Nx Rats Research Articles

An association of losartan-hydrochlorothiazide, but not losartan-furosemide, completely arrests progressive injury in the remnant kidney.

We have previously shown that an association of losartan and hydrochlorothiazide, initiated 1 mo after 5/6 nephrectomy (Nx), reversed hypertension and albuminuria and promoted lasting renoprotection. In this new study, we investigated whether equal or even better protection could be obtained by combining losartan and furosemide. Nx was performed in 58 Munich-Wistar rats. One month later, tail-cuff pressure and albuminuria were markedly elevated. At this time, Nx rats were distributed among the following four groups: untreated Nx rats, Nx rats that received losartan, Nx rats that received losartan + hydrochlorothiazide, and Nx rats that received losartan + furosemide. Seven months later, Nx rats exhibited high mortality, severe hypertension, albuminuria, glomerulosclerosis, and interstitial fibrosis. Losartan treatment limited mortality and attenuated the renal and hemodynamic abnormalities associated with Nx. As previously shown, the losartan + hydrochlorothiazide association normalized tail-cuff pressure and albumin, prevented renal injury, and reduced mortality to zero. The losartan + furosemide treatment failed to reduce tail-cuff pressure or albumin to normal and prevented renal injury less efficiently than the losartan and hydrochlorothiazide regimen. The reasons for the differing efficacies of the losartan + furosemide and losartan + hydrochlorothiazide schemes are unclear and may include beneficial nondiuretic actions of thiazides, such as vasorelaxation and antiproliferative activity. These results refute the established concept that thiazides and thiazide-like diuretics are ineffective at advanced chronic kidney disease stages. Rather, they suggest that, in view of their renoprotective action, these compounds may even be preferable to loop diuretics in the management of hypertension in advanced chronic kidney disease.

The role of adipose tissue asymmetric dimethylarginine/dimethylarginine dimethylaminohydrolase pathway in adipose tissue phenotype and metabolic abnormalities in subtotally nephrectomized rats.

The lipodystrophy-like phenotype has been suggested in early chronic kidney disease (CKD). It includes adipose tissue atrophy, systemic insulin resistance (IR), dyslipidemia and ectopic lipid accumulation. To elucidate its pathogenesis, we investigated the role of two uremic toxins that affect insulin sensitivity: an endogenous nitric oxide synthase inhibitor, and asymmetric dimethylarginine (ADMA) and indoxyl sulfate (IS). Six-week-old Sprague-Dawley rats were rendered CKD by subtotal nephrectomy (Nx) and compared with sham-operated rats. Cultured 3T3-L1 fibroblasts were differentiated into mature adipocytes with or without ADMA. Transgenic (Tg) mice overexpressing each isoform of ADMA degrading enzyme, dimethylarginine dimethylaminohydrolase 1 (DDAH1) and DDAH2 were subject to Nx and their phenotypes were investigated. In Nx rats, IR was evident and insulin stimulation failed to activate insulin signaling in adipose tissues. Adipose tissue weight, adipocyte size and adipocyte differentiation marker expressions decreased as a consequence of IR in Nx. Tissue lipid content in the liver and muscle increased in Nx rats. Tissue levels of ADMA, IS and oxidative stress increased in the adipose tissue of Nx rats. Both DDAH1 and DDAH2 expressions decreased, and a putative IS receptor, aryl hydrocarbon receptor, expression increased in the adipose tissue of Nx rats. ADMA inhibited adipocyte differentiation, triglyceride accumulation and insulin signaling, which were reversed by pretreatment with cGMP. In each type of Tg mice overexpressing DDAH1 or DDAH2, all lipodystrophy-like phenotypes induced by Nx were reversed. In mild CKD, dysregulation of the ADMA/DDAH pathway in adipose tissue triggers lipodystrophy-like phenotype changes, including ectopic fat depositions.

Gut Lactobacillus protects against the progression of renal damage by modulating the gut environment in rats.

