Nx Animals Research Articles

Abstract 9552: RNA Sequencing of Cardiac and Renal Tissue in a Rat Model of Chronic Kidney Disease Reveals Chronodisruption as Common Pathway

Introduction: Chronic kidney disease (CKD) is often accompanied by cardiovascular disease, which is referred to as type 4 cardiorenal syndrome. Management is challenging due to the multifactorial nature of the condition, and treatment of one organ may negatively impact the other. Hypothesis: More insights into the common molecular disease processes in the heart and kidney might provide a source for new therapies. We used RNA sequencing to identify pathways that were dysregulated in both organs. Methods: Cardiac and renal tissue samples from four 5/6 th partial nephrectomized (NX) and four sham-operated (SHAM) female Sprague-Dawley rats were analyzed using RNA sequencing. Strand-specific and rRNA depleted cDNA libraries were generated and were sequenced on NovaSeq (Ilumina) with 20 million reads per library. Results: In comparison to SHAM, the kidneys of NX animals had 290 differentially expressed genes (DEGs), showing increased expression of genes that map to Gene Ontology terms including collagen fibril organization , circadian regulation of gene expression , mitotic cell cycle , neutrophil chemotaxis , and inflammatory response . Within the hearts of NX animals, 8 DEGs were found, including those related to circadian regulation of gene expression (Per2, Per3, Arntl/Bmal1, BHLHE41) , as well as the Adra1d , C2cd4b , Ubtd1 , and Atp2b2 genes. Four genes were differentially expressed in both organs. Three of them were related to circadian rhythm, with Per2 and Per3 being downregulated and Arntl/Bmal1 being upregulated. Furthermore, Adra1d was downregulated in both organs. Conclusions: This comprehensive RNA sequencing in a rat model of type 4 cardiorenal syndrome identified chronodisruption as a common pathway, suggesting that circadian rhythms could be a novel target for intervention. Figure Legend. (A) Venn diagram with number of DEGs and vulcano plots for heart (left) and kidney (right). (B) GO terms for heart. (C) GO terms for kidney.

Dietary Phosphorus Differentially Alters Uremic Toxin Production in a Rat Model of Chronic Kidney Disease

ObjectivesChronic kidney disease (CKD) is characterized by declining kidney function, limiting the kidney’s ability to efficiently remove metabolic waste products from circulation. Byproducts of gut microbial protein metabolism, termed uremic retention solutes (URS), accumulate in CKD patients and are associated with accelerating kidney decline. The gut microbes responsible for generating URS are dependent upon phosphorus (P) for growth and survival. As dietary P restriction is a cornerstone of CKD treatment, we hypothesized that changes in dietary P loads would alter URS production. MethodsTo evaluate this, 8-week-old male Sprague Dawley rats underwent 5/6th nephrectomy (Nx, n = 24) or sham operation (n = 20) and were maintained on a 0.6% P diet (w/w) for three weeks. Animals were then randomized to receive either low (0.1% (w/w)) or high (1.2% (w/w)) P diets for 4h/d for 7d. Blood was collected at the start and end of the 7d diet (baseline and sacrifice, respectively). Serum was analyzed for blood urea nitrogen (BUN) and URS, including trimethylamine oxide (TMAO), indoxyl sulfate (IS), and p-cresol sulfate (pCS), via LC-MS. ResultsNx rats had significantly elevated BUN compared to sham controls (38.9 ± 5.9 vs 23.1 ± 5.1 mg/dL, p < 0.0001). Additionally, the presence of significantly enlarged kidney tissue in Nx animals verified the progression of kidney decline. At sacrifice, all URS were elevated in Nx animals as compared to sham controls (p < 0.0001), though changes in dietary P loads only affected IS production (low vs. high, p = 0.0003). When comparing baseline to sacrifice, TMAO decreased, IS remained consistent, and pCS increased in all rats. ConclusionsOur results indicate that dietary P loads may differentially affect the production of some URS in a rat model of CKD. As dietary P restriction is one of the cornerstones of CKD treatment, we posit that this dietary strategy influences URS production, CKD progression, and, ultimately, health outcomes. Funding SourcesASBMR, NIH K01.

