Photochemically Aided Arteriovenous Fistula Creation to Accelerate Fistula Maturation.

Rates of arteriovenous fistula maturation failure are still high, especially when suboptimal size veins are used. During successful maturation, the vein undergoes lumen dilatation and medial thickening, adapting to the increased hemodynamic forces. The vascular extracellular matrix plays an important role in regulating these adaptive changes and may be a target for promoting fistula maturation. In this study, we tested whether a device-enabled photochemical treatment of the vein prior to fistula creation facilitates maturation. Sheep cephalic veins were treated using a balloon catheter coated by a photoactivatable molecule (10-8-10 Dimer) and carrying an internal light fiber. As a result of the photochemical reaction, new covalent bonds were created during light activation among oxidizable amino acids of the vein wall matrix proteins. The treated vein lumen diameter and media area became significantly larger than the contralateral control fistula vein at 1 week (p = 0.035 and p = 0.034, respectively). There was also a higher percentage of proliferating smooth muscle cells in the treated veins than in the control veins (p = 0.029), without noticeable intimal hyperplasia. To prepare for the clinical testing of this treatment, we performed balloon over-dilatation of isolated human veins and found that veins can tolerate up to 66% overstretch without notable histological damage.

Creating a Natural Vascular Scaffold by Photochemical Treatment of the Extracellular Matrix for Vascular Applications.

The development of bioscaffolds for cardiovascular medical applications, such as peripheral artery disease (PAD), remains to be a challenge for tissue engineering. PAD is an increasingly common and serious cardiovascular illness characterized by progressive atherosclerotic stenosis, resulting in decreased blood perfusion to the lower extremities. Percutaneous transluminal angioplasty and stent placement are routinely performed on these patients with suboptimal outcomes. Natural Vascular Scaffolding (NVS) is a novel treatment in the development for PAD, which offers an alternative to stenting by building on the natural structural constituents in the extracellular matrix (ECM) of the blood vessel wall. During NVS treatment, blood vessels are exposed to a photoactivatable small molecule (10-8-10 Dimer) delivered locally to the vessel wall via an angioplasty balloon. When activated with 450 nm wavelength light, this therapy induces the formation of covalent protein-protein crosslinks of the ECM proteins by a photochemical mechanism, creating a natural scaffold. This therapy has the potential to reduce the need for stent placement by maintaining a larger diameter post-angioplasty and minimizing elastic recoil. Experiments were conducted to elucidate the mechanism of action of NVS, including the molecular mechanism of light activation and the impact of NVS on the ECM.

Photosensitized Oxidative Dimerization at Tyrosine by a Water-Soluble 4-Amino-1,8-naphthalimide.

The oxidation of proteins generates reactive amino acid (AA) residue intermediates, leading to protein modification and cross-linking. Aerobic studies with peptides and photosensitizers allow for the controlled generation of reactive oxygen species (ROS) and reactive AA residue intermediates, providing mechanistic insights as to how natural protein modifications form. Such studies have inspired the development of abiotic methods for protein modification and crosslinking, including applications of biomedical importance. Dityrosine linkages derived from oxidation at tyrosine (Tyr) residues represent one of the more well-understood oxidation-induced modifications. Here we demonstrate an aerobic, visible light-dependent oxidation reaction of Tyr-containing substrates promoted by a water-soluble 4-amino-1,8-naphthalimide-based photosensitizer. The developed procedure converts Tyr-containing substrates into o,o'-Tyr-Tyr linked dimers. The regioselectively formed o,o'-Tyr-Tyr linkage is consistent with dimeric standards prepared using a known enzymatic method. A crossover study with two peptides provides a statistical mixture of three distinct o,o'-Tyr-Tyr linked dimers, supporting a mechanism that involves Tyr residue oxidation followed by intermolecular combination.

Solvent-Dependent Photophysics and Reactivity of Monomeric and Dimeric 4-Amino-1,8-Naphthalimides.