The role of gut microbiota in the progression of chronic kidney disease (CKD) has not been fully elucidated. Renal failure was induced in 6-week-old spontaneously hypertensive rats by 5/6 nephrectomy (Nx). We analyzed the gut microbiota population to identify the relevant species potentially involved in inducing renal damage. Human colon Caco-2 cells were used to delineate the mechanism involved in the molecular changes in the gut of Nx rats. Nx rats showed an increase in Bacteroides (Bact) and a decrease in Lactobacillus (Lact) species compared with sham-operated rats. Lact, but not Bact, populations were significantly associated with urinary protein excretion. Treatment of Nx rats with 1 × 10(10) CFU/kg/day Lact ameliorated increased urinary protein excretion and higher serum levels of the uremic toxins, indoxyl sulfate and p-cresyl sulfate, and serum urea nitrogen levels. Lact also attenuated systemic inflammation in Nx rats, as evaluated by serum lipopolysaccharide, interleukin-6 and C-reactive protein levels. Histologically, renal sclerosis in Nx rats was restored by Lact treatment. A reduction in the expression of tight junction proteins and the Toll-like receptor 2 (TLR2), a putative Lact receptor, in the colons of Nx rats were mitigated by Lact. Treatment of Caco-2 cells with indole downregulated tight junction protein expression, which was abolished by exposure to Lact. The effects of Lact were reversed by treatment with OxPAPC, a TLR inhibitor. Similarly, the increase in the permeability of the Caco-2 cell monolayer was reversed by the administration of Lact. Lact upregulated TLR2 expression in Caco-2 cells. Lact also attenuated the increase in serum indoxyl sulfate and urea levels and urinary protein excretion in Nx rats even in the pseudogerm-free environment. Lact supplementation mitigated the systemic inflammation and proteinuria associated with renal failure, suggesting that in the gut microbiota, Lact plays a protective role against the progression of CKD.

Epoetin beta pegol prevents endothelial dysfunction as evaluated by flow-mediated dilation in chronic kidney disease rats

Chronic kidney disease (CKD) patients have a poor prognosis due to cardiovascular disease. Anemia and endothelial dysfunction are important risk factors for cardiovascular events in CKD patients, and treatment with erythropoiesis-stimulating agent (ESA) has been reported to improve the quality of life in CKD patients. In this study, we evaluated the effect of anemia correcting dose of epoetin beta pegol (continuous erythropoietin receptor activator; C.E.R.A.) on endothelial function in 5/6 nephrectomized rats (Nx rats). C.E.R.A. was subcutaneously administered once a fortnight, 5 times in total, from 1 week after nephrectomy. Twenty-four hours after last administration, endothelial function was evaluated by measuring flow-mediated dilation (FMD) in the femoral arteries of anesthetized Nx rats by ultrasound system. Femoral arteries were harvested for western blot analysis. C.E.R.A. significantly increased FMD of Nx rats. Endothelium-independent vasodilation induced by nitroglycerin injection was not influenced by C.E.R.A treatment. Nox4 expression and nitrotyrosine accumulation were significantly decreased, and phosphorylation of eNOS was significantly enhanced in the femoral arteries of C.E.R.A.-treated rats. C.E.R.A. normalized hemoglobin levels but did not affect body weight, systolic blood pressure, heart rate, urinary protein excretion and plasma creatinine. These results indicate that C.E.R.A. prevented endothelial dysfunction in Nx rats, possibly through reduction of local oxidative stress and enhancement of eNOS phosphorylation in the arteries. This study provides the first evidence that C.E.R.A. prevented endothelial dysfunction in CKD model rats under conditions of amelioration of anemia.

Slow- and fast-twitch hindlimb skeletal muscle phenotypes 12 wk after ⅚ nephrectomy in Wistar rats of both sexes.

This study describes fiber-type adaptations in hindlimb muscles, the interaction of sex, and the role of hypoxia on this response in 12-wk ⅚ nephrectomized rats (Nx). Contractile, metabolic, and morphological features of muscle fiber types were assessed in the slow-twitch soleus and the fast-twitch tibialis cranialis muscles of Nx rats, and compared with sham-operated controls. Rats of both sexes were considered in both groups. A slow-to-fast fiber-type transformation occurred in the tibialis cranialis of Nx rats, particularly in males. This adaptation was accomplished by impaired oxidative capacity and capillarity, increased glycolytic capacity, and no changes in size and nuclear density of muscle fiber types. An oxidative-to-glycolytic metabolic transformation was also found in the soleus muscle of Nx rats. However, a modest fast-to-slow fiber-type transformation, fiber hypertrophy, and nuclear proliferation were observed in soleus muscle fibers of male, but not of female, Nx rats. Serum testosterone levels decreased by 50% in male but not in female Nx rats. Hypoxia-inducible factor-1α protein level decreased by 42% in the tibialis cranialis muscle of male Nx rats. These data demonstrate that 12 wk of Nx induces a muscle-specific adaptive response in which myofibers do not change (or enlarge minimally) in size and nuclear density, but acquire markedly different contractile and metabolic characteristics, which are accompanied by capillary rarefaction. Muscle function and sex play relevant roles in these adaptations.

