wide range of animal (mostly rat) models of genetic or acquired bladder dysfunction. Such models included bladder irritation by intravesical instillation of acetic acid or prostaglandin E2, bladder outlet obstruction, stroke, diabetes, spontaneously hypertensive rats, and NO synthase inhibition. Across all of these models ß-adrenoceptor agonists had effects consistent with improved bladder storage function. ß3-adrenoceptor effects are resistant to agonist-induced desensitization in many cell types, but whether this also applies to the human bladder is unknown. The efficacy of ß-adrenoceptor agonists appears to be largely unaffected by common polymorphisms of the ß3-adrenoceptor gene. Taken together these findings suggest that ß3-adrenoceptor agonists may become useful drugs for the treatment of bladder storage dysfunction, a view supported by recent phase III clinical studies for one such agent, mirabegron.

Enalapril decreases cardiac mass and fetal gene expression without affecting the expression of endothelin-1, transforming growth factor ß-1, or cardiotrophin-1 in the healthy normotensive rat.

Angiotensin II can induce cardiac hypertrophy by stimulating the release of growth factors. ACE inhibitors reduce angiotensin II levels and cardiac hypertrophy, but their effects on the healthy heart are largely unexplored. We hypothesized that ACE inhibition decreases left ventricular mass in normotensive animals and that this is associated with altered expression of cardiac fetal genes, growth factors, and endothelial nitric oxide synthase (eNOS). Wistar rats (n = 7 per group) were orally administered with enalapril twice daily for a total daily dose of 5 mg·kg(-1)·d(-1) (ENAP5) or 15 mg·kg(-1)·d(-1) (ENAP15) or vehicle. Systolic blood pressure was measured by the tail-cuff method. Left ventricular expression of cardiac myosin heavy chain-α (MYH6) and -ß (MYH7), atrial natriuretic peptide (ANP), endothelin-1 (ET-1), transforming growth factor ß-1 (TGFß-1), cardiotrophin-1 (CT-1), and renal renin were examined by real-time PCR, and eNOS using Western blot. Blood pressure was decreased only in ENAP15 animals (p < 0.05 vs. Control), whereas left ventricular mass decreased after both doses of enalapril (p < 0.05 vs. Control). MYH7 and ANP were reduced in ENAP15, while no changes in ET-1, TGFß-1, CT-1, and MYH6 mRNA or eNOS protein were observed. Renal renin dose-dependently increased after enalapril treatment. Enalapril significantly decreased left ventricular mass even after 1 week treatment in the normotensive rat. This was associated with a decreased expression of the fetal genes MYH7 and ANP, but not expression of ET-1, CT-1, or TGFß-1.

Desirable properties of ß3-adrenoceptor agonists: implications for the selection of drug development candidates.

ß3-adrenoceptor agonists are currently in clinical development for the treatment of overactive bladder and considered for several other indications. This Perspective discusses desirable properties of such drugs mainly based on the example of overactive bladder, but at least partly they should also be applicable to other indications of ß(3)-adrenoceptor agonists or other drug classes and therapeutic areas. These include degree of selectivity for the molecular target in terms of affinity, intrinsic efficacy and ligand-directed signaling. The ability to cause agonist-induced desensitization and the potential impact of gene polymorphisms also need to be considered. Depending on intended indication, specific pharmacokinetic considerations may also apply. These findings challenge the usefulness of high-throughput screening assays based upon a single molecular response in an artificial system and emphasize the need for early use of in vivo testing in species considered to be predictive for the human situation.

Pycnogenol improves left ventricular function in streptozotocin-induced diabetic cardiomyopathy in rats.

We studied whether Pycnogenol (PYC) may attenuate the development of experimental streptozotocin-induced diabetic cardiomyopathy in rat. In addition, we aimed to study whether PYC affects cardiac oxidative stress and the protein expression of reactive oxygen species (ROS)-producing molecules (gp91(phox)-containing NADPH oxidase and NO-signalling proteins). Experimental diabetes mellitus was manifested by hyperglycaemia and impaired cardiac function estimated using left ventricular catheterisation in vivo. PYC lowered fasting plasma glucose and normalized basal cardiac function. Excessive oxidative stress in streptozotocin (STZ) hearts, evidenced by 40% increase (P < 0.05) of thiobarbituric acid reactive substances (TBARS) concentration, was associated with increased expression of gp91(phox) (by 75%, P < 0.05), iNOS (by 40%, P < 0.05) and alpha-tubulin (by 49%, P < 0.05), but unchanged expression of eNOS and its alosteric regulators, as compared to CON. PYC failed to affect these expression abnormalities. Our study shows that PYC corrects diabetic cardiac dysfunction, probably by its metabolic and direct radical scavenging activity without affecting the molecular maladaptations of ROS-producing enzymes and cytoskeletal components.

Renal vascular dysfunction precedes the development of renal damage in the hypertensive Fawn-Hooded rat.

It is unknown whether generalized vascular dysfunction precedes the development of kidney disease. Therefore, we studied myogenic constriction and endothelium-mediated dilatory responses in two inbred Fawn-Hooded (FH) rat strains, one of which spontaneously develops hypertension, proteinuria, and glomerulosclerosis (FHH), whereas the other (FHL) does not. Small renal, mesenteric resistance arteries and thoracic aorta isolated from FH rats before (7 wk old) and after the development of mild proteinuria (12 wks old) were mounted in perfused and isometric set-ups, respectively. Myogenic response, endothelium-dependent relaxation, and the contribution of endothelium-mediated dilatory compounds were studied using their respective inhibitors. Myogenic reactivity was assessed constructing pressure-diameter curves in the presence and absence of calcium. At the age of 7 wk, renal arteries isolated from kidneys of FHH rats developed significantly lower myogenic tone compared with FHL, most likely because of excessive cyclo-oxygenase 1-mediated production of constrictive prostaglandins. Consequently, young FHH demonstrated reduced maximal myogenic tone (22 +/- 4.8 vs. 10.8 +/- 2.0%, P = 0.03) and the peak myogenic index (-6.9 +/- 4.8 vs. 0.6 +/- 0.8%/mmHg, P = 0.07 for FHL vs. FHH, respectively). Active myogenic curves obtained in mesenteric arteries isolated from 7-wk-old rats did not differ between either strain, demonstrating a similar level of systemic myogenic tone in FHL and FHH rats. Therefore, before any renal end-organ damage is present, myogenic response seems selectively impaired in renal vasculature of FHH rats. Aortic reactivity did not differ between FHL and FHH at the time points studied. The present study shows that vascular dysfunction in both small renal and systemic arteries precedes renal end-organ damage in a spontaneous model of hypertension-associated renal damage. These early vascular changes might be potentially involved in the increased susceptibility of FHH rats to renal injury.