The solvent-dependent photophysics of two 4-amino-substituted 1,8-naphthalene imides (AIs) were studied using fluorescence spectroscopy and laser flash photolysis. The compounds were functionalized with water-soluble 2,2'(ethylenedioxy) diethylamine groups, yielding a monomer (AI3) and a dimer (AI4). The radiative and nonradiative singlet-state deactivation processes of AI3 and AI4 were quantified in 10 solvents and at different pH values. The fluorescence quantum yield for the AI4 dimer in water was more than 100× lower than in other solvents, or for the monomeric AI3. The enhanced nonradiative decay of aqueous solutions of dimeric AI4 is accompanied by biexponential decay kinetics, suggesting equilibration with a dark excited state. An oxygen-quenchable triplet state (T1) of AI3 was produced upon 416 nm excitation in both water and n-octanol. In water, the T1 state evolved into a long-lived transient that was unreactive toward oxygen or several electron donors. This species was not observed in n-octanol. The transient observed upon 416 nm excitation of AI4 in water was extremely weak. However, production of T1 in both AI3 and AI4 was evidenced by the photoinduced electron transfer to methyl viologen, albeit in low quantum yield (0.0503 and 0.00778 for AI3 and AI4, respectively). The photophysics and reactivity are consistent with the production of an intramolecular charge transfer (ICT) state that is stabilized in water. Significantly, this stabilization enhances nonradiative decay pathways, particularly in the AI4 dimer. The results indicate that the photochemistry of these compounds can be environmentally mediated, switching from radical- to triplet-initiated processes.

In Vivo Target Validation Using Biological Molecules in Drug Development.

Drug development is a resource-intensive process requiring significant financial and time investment. Preclinical target validation studies and in vivo testing of the therapeutic molecules in clinically relevant disease models can accelerate and significantly de-risk later stage clinical development. In this chapter, we will focus on (1) in vivo animal models and (2) pharmacological tools for target validation.

Photochemical Modification of the Extracellular Matrix to Alter the Vascular Remodeling Process.

Therapeutic interventions for vascular diseases aim at achieving long-term patency by controlling vascular remodeling. The extracellular matrix (ECM) of the vessel wall plays a crucial role in regulating this process. This study introduces a novel photochemical treatment known as Natural Vascular Scaffolding, utilizing a 4-amino substituted 1,8-naphthimide (10-8-10 Dimer) and 450 nm light. This treatment induces structural changes in the ECM by forming covalent bonds between amino acids in ECM fibers without harming vascular cell survival, as evidenced by our results. To further investigate the mechanism of this treatment, porcine carotid artery segments were exposed to 10-8-10 Dimer and light activation. Subsequent experiments subjected these segments to enzymatic degradation through elastase or collagenase treatment and were analyzed using digital image analysis software (MIPAR) after histological processing. The results demonstrated significant preservation of collagen and elastin structures in the photochemically treated vascular wall, compared to controls. This suggests that photochemical treatment can effectively modulate vascular remodeling by enhancing the resistance of the ECM scaffold to degradation. This approach shows promise in scenarios where vascular segments experience significant hemodynamic fluctuations as it reinforces vascular wall integrity and preserves lumen patency. This can be valuable in treating veins prior to fistula creation and grafting or managing arterial aneurysm expansion.

Differential effects of Selexipag [corrected] and prostacyclin analogs in rat pulmonary artery.