Abstract 61: miRNA-21 Targets Left Ventricular Peroxisome Proliferator-activated Receptor Alpha in a Rat Model of Type 4 Cardiorenal Syndrome

Type 4 cardiorenal syndrome (CRS4) is a condition in which chronic kidney disease (CKD) contributes to cardiovascular pathology including cardiac dysfunction, left ventricular (LV) hypertrophy, atherosclerosis, and heart failure. We have used a rat model of CKD, the 5/6 nephrectomy (5/6 NX), to study molecular mechanisms that mediate the development of cardiac pathology in CRS4. We previously reported that the upregulation of microRNA miR-21-5p (miR-21) in the left ventricle (LV) was accompanied by pathological remodeling and a drop in fractional shortening in adult male Sprague Dawley rats 7 weeks after 5/6 NX. Systemic knockdown of miR-21 in 5/6 NX rats with LNA-modified anti-miR-21 improved cardiac function, however it did not reduce the modest fibrosis observed with our 5/6 NX model or upregulate miR-21 targets identified in other models of cardiac pathology, suggesting a novel cardiac target for miR-21 in this model. Through next-generation mRNA sequencing of LV tissues from anti-miR-21 treated rats, and subsequent Ingenuity Pathway Analysis, we have found cardioprotective alterations in genes related to cardiac hypertrophy, metabolism, immune and inflammatory signaling, and atherosclerosis. Suppression or reduction of miR-21 target peroxisome proliferator-activated receptor alpha (PPARa), a master regulator of fatty acid oxidation, has been reported to be involved in all of these processes. Translational suppression of PPARa through miR-21 has been confirmed in other tissues, but not in the myocardium. The average LV PPARa protein expression level was significantly reduced (-37.7 ± 5.4%) in the 5/6 NX model and restored by miR-21 knockdown (Western blot; n=5-6/group; mean ± SEM). Immunohistochemistry revealed that the pronounced alterations in PPARa expression occurred within cardiomyocytes (CMs) in these samples. Transfection of neonatal CMs with pre-miR-21, significantly reduced PPARa protein expression within 48 hours (-23.0 ± 0.9%; n=3/group). These data indicate that PPARa suppression by miR-21 occurs within LV in 5/6 NX model of CKD and that miR-21 can regulate PPARa in CMs. This regulation may be relevant in other models of chronic cardiac disease where increased miR-21 and suppression of PPARa have been independently reported.

Vasopeptidase Inhibition Corrects the Structure and Function of the Small Arteries in Experimental Renal Insufficiency

Background: We studied whether vasopeptidase inhibition corrects the structure and function of the small arteries in experimental chronic renal insufficiency (CRI). Methods: After 5/6 nephrectomy (NX) surgery was performed on rats, there was a 14-week follow-up, allowing CRI to become established. Omapatrilat (40 mg/kg/day in chow) was then given for 8 weeks, and the small mesenteric arterial rings were investigated in vitro using wire and pressure myographs. Results: Plasma and ventricular B-type natriuretic peptide (BNP) concentrations were increased 2- to 2.7-fold, while systolic blood pressure (BP) increased by 32 mm Hg after NX. Omapatrilat treatment normalized the BNP and reduced the BP by 45 mm Hg in the NX rats. Endothelium-dependent vasorelaxation was impaired but the response to acetylcholine was normalized after omapatrilat treatment. Vasorelaxations induced by nitroprusside, isoprenaline and levcromakalim were enhanced after omapatrilat, and the responses were even more pronounced than in untreated sham-operated rats. Arterial wall thickness and wall-to-lumen ratio were increased after NX, whereas omapatrilat normalized these structural features and improved the strain-stress relationship in the small arteries; this suggests improved arterial elastic properties. Conclusion: Omapatrilat treatment reduced BP, normalized volume overload, improved vasorelaxation and corrected the dimensions and passive elastic properties of the small arteries in the NX rats. Therefore, we consider vasopeptidase inhibition to be an effective treatment for CRI-induced changes in the small arteries.