P0094KLOTHO DECLINE IS ASSOCIATED WITH PRO-HYPERTROPHIC SIGNALING IN MYOCARDIUM OF NEPHRECTOMIZED SPONTANEOUSELY HYPERTENSIVE RATS (PILOT SYUDY)

Abstract Background and Aims Klotho deficiency is suggested to be involved in cardiac complications in chronic kidney disease (CKD) through modulation of intracellular pro-hypertrophic signaling pathways. The aim of this study was to investigate the molecular mechanisms of myocardial remodeling in the settings of Klotho decline in experimental CKD. Method Systolic blood pressure (BP), myocardial mass index (MMI), serum and urinary creatinine (Cr), serum urea (Ur), inorganic phosphate (Pi), 25-hydroxyvitamin D (25OHD), parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), Klotho, myocardial Klotho, calcineurin/ NFAT and beta-catenin expression (IHC), quantitative morphometry of cardiomyocite (CM) thickness and nuclei area (hematoxylin and eosin), myocardial perivascular (PF) and interstitial fibrosis (IF), renal fibrosis (RF) (Masson’s trichrome) and Klotho expression (rKl, IHC) were analyzed in spontaneously hypertensive rats with 3/4 or 5/6 nephrectomy (Nx) and duration of experimental exposure 1 and 2 mo (Nx1, n = 9; Nx2, n = 8). Controls were sham operated (SO) animals (SO1, n = 9; SO2, n = 9). Results The experimental model was corresponded to the initial stages of CKD (Ur level was between 6.64 and 13.36 mmol/L, serum Cr increased less than 50% vs SO, RF did not exceed 22 % FOV). Serum concentration of Klotho and rKl expression decreased, while MMI, CM thickness increased in both Nx1 and Nx2 (Table). There were no differences in serum Pi, FGF23 and PTH levels in Nx1 vs SO (Table). In Nx2 Klotho decline was accompanied by the development of IF and PF, serum 25OHD decrease, Pi and FGF23 increase vs SO2 (Table). In multiple regression analyzes including SO and Nx animals rKl was independently associated with MMI (β = -0.38 ± 0.16, p = 0.026), and CM thickness (β = -0.64 ± 0.14, p &amp;lt;0.001). Myocardial fibrosis was associated with rKl level (IF: β = -0.87 ± 0.19, p = 0.001) and serum 25OHD (PF: β =-0.34 ± 0.15, p=0.032). According to pilot data of IHC, in Nx rat myocardium Klotho protein expression decreased, cytoplasmic calcineurin and nuclear NFAT in CM were observed (Figure), suggesting an increase in the activity of the calcineurin / NFAT signaling pathway. An intracellular redistribution of β-catenin from the cytoskeleton to the cytoplasm and nuclei were observed in CM of Nx rats (Figure), likely corresponding to increase in the activity of Wnt signaling. Conclusion Associations of CM hypertrophy, myocardial fibrosis and Klotho decline in CKD may be mediated by calcineurin / NFAT and Wnt signaling in myocardium.

Vitamin D and fibroplastic processes in myocardium in spontaneously hypertensive rats with initial kidney dysfunction