{4-[(5,6-Diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}acetic acid (ACT-333679) is the main metabolite of the selective prostacyclin (PGI(2)) receptor (IP receptor) agonist selexipag. The goal of this study was to determine the influence of IP receptor selectivity on the vasorelaxant efficacy of ACT-333679 and the PGI(2) analog treprostinil in pulmonary artery under conditions associated with pulmonary arterial hypertension (PAH). Selexipag and ACT-333679 evoked full relaxation of pulmonary artery from control and monocrotaline (MCT)-PAH rats, and ACT-333679 relaxed normal pulmonary artery contracted with either endothelin-1 (ET-1) or phenylephrine. In contrast, treprostinil evoked weaker relaxation than ACT-333679 of control pulmonary artery and failed to induce relaxation of pulmonary artery from MCT-PAH rats. Treprostinil did not evoke relaxation of normal pulmonary artery contracted with either ET-1 or phenylephrine. Expression of prostaglandin E(3) (EP(3)) receptor mRNA was increased in pulmonary artery from MCT-PAH rats. In contraction experiments, the selective EP(3) receptor agonist sulprostone evoked significantly greater contraction of pulmonary artery from MCT-PAH rats compared with control rats. The presence of a threshold concentration of ET-1 significantly augmented the contractile response to sulprostone in normal pulmonary artery. ACT-333679 did not evoke direct contraction of rat pulmonary artery, whereas treprostinil evoked concentration-dependent contraction that was inhibited by the EP(3) receptor antagonist (2E)-3-(3',4'-dichlorobiphenyl-2-yl)-N-(2-thienylsulfonyl)acrylamide. Antagonism of EP(3) receptors also revealed a relaxant response to treprostinil in normal pulmonary artery contracted with ET-1. These data demonstrate that the relaxant efficacy of the selective IP receptor agonist selexipag and its metabolite ACT-333679 is not modified under conditions associated with PAH, whereas relaxation to treprostinil may be limited in the presence of mediators of disease.

Retained Functionality of Atherosclerotic Human Arteries Following Photoactivated Linking of the Extracellular Matrix by Natural Vascular Scaffolding Treatment.

In this study, we investigated natural vascular scaffolding (NVS) treatment on vascular functionality using freshly isolated human popliteal arteries in vitro. Arteries were exposed to intraluminal NVS treatment consisting of a compound (4 amino-1,8-naphthalimide) photoactivated by a 450-nm light-emitting light fiber placed inside the artery. This procedure results in covalent linking between the extracellular matrix proteins to achieve a larger vessel diameter post-angioplasty and minimizing elastic recoil. Immediately following NVS treatment, rings were cut from the treated arteries and mounted in organ baths for contractility testing in response to U46619 and sodium nitroprusside. We also investigated the effect of NVS treatment on IL-6 cytokine release from vascular rings following a 4-h organoculture post-NVS treatment. Based on our results, we conclude that exposure of the vessels to NVS treatment does not adversely affect the contractile responsiveness of the vascular smooth muscle and exerts no pro-inflammatory effect. Graphical abstract.

Natural Vascular Scaffolding Treatment Promotes Outward Remodeling During Arteriovenous Fistula Development in Rats.

Following creation, an arteriovenous fistula (AVF) must mature (i.e., enlarge lumen to allow high blood flow) before being used for hemodialysis. AVF maturation failure rates are high, and currently, there are no effective therapy to treat this problem. The maturation process is likely affected by the integrity of the vascular extracellular matrix (ECM). Natural Vascular Scaffolding (NVS) Therapy is a new technology that interlinks collagen and elastin via photoactivation of a locally delivered small molecule (4-amino-1,8-naphtalamide). We hypothesized that NVS Therapy may improve AVF remodeling by preserving ECM integrity. AVFs were created in Wistar male rats by connecting the femoral vein (end) to femoral artery (side) in the same limb. Immediately after blood flow was restored to dilate the femoral vein by arterial pressure, a 10 µl-drop of the NVS compound (2 mg/ml) was placed on the anastomosis perivascularly. Following 5-min incubation, the NVS treated area was exposed to 1-min illumination by 450-nm light. The control group received 10 µl-drop of phosphate buffered saline (PBS) and the same light activation. The skin was closed, and rats were euthanized 4 weeks (n = 6-9 per group) post-AVF creation for histology, morphometry, immunohistochemistry (IHC), and multiphoton microscopy for second-harmonic-generation evaluation of collagen fibers. The vascular thickness was similar in both groups. The AVF vein's open lumen area and % open lumen area in NVS-treated rats were significantly larger than in PBS-treated rats (4.2-fold p = 0.014 and 2-fold p = 0.009, respectively). The inflammatory markers IL-6 and MMP-9 in the AVF walls were significantly decreased in the NVS group than the PBS group. Collagen fibers in the vascular wall trended toward perpendicular alignment to the lumen circumference in the NVS-treated AVFs, with more defined shape but less area than in the PBS-treated AVFs. These results indicate that the NVS Therapy exerted changes in collagen, which may influence AVF maturation. Rats tolerated the NVS treatment well, and the lack of cell death by the treatment was confirmed in cell culture experiments. These results suggest that NVS treatment is safe and may have therapeutic potential by facilitating lumen expansion to enhanced AVF maturation in patients.