5/6 Nephrectomy Induces Changes in Immune and Inflammatory Gene Expression in the Left Ventricle

Chronic kidney disease (CKD) patients have increased cardiovascular risk. We have used 5/6 nephrectomy (5/6 NX) in Sprague Dawley rats to investigate the molecular mediators of cardiac pathology in this condition. Understanding gene expression changes in the heart could yield valuable information about pathology mediators and identify therapeutic targets. The goal of this study was to characterize changes in left ventricle (LV) gene expression following 5/6 NX. Reproducible alterations in LV structure and function were observed in the weeks following 5/6 NX including enhanced function (2 weeks), hypertrophic remodeling (4 and 5 weeks), and dilation with reduced function (7 weeks post). We performed next‐generation sequencing of mRNA extracted from the LV of 5/6 NX and sham rats at these time points (2 pools/group, 2‐3 animals/pool). Genes from our data set with an average fold change of >1.5 (vs. sham) were analyzed using DAVID and Ingenuity Pathway Analysis. Several signaling pathways were enriched with identified genes in a time dependent manner, corresponding with pathological LV changes. However, we found enrichment of altered genes in numerous immune system and inflammatory pathways throughout the study, including pathways related to atherosclerosis, granulocyte and agranulocyte movement, and innate and adaptive immune signaling. This study provides the first gene expression data showing that a reduction in renal mass alone can drive broad immune related gene expression changes in LV tissue. Further, these genes are altered beginning as early as 2 weeks after 5/6 NX, long before cardiac remodeling or dysfunction are observed. Support 13SDG17100095 and 8UL1TR000055.

Antioxidant and Antiinflammatory Therapies Preserve Renal Autoregulation in 5/6 Nx Rats

The aim of this study was to test whether antiinflammatory or antioxidant therapies preserve autoregulation in a model of chronic renal disease (5/6 Nx) We studied the following groups (n=6-7 each): Control sham, 5/6 Nx, 5/6 Nx + mycophenolate mofetil (MMF) and 5/6 Nx+Curcumin (Cur). Daily gavage dosed C (120 mg/k BW) and MMF (30 mg/k BW). Animals were followed during one month and SBP, by tail-cuff, measured. At the end of the study baseline renal blood flow and autoregulation (at fixed BP 100, 130 and 150 mmHg) were evaluated in anesthetized animals. Arterial pressure was 44% higher in 5/6 Nx compared to C. Cur partially prevented HT, while MMF further reduced BP. Absolute RBF was reduced in 5/6 Nx groups by 50% compared to control sham. Despite this effect, renal autoregulatory response to fixed increments in BP was preserved in Cur and MMF treated animals (fig 1). Co-treatment with Cur+MMF did not produce a synergy effect (3 rats). Moreover, acute treatment with TEMPOL to 5/6 Nx rats reduced systemic BP by 30%, however did not restore autoregulatory response, therefore RBF also decreased by 30% in those animals (4 rats) In conclusion, antioxidant and anti-inflammatory therapies preserve renal autoregulation likely inhibiting a common pathogenic pathway since the co-treatment did not provide further benefit. The salutary effect afforded by the two therapies may also be related to the preservation of vascular structure since acute relive of oxidative stress with TEMPOL did not restore autoregulatory response.

Aliskiren Modifies Canonical Wnt/β‐Catenin Signaling Pathway Associated to (Pro)renin Receptor on Heart and Kidney in 5/6 Nephrectomy‐Induced Hypertension in Rats

The Wnt/frizzled receptor signaling pathways are important in morphogenesis and development of essential organs. (Pro)renin receptor (PRR) has been characterized as a multifunctional protein associated with vacuolar H+‐ATPase (v‐H+‐ATPase) (independently of renin) and is required for canonical Wnt/β‐catenin signaling activation. Aliskiren, a direct renin inhibitor, has been used to treat cardiovascular disorders. The aim of this work was to study Aliskiren effects upon PRR and canonical Wnt/β‐catenin pathway in cardiac and renal tissue during 5/6 nephrectomy (5/6 Nx)‐induced hypertension. Male Wistar rats underwent 5/6 Nx or sham operation (S), and were treated with Aliskiren, 10 mg/kg/day/7 days. Control groups received vehicle. 24‐hour urine was collected and blood pressure was measured at the end of experiment. PRR, β‐catenin and dishevelled (Dvl‐1) expression was determined by immunoblot. Our results showed that, compared to sham animals, 5/6 Nx rats treated with Aliskiren had lowered blood pressure and improved seric creatinine, proteinuria and creatinine clearance. We found in nephrectomized rats that PRR expression increased in heart and kidney seven days after surgery, and aliskiren reduced PRR, β‐catenin and Dvl‐1 expression in both tissues. These results confirm the antihypertensive and renoprotective effects of aliskiren and interestingly, showed that aliskiren modifies PRR expression as well as canonical Wnt/β‐catenin signaling expression. Supported by SIP20140636 grant