Background. Even a moderate decrease in glomerular filtration rate leads to an increased risk of cardiovascular diseases (CVD), which is the leading cause of mortality in patients with chronic kidney disease (CKD). Left ventricular hypertrophy (LVH) underlies CVD development in renal dysfunction. The prevalence of LVH in patients with CKD stages 2–4 is 50–70 % and reaches 95 % at the beginning of dialysis, which significantly exceeds the number of cases in general population (15–21 %). Common hemodynamic factors associated with chronic kidney damage —hypertension (HTN), activation of the renin-angiotensin system, anemia, fluid and sodium retention, and others largely explain the high prevalence of LVH among patients with CKD. Nevertheless, the existence of additional non-hemodynamic mechanisms of myocardial remodeling (MR) is evident.Objective. To investigate the associations between the MR physiological/histological characteristics and laboratory parameters of calcium-phosphate metabolism in the initial stages of experimental CKD. Design and methods. Four groups of spontaneously hypertensive rats (SHR) were studied (n = 35): 3/4 nephrectomized rats (Nx) one month exposed after surgery (Nx(1), n = 9), 5/6 Nx two months after surgery (Nx(2), n = 8), sham operated rats one month after surgery (SO(1), n = 9) and two months after surgery (SO(2), n = 9). Myocardial mass index (MMI), systolic blood pressure (BP), proteinuria, creatinine (Cr) concentration, total calcium (Ca) and inorganic phosphate (Pi), 25-OH vitamin D (25OHD) and parathyroid hormone (PTH) in serum, myocardial morphology were studied in all experimental animals.Results. The models corresponded to the 1–3 stages CKD. There were no significant changes in serum total Ca (p = 0,066), Pi (p = 0,051) and PTH (p = 0,015) concentrations, the level of 25OHD was significantly lower in Nx(2) rats vs control (p = 0,015). MMI increased in all nephrectomized rats (p = 0,008). The cardiomyocytes (CM) thickness increased in Nx(1) and Nx(2) animals compared to the corresponding controls (p = 0,010, p = 0,002). A significant increase in interstitial (IF) and perivascular (PF) fibrosis occurred in Nx(2) rats with more damaging influence (p = 0,017, p = 0,004). CM thickness, IF and PF increased with the elevation of BP (r = 0,39, p = 0,038, r = 0,47, p = 0,026, r = 0,49, p = 0,031) and serum Cr (r = 0,68, p = 0,001, r = 0,61, p = 0,003, r = 0,69, p = 0,001), and the decrease in serum 25OHD concentration (r = –0,045, p = 0,047, r = –0,50, p = 0,020, r = –0,52, p = 0,012). Multiple linear regression analysis showed, that 25OHD is an independent predictor of myocardial fibrosis (IF: β = –0,38 ± 0,18, p = 0,047, PF: β = –0,34 ± 0,15, p = 0,032).Conclusions. The initial stages of CKD accompanied with HTN are associated with serum 25OHD concentration decrease CM hypertrophy and myocardial fibrosis. The CM growth is an earlier event in relation to the interstitial fibrosis. The obtained data suggest a possible role of vitamin D deficiency in the development of myocardial fibrotic lesions.

Resistance training downregulates macrophages infiltration in the kidney of 5/6 nephrectomized rats

IntroductionChronic kidney disease (CKD) is considered a significant world health problem with elevated mortality rates. Patients with CKD are restricted to mild physical activity, present chronic inflammatory state and loss of muscle strength. Currently, the influence of resistance exercise (RE) on the progression of renal disease has not being fully elucidated. PurposeTo evaluate the effects of RE on the progression of CKD in a remnant kidney model (5/6Nx) in rats. MethodsEight-week-old Wistar rats were submitted to 5/6 nephrectomy and were divided into four groups: Sham sedentary (Sham SD); Sham RE (Sham RE); 5/6Nx SD and 5/6Nx RE. The animals were trained for 8 weeks in a vertical climbing ladder for 3 days per week, on non-consecutive days. ResultsAs expected, 5/6Nx SD group presented a markedly loss of renal function, increased plasma inflammatory cytokines and increased oxidative stress with a reduced activity of nitric oxide. The higher macrophage infiltration and fibrosis confirmed these conditions. RE attenuated systolic blood pressure and renal function decrease and also improved serum lipid parameters in 5/6 Nx animals. It was evident the increase of muscle strength and mass in the trained groups while the sedentary group showed reduced muscle weight and strength compared to Sham SD. ConclusionsRE implemented following 5/6Nx retard the progression of chronic kidney injury while simultaneously allowed the maintenance of skeletal muscle strength.

Sodium butyrate ameliorates insulin resistance and renal failure in CKD rats by modulating intestinal permeability and mucin expression.

BackgroundThe associated increase in the lipopolysaccharide (LPS) levels and uremic toxins in chronic kidney disease (CKD) has shifted the way we focus on intestinal microbiota. This study shows that a disruption of the intestinal barrier in CKD promotes leakage of LPS from the gut, subsequently decreasing insulin sensitivity. Butyrate treatment improved the intestinal barrier function by increasing colonic mucin and tight junction (TJ) proteins. This modulation further ameliorated metabolic functions such as insulin intolerance and improved renal function.MethodsRenal failure was induced by 5/6th nephrectomy (Nx) in rats. A group of Nx and control rats received sodium butyrate in drinking water. The Nx groups were compared with sham-operated controls.ResultsThe Nx rats had significant increases in serum creatinine, urea and proteinuria. These animals had impaired glucose and insulin tolerance and increased gluconeogenesis, which corresponded with decreased glucagon-like peptide-1 (GLP-1) secretion. The Nx animals suffered significant loss of intestinal TJ proteins, colonic mucin and mucin 2 protein. This was associated with a significant increase in circulating LPS, suggesting a leaky gut phenomenon. 5′adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, known to modulate epithelial TJs and glucose metabolism, was significantly reduced in the intestine of the Nx group. Anti-inflammatory cytokine, interleukin 10, anti-bacterial peptide and cathelicidin-related antimicrobial peptide were also lowered in the Nx cohort. Butyrate treatment increased AMPK phosphorylation, improved renal function and controlled hyperglycemia.ConclusionsButyrate improves AMPK phosphorylation, increases GLP-1 secretion and promotes colonic mucin and TJ proteins, which strengthen the gut wall. This decreases LPS leakage and inflammation. Taken together, butyrate improves metabolic parameters such as insulin resistance and markers of renal failure in CKD animals.