Telmisartan regresses left ventricular hypertrophy in caveolin-1-deficient mice.

The role of angiotensin II (Ang II) in promoting cardiac hypertrophy is well known; however, its role in a spontaneous model of hypertrophy in mice lacking the protein caveolin-1 (Cav-1 KO) has not been explored. In this study, WT and Cav-1 KO mice were treated with angiotensin receptor blocker (ARB), telmisartan (Telm), and cardiac function was assessed by echocardiography. Treatment of Cav-1 KO mice with Telm significantly improved cardiac function compared with age-matched vehicle-treated Cav-1 KO mice, whereas Telm did not affect cardiac function in WT mice. Both left ventricular (LV) weight to body weight ratios and LV to tibial length ratios were also reverted by Telm in Cav-1 KO but not in WT mice. LV hypertrophy was associated with increased expression of natriuretic peptides A and B, ß-myosin heavy chain and TGF-ß, and Telm treatment normalized the expression of these genes. Telm reduced the expression of collagen genes (Col1A and Col3A) and associated perivascular fibrosis in intramyocardial vessels in Cav-1 KO mice. In conclusion, Telm treatment reduces indexes of cardiac hypertrophy in this unique genetic model of spontaneous LV hypertrophy.

Blockade of placental growth factor reduces vaso-occlusive complications in murine models of sickle cell disease.

Vaso-occlusive crisis (VOC) is the most common and debilitating complication of sickle cell disease (SCD); recurrent episodes cause organ damage and contribute to early mortality. Plasma placental growth factor (PlGF) levels are elevated in SCD and can further increase under hypoxic conditions in SCD mice. Treatment with a PlGF-neutralizing antibody (anti-PlGF Ab) in SCD mice reduced levels of monocyte chemoattractant protein-3, eotaxin, macrophage colony-stimulating factor, and plasminogen activator inhibitor-1 significantly, and of macrophage-derived chemokine and macrophage inflammatory protein-3ß moderately; this may contribute to inhibition of leukocyte recruitment, activation, and thrombosis. In subsequent experiments, anti-PlGF Ab treatment significantly reduced plasma lactate dehydrogenase levels, indicating possible reduction in cellular destruction and/or hemolysis. Histopathology studies revealed decreased incidence and severity of congestion in the lungs and spleen with repeated anti-PlGF Ab treatment. Furthermore, anti-PlGF Ab significantly reduced vaso-occlusion events under hypoxic conditions in a modified dorsal skinfold chamber model in SCD mice. Therefore, elevated PlGF levels may contribute to recruitment and activation of leukocytes. This can subsequently lead to increased pathology of affected organs in addition to mediating acute hypoxia/reoxygenation-triggered vaso-occlusion under SCD conditions. Thus, targeting PlGF may offer a therapeutic approach to reduce acute VOC and possibly alleviate long-term vascular complications in patients with SCD.

Fasudil, a Rho-kinase inhibitor, attenuates angiotensin II-induced abdominal aortic aneurysm in apolipoprotein E-deficient mice by inhibiting apoptosis and proteolysis.