The Effects of Losartan on Expressions of Klotho and Wnt in the 5/6 Nephrectomy Model of Progressive Chronic Kidney Disease

Klotho is a transmembrane protein expressed in different tissues but in particularly high levels in the kidney. Soluble klotho acts an endocrine factor with diverse functions including endothelium protection and reducing inflammation and fibrosis through mechanisms involving Wnt and TGFbeta1 signaling. Renal fibrosis is the end stage of the progressive chronic kidney disease (CKD) and renal klotho is markedly decreased in CKD patients. Renin angiotensin system (RAS) blockade is a relevant therapy to reduce CKD progression since it has a pivotal role in renal fibrogenesis. We investigated the effects of losartan on Klotho/Wnt signaling in 5/6 nephrectomy model of CKD in rats. Adult male Wistar rats underwent 5/6 nephrectomy (NX) were separated into 2 groups, NX control and NX treated with losartan (LOS, 25 mg/kg) in the drinking water from day 1 after nephrectomy. Blood pressure (MAP) was measured weekly. Urine was colected to determine proteinuria. After 8 weeks, rats were sacrificed and the remmnant kidney was removed and prepared to estimate the renal expression of collagen, epithelial‐to‐mesenchymal transition (EMT) marker (FSP1) and Klotho signaling (Klotho, Wnt and GSK3β) by real time PCR. NX rats presented hypertension and proteinuria that were blunted by LOS treatment. LOS also induced a reduction in the expression of the fibrosis markers collagen and FSP1. Klotho was reduced in NX animals and LOS significantly increased the expression. The LOS treatment increased Wnt3 expression and did not change the Wnt7b and GSK3β. These data suggest that besides the reduction in the MAP, the benefical renal effect of losartan may be at least in part due to upregulation of klotho expression.

Effects of oxonic acid-induced hyperuricemia on mesenteric artery tone and cardiac load in experimental renal insufficiency.

BackgroundRecent studies suggest a causal role for increased plasma uric acid in the progression of chronic renal insufficiency (CRI). However, uric acid also functions as an antioxidant with possible beneficial effects.MethodsWe investigated the influence of hyperuricemia on mesenteric arterial tone (main and second order branch) and morphology in experimental CRI. Forty-four Sprague–Dawley rats were 5/6 nephrectomized (NX) or Sham-operated and fed 2.0% oxonic acid or control diet for 9 weeks.ResultsOxonic acid feeding elevated plasma uric acid levels 2.4 and 3.6-fold in the NX and Sham groups, respectively. Plasma creatinine and urea were elevated 2-fold and blood pressure increased by 10 mmHg in NX rats, while hyperuricemia did not significantly influence these variables. Right and left ventricular weight, and atrial and B-type natriuretic peptide mRNA content were increased in NX rats, but were not affected by hyperuricemia. In the mesenteric artery, hyperuricemia did not influence vasoconstrictor responses in vitro to norepinephrine or potassium chloride. The small arteries of NX rats featured hypertrophic remodeling independent of uric acid levels: wall to lumen ratio, wall thickness and cross-sectional area were increased without changes in lumen diameter. In the main branch, vasorelaxations to acetylcholine were impaired in NX rats, but were not affected by hyperuricemia. In contrast, relaxations to the large-conductance Ca2+-activated K+-channel (BKCa) opener NS-1619 were reduced by oxonic acid feeding, whereas responses to nitroprusside were not affected.ConclusionsExperimental hyperuricemia did not influence cardiac load or vascular remodeling, but impaired BKCa -mediated vasorelaxation in experimental CRI.

Effect of Shenkang granules on the progression of chronic renal failure in 5/6 nephrectomized rats