Tangeretin ameliorates renal failure via regulating oxidative stress, NF-κB-TNF-α/iNOS signalling and improves memory and cognitive deficits in 5/6 nephrectomized rats.

Chronic kidney disease (CKD) is a significant global health concern with limited treatment options. Oxidative stress and inflammatory responses have been implicated in the pathology of CKD. Patients with CKD are frequently affected with neurological complications that affect both the central and peripheral nervous system. Identification of effective treatment strategies are of much clinical value in the therapy of CKD. Tangeretin, a plant-derived flavonoid has been described to retain extensive pharmacological properties. In the present study, we explored whether tangeretin exerted protective effects in 5/6 nephrectomized rats. CKD was induced in Sprague-Dawley rats by 5/6 nephrectomy (Nx). Separate groups of 5/6 Nx rats were treated with tangeretin (50, 100 or 200mg/kg b.wt.) or enalapril for 30days (starting 5days after surgery for 35days). Control animals were not subjected to Nx nor were treated with tangeretin or enalapril. Renal dysfunction, as evinced by raised serum urea, serum creatinine, proteinuria, and histological alterations were significantly reduced by tangeretin and enalapril treatment. 5/6 Nx animals exhibited raised levels of malondialdehyde (MDA) and reactive oxygen species. Elevated TNF-α, nitric oxide (NO) and cytokines-IL-6 and IL-1β with upregulated NF-κB/TNF-α/iNOS signalling pathways were effectively down-regulated by tangeretin. Cognitive disturbances and memory impairments observed in Nx rats were substantially improved by tangeretin. Collectively, the experimental data indicate that the anti-oxidant and anti-inflammatory effects of tangeretin effectively improved renal function and reduced the cognitive and memory impairments in CKD-induced animals.

Corrigendum.

CorrigendumCorrigendumPublished Online:10 Aug 2017https://doi.org/10.1152/ajprenal.zh2-8242-corr.2017Original articleMoreSectionsPDF (32 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Volume 302, February 15, 2012Pages F439–F454. Ghosh SS, Kreig R, Massey HD, Sica DA, Fakhry I, Ghosh S, and Gehr TW. Curcumin and enalapril ameliorate renal failure by antagonizing inflammation in 5/6 nephrectomized rats: role of phospholipase and cyclooxygenase. Am J Physiol Renal Physiol 302: F439–F454. First published October 26, 2011. doi:—The above-noted Research Article was published incorrectly. In the abstract, the following sentence appears: “Curcumin (75 mg/kg) and enalapril (10 mg/kg) were administered for 10 wk.” However, under materials and methods, the description of Group V erroneously states the dosage for enalapril as 75 mg/kg. The correct version of the description should read as follows: “Group V: enalapril-treated (10 mg/kg) Nx animals; treatment started 6 wk after second Nx and animals were euthanized at week 17.”This article has no references to display. Download PDF Previous Back to Top FiguresReferencesRelatedInformation Related ArticlesCurcumin and enalapril ameliorate renal failure by antagonizing inflammation in ⅚ nephrectomized rats: role of phospholipase and cyclooxygenase 15 Feb 2012American Journal of Physiology-Renal Physiology More from this issue > Volume 313Issue 2August 2017Pages F560-F560 Copyright & PermissionsCopyright © 2017 the American Physiological Societyhttps://doi.org/10.1152/ajprenal.zh2-8242-corr.2017PubMed28798135History Published online 10 August 2017 Published in print 1 August 2017 Metrics

Role of mitochondrial dysfunction in renal fibrosis promoted by hypochlorite-modified albumin in a remnant kidney model and protective effects of antioxidant peptide SS-31