BACKGROUND: Angiotensin II (Ang II) accelerates atherosclerosis and induces abdominal aortic aneurysm (AAA) in an experimental mouse model. Agonism of a G protein-coupled receptor by Ang II activates Rho-kinase and other signaling pathways and results in activation of proteolysis and apoptosis. Enhanced proteolysis and smooth muscle cell apoptosis are important mechanisms associated with AAA. In this study, we tested the hypothesis that fasudil, a Rho-kinase inhibitor, could attenuate Ang II-induced AAA formation by inhibiting vascular wall apoptosis and extracellular matrix proteolysis. METHODS AND RESULTS: Six-month-old apolipoprotein E-deficient mice were infused with Ang II (1.44 mg x kg(-1) x d(-1)) for 1 month. Animals were randomly assigned to treatment with fasudil (136 or 213 mg x kg(-1) x d(-1) in drinking water) or tap water. Ang II infusion induced AAA formation in 75% of the mice, which was accompanied by an increase in proteolysis detected by zymographic analysis and quantified by active matrix metalloproteinase-2 activity, as well as apoptosis detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and quantified by both caspase-3 activity and histone-associated DNA fragmentation. The level of DNA fragmentation in the suprarenal aorta correlated with AAA diameter. Ang II also increased atherosclerotic lesion area and blood pressure. Fasudil treatment resulted in a dose-dependent reduction in both the incidence and severity of AAA. At the higher dose, fasudil decreased AAA by 45% while significantly inhibiting both apoptosis and proteolysis, without affecting atherosclerosis or blood pressure. CONCLUSIONS: These data demonstrate that inhibition of Rho-kinase by fasudil attenuated Ang II-induced AAA through inhibition of both apoptosis and proteolysis pathways.

Regulation of cardiac and renal mineralocorticoid receptor expression by captopril following myocardial infarction in rats.

Myocardial infarction (MI) activates the renin-angiotensin system in the heart and increases local production of aldosterone. This hormone may increase reactive fibrosis in the myocardium favoring heart failure development. To elucidate the potential contribution of aldosterone to cardiac remodeling following MI, we evaluated the expression of mineralocorticoid receptors (MCR) in the left ventricle (LV) and kidney of rats after MI and captopril treatment. MI was induced by ligation of the coronary artery in Wistar rats, which were separated into (1) sham-operated group, (2) MI group, (3) MI-captopril treated group (cap, 50 mg kg(-1) day(-1)). One month later angiotensin converting enzyme (ACE) activity was assayed in the plasma, LV and kidney. Cardiac and renal angiotensin II (Ang II) levels were determined by ELISA and MCR mRNA expression and protein were measured by Taqman RT-PCR and Western blot, respectively. Cardiac MCR mRNA and protein levels increased nearly by 80% after MI and Cap treatment normalized cardiac MCR protein and mRNA expression. Kidney MCR expression was not affected. ACE activity increased 34% in the plasma and 83% in the LV after MI. This increase was prevented by Cap. Ang II concentration increased 225% in the LV and 193% in kidney, which was partially attenuated by Cap. Our data demonstrate upregulation of MCR in the heart following MI what may facilitate the effects of aldosterone in the ventricular remodeling process. ACE inhibitors may reduce reactive fibrosis not only by decreasing Ang II production but also by attenuating the aldosterone-signaling pathway by decreasing the expression of MCR receptors.

Estrogen inhibits the initiation of fatty streaks throughout the vasculature but does not inhibit intra-plaque hemorrhage and the progression of established lesions in apolipoprotein E deficient mice.

Estrogen has previously been shown to inhibit development of early atherosclerotic lesions in hyperlipidemic mice. However, it is still not known whether estrogen also inhibits progression and destabilization of lesions once established and whether there are other effects of long-term hormone therapy in mice. To address this question, male, 20-week old, apolipoprotein E deficient mice were administered 17-beta estradiol or placebo subcutaneously for between 4 and 40 weeks. Estrogen administration did not cause regression of established lesions in the carotid arteries, aortic arch and thoracic aorta but prevented the initiation of new lesions in the abdominal aorta and iliac, femoral and popliteal arteries. Although the established lesions were slightly smaller in the innominate artery of the estrogen treated mice, estrogen did not prevent lesion progression. Estrogen administration also had no effect on the frequency of intra-plaque hemorrhage, atrophy of the fibrous cap, medial erosion, and fibro-fatty nodules, but did reduce the frequency of fatty streaks that form on top of or adjacent to the established lesions in the innominate artery. These data suggest that estrogen inhibits the initiation of the fatty streak but does not alter the progression of established lesions through stages of instability and healing.