Shenkang granules (SKGs) are a Chinese herbal medicinal formula, consisting of rhubarb (Rheum palmatum L.), Salvia miltiorrhiza, milkvetch root [Astragalus membranaceus (Fisch.) Bunge] and safflower (Carthamus tinctorius L.). The aim of the present study was to investigate the effect of SKG on chronic renal failure (CRF) in 5/6 nephrectomized (5/6 Nx) rats. The rats were randomly divided into seven groups (n=10 per group) as follows: (i) 5/6 Nx (model group; 2.25 ml/kg/day normal saline); (ii) SKGL (low dose; 5/6 Nx treated with 2 g crude drug/kg/day SKG); (iii) SKGM (moderate dose; 5/6 Nx treated with 4 g crude drug/kg/day SKG); (iv) SKGH (high dose; 5/6 Nx treated with 8 g crude drug/kg/day SKG); (v) benazepril treatment group (5/6 Nx treated with 5 mg/kg/day benazepril); (vi) Shenkang injection (SKI) group (5/6 Nx with 13.3 ml/kg/day SKI); and (vii) sham-operated group (2.25 ml/kg/day normal saline). After 30 days, the levels of microalbumin, total protein, serum creatinine, blood urea nitrogen and serum lipid were found to be significantly decreased in the SKGL and SKGM rats, showing histological improvement compared with the untreated 5/6 Nx rats, as determined by hematoxylin and eosin, and Masson's trichrome staining. In addition, SKG was found to significantly improve the levels of glutathione peroxidase and reduce the damage caused by free radicals to the kidney tissues. Furthermore, SKG prevented the accumulation of extracellular matrix by decreasing the expression of collagen I and III and inhibiting the expression of matrix metalloproteinases-2 and -9 in the renal tissue, as determined by western blot analysis. SKG was also shown to decrease the concentrations of serum transforming growth factor-β1, as determined by ELISA, and kidney angiotensin II, as determined using a radioimmunoassay kit. In conclusion, SKG was demonstrated to ameliorate renal injury in a 5/6 Nx rat model of CRF. Thus, SKG may exert a good therapeutic effect on CRF.

Caveolae are involved in stretch-induced Ca2+ signaling in pulmonary hypertension

Introduction Vascular smooth muscle cells are submitted to stretch forces exerted by the blood pressure. Pulmonary arteries can transduce a mechanical stimulus of stretch into a biological response of contraction, a mechanism called myogenic tone, which involves plasma membrane Ca 2+ stretch-activated channels (SAC). As membrane plasticity and shape are important for the SAC activity, we investigate the involvement of caveolae in the Ca 2+ and contraction response to stretch of pulmonary arterial smooth muscle cells (PA-SMC). Methods Isometric contractions were performed in pulmonary arterial vessels from normoxic rats (Nx rats) and rats with a pulmonary hypertension induced by a chronic hypoxia of 3 weeks (CH rats). Inward currents from SAC were recorded on freshly isolated PA-SMC after a negative pressure applied by a patch-clamp pipette and Ca 2+ variations were simultaneously recorded with the fluorescent probe indo-1. Sarcoplasmic reticulum Ca 2+ was measured by a confocal microscope with the fluo-5N probe. Finally, a pharmacological approach using methyl-β-cyclodextrin (MβCD, a caveolae disrupter) coupled with different immunolabelings of caveolin-1 and sarcoplasmic Ca 2+ stores were used to investigate the role of caveolae. Results We show that caveolae are present and caveolin-1 expressed in PA-SMC from both normal and pulmonary hypertensive rats. Isolated PA-SMC and pulmonary arteries exhibit a higher SAC activity, Ca 2+ response and contraction to stretch in CH rats than in Nx rats. These responses are reduced by MβCD only in CH rats. In the absence of extracellular calcium, a stretch induces a Ca 2+ and contraction response only in CH rats but not in Nx rats. This phenomenon involves a Ca 2+ release from the sarcoplasmic reticulum and is fully inhibited by MβCD. Conclusion Caveolae are important for stretch-induced calcium response in CH rats via a new subcellular organization between caveolae and intracellular calcium stores.

Brazilian red propolis attenuates hypertension and renal damage in 5/6 renal ablation model.

The pathogenic role of inflammation and oxidative stress in chronic kidney disease (CKD) is well known. Anti-inflammatories and antioxidant drugs has demonstrated significant renoprotection in experimental nephropathies. Moreover, the inclusion of natural antioxidants derived from food and herbal extracts (such as polyphenols, curcumin and lycopene) as an adjuvant therapy for slowing CKD progression has been largely tested. Brazilian propolis is a honeybee product, whose anti-inflammatory, antimicrobial and antioxidant effects have been widely shown in models of sepsis, cancer, skin irritation and liver fibrosis. Furthermore, previous studies demonstrated that this compound promotes vasodilation and reduces hypertension. However, potential renoprotective effects of propolis in CKD have never been investigated. The aim of this study was to evaluate the effects of a subtype of Brazilian propolis, the Red Propolis (RP), in the 5/6 renal ablation model (Nx). Adult male Wistar rats underwent Nx and were divided into untreated (Nx) and RP-treated (Nx+RP) groups, after 30 days of surgery; when rats already exhibited marked hypertension and proteinuria. Animals were observed for 90 days from the surgery day, when Nx+RP group showed significant reduction of hypertension, proteinuria, serum creatinine retention, glomerulosclerosis, renal macrophage infiltration and oxidative stress, compared to age-matched untreated Nx rats, which worsened progressively over time. In conclusion, RP treatment attenuated hypertension and structural renal damage in Nx model. Reduction of renal inflammation and oxidative stress could be a plausible mechanism to explain this renoprotection.