Oxidative stress aggravates renal fibrosis, a pathway involved in almost all forms of chronic kidney disease (CKD). However, the underlying mechanism involved in the pathogenesis of renal oxidative stress has not been completely elucidated. In this study, we explored the role and mechanism of hypochlorite-modified albumin (HOCl-alb) in mediating oxidative stress and fibrotic response in a remnant-kidney rat model. Five-sixths nephrectomy (5/6 NX) was performed on the rats and then the animals were randomly assigned to intravenous treatment with either vehicle alone, or HOCl-rat serum albumin (RSA) in the presence or absence of SS-31 (administered intraperitoneally). A sham-operation control group was set up concurrently. Compared with the control group, 5/6 NX animals displayed marked mitochondrial (mt) dysfunction, as evidenced by decrease of mitochondrial membrane potential (MMP), ATP production, mtDNA copy number alterations and manganese superoxide dismutase (MnSOD) activity, release of cytochrome C (Cyto C) from mitochondria to the cytoplasm, and increase of mitochondrial reactive oxygen species in renal tissues. They also displayed increased levels of HOCl-alb in both plasma and renal tissues. These changes were accompanied by accumulation of extracellular matrix, worsened proteinuria, deteriorated renal function, and a marked increase of macrophage infiltration along with up-regulation of monocyte chemoattractant protein (MCP)−1 and transforming growth factor (TGF)-β1 expression. HOCl-alb challenge further exacerbated the above biological effects in 5/6 NX animals, but these adverse effects were prevented by administration of SS-31, a mitochondrial targeted antioxidant peptide. These data suggest that accumulation of HOCl-alb may promote renal inflammation and fibrosis, probably related to mitochondrial oxidative stress and dysfunction and that the mitochondrial targeted peptide SS-31 might be a novel therapy for renal fibrosis and chronic renal failure (CRF).

High Fat High Cholesterol Diet (Western Diet) Aggravates Atherosclerosis, Hyperglycemia and Renal Failure in Nephrectomized LDL Receptor Knockout Mice: Role of Intestine Derived Lipopolysaccharide.

A high fat meal, frequently known as western diet (WD), exacerbates atherosclerosis and diabetes. Both these diseases are frequently associated with renal failure. Recent studies have shown that lipopolysaccharide (LPS) leaks into the circulation from the intestine in the setting of renal failure and after WD. However, it is not clear how renal function and associated disorders are affected by LPS. This study demonstrates that circulatory LPS exacerbates renal insufficiency, atherosclerosis and glucose intolerance. Renal insufficiency was induced by 2/3 nephrectomy in LDL receptor knockout mice. Nx animals were given normal diet (Nx) or WD (Nx+WD). The controls were sham operated animals on normal diet (control) and WD (WD). To verify if LPS plays a role in exaggerating renal insufficiency, polymyxin (PM), a known LPS antagonist, and curcumin (CU), a compound known to ameliorate chronic kidney disease (CKD), was given to Nx animals on western diet (Nx+WD+PM and Nx+WD+CU, respectively). Compared to control, all other groups displayed increased circulatory LPS. The Nx+WD cohort had the highest levels of LPS. Nx group had significant renal insufficiency and glucose intolerance but not atherosclerosis. WD had intense atherosclerosis and glucose intolerance but it did not show signs of renal insufficiency. Compared to other groups, Nx+WD had significantly higher cytokine expression, macrophage infiltration in the kidney, renal insufficiency, glucose intolerance and atherosclerosis. PM treatment blunted the expression of cytokines, deterioration of renal function and associated disorders, albeit not to the levels of Nx, and was significantly inferior to CU. PM is a non-absorbable antibiotic with LPS binding properties, hence its beneficial effect can only be due to its effect within the GI tract. We conclude that LPS may not cause renal insufficiency but can exaggerate kidney failure and associated disorders following renal insufficiency.

Altered KLOTHO and NF-κB-TNF-α Signaling Are Correlated with Nephrectomy-Induced Cognitive Impairment in Rats.