Anti-atherosclerotic effect of simvastatin depends on the presence of apolipoprotein E.

Low density lipoprotein receptor deficient (LDLR-KO) and apolipoprotein E deficient (apo E-KO) mice both develop hyperlipidemia and atherosclerosis by different mechanisms. The aim of the present study was to compare the effects of simvastatin on cholesterol levels, endothelial dysfunction, and aortic lesions in these two models of experimental atherosclerosis. Male LDLR-KO mice fed a high cholesterol (HC; 1%) diet developed atherosclerosis at 8 months of age with hypercholesterolemia. The addition of simvastatin (300 mg/kg daily) to the HC diet for 2 more months lowered total cholesterol levels by approximately 57% and reduced aortic plaque area by approximately 15% compared with the LDLR-KO mice continued on HC diet alone, P<0.05. Simvastatin treatment also improved acetylcholine (ACh)-induced endothelium-dependent vasorelaxation in isolated aortic rings, which was associated with an increase in NOS-3 expression by approximately 88% in the aorta measured by real time polymerase chain reaction (PCR), P<0.05. In contrast, in age-matched male apo E-KO mice fed a normal diet, the same treatment of simvastatin elevated serum total cholesterol by approximately 35%, increased aortic plaque area by approximately 15%, and had no effect on endothelial function. These results suggest that the therapeutic effects of simvastatin may depend on the presence of a functional apolipoprotein E.

Role of estrogen receptors in the vascular system.

Estrogens have been shown to exert significant benefits on the cardiovascular system both in animals and in postmenopausal women. However, the exact mechanism of these effects are, for the most part, still unknown. The goal of this paper is to evaluate the role of estrogen receptors (ER) in mediating some of the cardiovascular beneficial actions of 17 beta-estradiol (E2). This analysis was possible because of the availability of ER alpha (ER alpha KO) and ER beta-deficient (ER beta KO) mice, and access to a patient with ER alpha-deficiency. Experimental results obtained in our laboratory demonstrated that the ER alpha subtype mediates E2-induced increase in endothelial nitric oxide production and facilitation of fibroblast growth factor-elicited angiogenesis in vivo. Others have confirmed these findings. Experiments using a novel ER-antagonist and ApoExER alpha double-knockout mice proved that ER alpha mediates some of the antiatherosclerotic effects of E2 as well. In contrast, both the ER alpha and ER beta subtypes appear to mediate the beneficial effects of E2 on vascular smooth muscle proliferation after vessel injury. The young male patient with ER alpha-deficiency exhibited reduced endothelial nitric oxide production and premature coronary arteriosclerosis. These studies in mice and a male human subject suggest that absence of functional ER may represent a novel risk factor for cardiovascular diseases.

Effect of estrogen on endothelial function and angiogenesis.