Comparative proteomic analysis of rat left ventricle in a subtotal nephrectomy model

BackgroundChronic kidney disease (CKD) is associated with cardiac hypertrophy that leads to increased cardiovascular morbidity and mortality. To date, use of the renin–angiotensin–aldosterone system blockade has been the main treatment modality. However, renin–angiotensin–aldosterone system blockade by the angiotensin converting enzyme inhibitors (ACEi) can only partially reverse the cardiac hypertrophy without having a significant impact on all-cause mortality as evidenced by meta-analyses from clinical trials. It is imperative to elucidate the molecular pathogenesis of CKD-related cardiomyopathy for potential targets in further treatment. MethodsMale Sprague–Dawley rats that underwent subtotal nephrectomy (SNX) rats were established as the CKD model. A hemodynamic study was used to evaluate the left ventricle (LV) structural and functional alterations. We used proteomic techniques to profile the LV protein changes among sham-operated rats, SNX rats, and SNX rats with 6 months of ACEi enalapril interventions. The differentially expressed proteins were further annotated by functional and network analyses. ResultsAs compared to the sham-operated rats, the SNX rats had 25 upregulated and 46 decreased protein expression. The top canonical pathways identified by ingenuity pathway analysis for the CKD cardiomyopathy were mitochondrial dysfunction, oxidative phosphorylation, fatty acid β oxidation, protein ubiquitination, and ketolysis. The most relevant functions extracted from these networks contained 27 and 23 focused proteins, respectively. They were related to cellular assembly and organization, RNA posttranscriptional modification, and protein synthesis. After ACEi intervention for 6 weeks, the residual canonical pathways identified by ingenuity pathway analysis that mediated the CKD-related cardiomyopathy were mitochondrial dysfunction, ketolysis, phenylalanine degradation IV, and putrescine degradation III. There were decreased Sirt3 and SNRNP, and increased monoamine oxidase and SAHH expression in the LV of SNX rats that could not be reversed by the ACEi. ConclusionOur studies provide a repertoire of potential biomarkers related to cardiac hypertrophy in CKD. There are still residual disturbed molecules/pathways despite ACEi intervention. Further studies are warranted to investigate these potential novel targets to alleviate CKD-related cardiomyopathy.

Angiotensin II-mediated GFR decline in subtotal nephrectomy is due to acid retention associated with reduced GFR.

Angiotensin II (AII) mediates glomerular filtration rate (GFR) decline in animals with subtotal nephrectomy (Nx), but the mechanisms for increased AII activity are unknown. Because reduced GFR of Nx is associated with acid (H(+)) retention that increases kidney AII, AII-mediated GFR decline might be induced by H(+) retention. We measured GFR and kidney microdialyzate H(+) and AII content in Sham and 2/3 Nx rats in response to amelioration of H(+) retention with dietary NaHCO3, to AII receptor antagonism and to both. GFR was lower in Nx than that in Sham. Nx but not Sham GFR was lower at Week 24 than that at Week 1. Despite no differences in plasma acid-base parameters or urine net acid excretion, kidney H(+) content was higher in Nx than that in Sham, consistent with H(+) retention. Plasma and kidney microdialyzate AII were higher in Nx than that in Sham and dietary NaHCO3 reduced each in Nx but not in Sham. AII receptor antagonism was associated with higher Week 24 GFR in Nx with H(+) retention but not in Sham or in Nx in which H(+) retention had been corrected with dietary NaHCO3. Week 24 GFR after dietary NaHCO3 was higher than after AII receptor antagonism. Week 24 GFR was not different after adding AII receptor antagonism to dietary NaHCO3. AII-mediated GFR decline in 2/3 Nx was induced by H(+) retention and its amelioration with dietary HCO3 conserved GFR better than AII receptor antagonism in this CKD model. H(+) retention might induce AII-mediated GFR decline in patients with reduced GFR, even without metabolic acidosis.

Vitamin D receptor agonist VS-105 improves cardiac function in the presence of enalapril in 5/6 nephrectomized rats.

Vitamin D receptor (VDR) agonists (VDRAs) are commonly used to manage hyperparathyroidism secondary to chronic kidney disease (CKD). Patients with CKD experience extremely high risks of cardiovascular morbidity and mortality. Clinical observations show that VDRA therapy may be associated with cardio-renal protective and survival benefits in patients with CKD. The 5/6 nephrectomized (NX) Sprague-Dawley rat with established uremia exhibits elevated serum parathyroid hormone (PTH), hypertension, and abnormal cardiac function. Treatment of 5/6 NX rats with VS-105, a novel VDRA (0.05 and 0.5 μg/kg po by gavage), once daily for 8 wk in the presence or absence of enalapril (30 mg/kg po via drinking water) effectively suppressed serum PTH without raising serum calcium. VS-105 alone reduced systolic blood pressure (from 174 ± 6 to 145 ± 9 mmHg, P < 0.05) as effectively as enalapril (from 174 ± 6 to 144 ± 7 mmHg, P < 0.05). VS-105 improved cardiac functional parameters such as E/A ratio, ejection fraction, and fractional shortening with or without enalapril. Enalapril or VS-105 alone significantly reduced left ventricular hypertrophy (LVH); VS-105 plus enalapril did not further reduce LVH. VS-105 significantly reduced both cardiac and renal fibrosis. The lack of hypercalcemic toxicity of VS-105 is due to its lack of effects on stimulating intestinal calcium transport and inducing the expression of intestinal calcium transporter genes such as Calb3 and TRPV6. These studies demonstrate that VS-105 is a novel VDRA that may provide cardiovascular benefits via VDR activation. Clinical studies are required to confirm the cardiovascular benefits of VS-105 in CKD.

Histopathological characterization of renal tubular and interstitial changes in 5/6 nephrectomized marmoset monkeys (Callithrix jacchus)

Common marmosets (Callithrix jacchus) have become a useful animal model, particularly for development of biopharmaceuticals. While various renal failure models have been established in rodents, there is currently no acceptable model in marmosets. We analyzed the damaged renal tubules and tubulointerstitial changes (inflammation and fibrosis) of 5/6 nephrectomized (Nx) common marmosets by histopathological/immunohistochemical methods, and compared these findings to those in 5/6 Nx SD rats. In Nx marmosets and rats sacrificed at 5 and 13 weeks after Nx, variously dilated and atrophied renal tubules were seen in the cortex in common; however, the epithelial proliferating activity was much less in Nx marmosets. Furthermore, the degrees of inflammation and fibrosis seen in the affected cortex were more severe and massive in Nx marmosets with time-dependent increase. Interestingly, inflammation in Nx marmosets, of which degree was less in Nx rats, consisted of a large number of CD3-positive T cells and CD20-positive B cells (occasionally forming follicles), and a few CD68-positive macrophages. Based on these findings, lymphocytes might contribute to the progressive renal lesions in Nx marmosets. Fibrotic areas in Nx marmosets comprised myofibroblasts expressing vimentin and α-smooth muscle actin (α-SMA), whereas along with vimentin and α-SMA expressions, desmin was expressed in myofibroblasts in Nx rats. This study shows that there are some differences in renal lesions induced by Nx between marmosets and rats, which would provide useful, base-line information for pharmacology and toxicology studies using Nx marmosets.

Chronic renal insufficiency does not induce behavioral and cognitive alteration in rats

In humans, chronic kidney disease (CKD) is associated with cognitive decline, increase in anxiety, or depression. The underlying mechanisms of these changes remain unclear. The aim of this study was to elucidate whether and how experimentally induced long-term CKD affects cognitive functions in rats.Thirty male Wistar rats underwent 5/6 nephrectomy (5/6 Nx), an established model of CKD, or sham surgery. Development of CKD was monitored using biochemical analyses and confirmed by renal histology. Behavioral tests of anxiety, depression and spatial behavior were performed before, and at 3 and 9months after the surgery.CKD in 5/6 Nx rats was characterized by significant decrease of renal function, e.g., glomerular filtration rate, and progressive glomerulosclerosis, tubular atrophy, and interstitial fibrosis; and increased plasma uremic toxins. Mortality was higher in 5/6 Nx rats in comparison with controls. Compared to control group, the surviving 5/6 Nx rats presented similar general locomotor activity, depression traits, and spatial abilities (p=0.43, p=0.84, p=0.71, respectively). At 9months, lower anxiety in the light–dark box test was observed in 5/6 Nx rats if compared with the control group (p=0.02).Despite the development of progressive CKD in 5/6 Nx rats, no expected behavioral changes were observed. Further experimental studies associating behavioral responses to severity of CKD are definitely needed to confirm the solely psychosocial aspect background of CKD-associated cognitive impairment in humans.