Animal studies evaluating gender difference, the effects of gonadectomy and estrogen replacement and clinical studies in post-menopausal women with and without estrogen replacement therapy (ERT) proved that estrogen exerts significant benefits on the cardiovascular system. Since effects on the plasma lipoprotein profile is responsible for only approximately 25-40% of the cardiovascular protection exerted by estrogens, it is postulated that direct effects of estrogen on the vascular wall must play an important role. Indeed, experimental and clinical evidence accumulated over the past decade, and reviewed briefly here, indicate that at least a part of cardiovascular benefits of 17 beta-estradiol can be attributed to the direct effect of the ovarian sex steroid hormone on vascular endothelial cells. Maintenance and upregulation of endothelial nitric oxide production and suppression of EDCF generation by 17 beta-estradiol may play an important role in preventing or reversing endothelial dysfunction, associated with atherosclerosis, hypertension and other cardiovascular diseases. Stimulation of angiogenesis (especially collateral vessel formation in ischemic tissues) by the ovarian steroid hormone could be beneficial in coronary artery disease, peripheral vascular disease, cerebral ischemia (stroke) and congestive heart failure. Despite these indisputable beneficial effects, several key questions remain to be answered in the future, including the better understanding of the apparently opposite effects of estrogen on prevention of cardiovascular disease vs. treatment of existing disease.

Amlodipine Reduces Inflammation despite Promoting Albuminuria in the Streptozotocin-Induced Diabetic Rat.

Amlodipine reduces blood pressure; however, its effect in the diabetic kidney irrespective of its blood pressure-lowering effects is unclear. This study examined the effects of amlodipine (0, 5, 10 and 20 mg/kg; D(A0), D(A5), D(A10) and D(A20), respectively) for 12 weeks on renal functional and structural changes in the streptozotocin-induced diabetic rat, a nonhypertensive model of diabetes-associated hyperfiltration. Compared with nondiabetic rats, diabetes (D) was associated with increased urine albumin excretion (UAE, 12.6 ± 3.40 vs. 3.73 ± 1.14 mg/day), glomerular filtration rate (2.17 ± 0.09 vs. 1.64 ± 0.12 ml/min/g kidney weight), glomerulosclerosis (0.21 ± 0.03 vs. 0.05 ± 0.01 AU) and infiltration of inflammatory cells (18.5 ± 2.78 vs. 6.92 ± 0.70 cells/cm(2)), but did not affect mean arterial pressure (MAP, 110 ± 4.70 vs. 109 ± 5.33 mm Hg). While D(A20) abolished glomerular hyperfiltration (1.49 ± 0.05 ml/min/g kidney weight) and inflammatory cell abundance (6.0 ± 0.79 cells/cm(2)), it exacerbated UAE (43.5 ± 8.49 mg/day) and increased MAP (132 ± 3.76 mm Hg), but had no effect on renal pathology. These data suggest that amlodipine reduces renal inflammation and abolished glomerular hyperfiltration, but increases blood pressure and exacerbates albuminuria in the rat model of normotensive diabetic kidney disease. We conclude that amlodipine may have limited renoprotective effects in the face of hyperfiltration and absence of elevated blood pressure.

Identification of cathepsin L as a potential sex-specific biomarker for renal damage.

The renin-angiotensin system is a well-known regulator of blood pressure and plays an important role in the pathogenesis of cardiovascular disease and renal damage. Genetic factors, including single nucleotide polymorphisms and sex, are increasingly recognized as potential risk factors for the development of cardiovascular disease. Double transgenic rats (dTGRs), harboring human renin and angiotensinogen genes, were used in this study to investigate potential sex differences influencing renal function and renal gene expression. dTGR males and females had comparable increases in blood pressure, whereas body weight, albuminuria/proteinuria, and urine flow rate were higher in males. At 8 weeks of age, renal plasma flow and glomerular filtration rate were proportionally lower in males, and renal vascular resistance tended to be higher. Males developed more severe tubulointerstitial and vascular lesions. By the end of week 8, 40%of the males but none of the females had died. Genome expression studies were performed with RNA from kidneys of 7-week-old male and female dTGRs and control rats to further investigate the sex-related differences on a molecular level. Forty-five genes showed sex-dependent expression patterns in dTGRs that were significantly different compared to controls. Cathepsin L, one of the genes differentially expressed between the sexes, was also shown to be strongly associated with the degree of renal injury. In dTGRs, urinary cathepsin L at week 7 was higher in males (nanograms per 24 hours: male, 512±163; female, 132±70). These results reveal a potential new biomarker for the personalized diagnosis and management of chronic kidney disease.