Renal insufficiency can have a negative impact on cognitive function. Neuroinflammation and changes in klotho levels associate with chronic kidney disease (CKD) and may play a role in the development of cognitive impairment (CI). The present study evaluates the correlation of cognitive deficits with neuroinflammation and soluble KLOTHO in the cerebral spinal fluid (CSF) and brain tissue of nephrectomized rats (Nx), with 5/6 renal mass ablation. Nx and sham Munich Wistar rats were tested over 4 months for locomotor activity, as well as inhibitory avoidance or novel object recognition, which started 30 days after the surgery. EMSA for Nuclear factor-κB and MILLIPLEXMAP or ELISA kit were used to evaluate cytokines, glucocorticoid and KLOTHO levels. Nx animals that showed a loss in aversive-related memory and attention were included in the CI group (Nx-CI) (n=14) and compared to animals with intact learning (Nx-M n=12 and Sham n=20 groups). CSF and tissue samples were collected 24 hours after the last behavioral test. The results show that the Nx-groups have increased NF-κB binding activity and tumor necrosis factor-alpha (TNF-α) levels in the hippocampus and frontal cortex, with these changes more pronounced in the Nx-CI group frontal cortex. In addition, the Nx-CI group showed significantly increased CSF glucocorticoid levels and TNF-α /IL-10 ratio compared to the Sham group. Klotho levels were decreased in Nx-CI frontal cortex but not in hippocampus, when compared to Nx-M and Sham groups. Overall, these results suggest that neuroinflammation mediated by frontal cortex NF-κB, TNF-α and KLOTHO signaling may contribute to Nx-induced CI in rats.

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.

Tempol, a Superoxide Dismutase-Mimetic Drug, Ameliorates Progression of Renal Disease in CKD Mice

Background: Oxidative stress has been implicated in the pathogenesis of chronic kidney disease (CKD) and antioxidants may ameliorate disease progression. We investigate the beneficial effect of Tempol, a superoxide dismutase-mimetic drug, on progression of disease in a mouse model of CKD. Methods: CKD was surgically induced in c57BL/6 mice by 5/6 nephrectomy. Mice were randomly divided into 3 groups: sham group, 5/6 nephrectomized group (Nx) and Nx+Tempol (2 mmol/l in drinking water). Mice were sacrificed at the end of 12 weeks. Renal function, structure as well as expression of key molecules involved in the pathogenesis of inflammation, fibrosis and progression in mice were measured. Results: Reduced body weight and impaired renal function (elevation on serum creatinine, blood urea nitrogen, urine albumin, segmental sclerosis and tubulointerstitial damage) was demonstrated in Nx mice but was significantly improved by Tempol administration. Nx animals exhibited significantly elevated proinflammatory and profibrotic factors, activation of NF-κB, increased expression of NADPH oxidase related subunits (p47phox, p67phox, gp91phox), and elevated activation of TGF-ß/Smad3, EGFR, MAPK signaling pathway. Tempol inhibited NF-κB mediated inflammation, TGF-ß/Smad3-induced renal fibrosis as well as EGFR and MAPK signaling pathway activation. Conclusions: Tempol administration attenuated renal injury in CKD mice through NF-κB, TGF-ß/Smad3, redox-senstive EGFR activation and c-Raf/MEK/ERK pathways.

Influence of dietary phosphorus in the regulation of Sodium phosphate cotransporters in uremic rats (LB717)

Hyperphosphatemia is a common and serious complication of chronic kidney disease, contributing to the increased cardiovascular mortality seen in this patient group. The sodium phosphate (P) cotransporters system is involved in intestinal P absorption and is regulated by several factors. We investigate the effect of diets with distinct concentrations of P in the regulation of cotransporters NaPIIb and PiT1 in the intestine of rats. Male Wistar rats (n=66) were used, 33 of which were submitted to nephrectomy 5/6 (Nx) and the others were used as controls (C). The animals received a standard (ST) diet (20% protein and 0.54% P) for 30 days, after they were divided into 2 sets of 3 groups in accordance with the concentration of P in the diet (0.2%, 0.54% or 0.9%) and fed for 2 days. Blood samples, 24h urine, and intestine (duodenum, ileum and jejunum) were accordingly collected. Immunofluorescence, Western blot or ELISA were used to analyze the expressions of the cotransporters and qRT-PCR to evaluate the gene expression of NaPIIb and PiT1. The concentrations of Cr, Pi, FGF23, PTH and Albu were higher in Nx animals. Pu decreased in Nx animals and some of these parameters were modified by the concentration of dietary P. There were significant differences in the expression of PiT1 in jejunum and ileum of Nx animals in diet 0.2% P relative to their respective controls, normalized to B-actin (p=0.01 and p=0.02, respectively). The results demonstrate alteration in P homeostasis in Nx animals impaired upregulation of cotransporters in the P low diet and downregulation in the P high diet differently of the controls, as observed in PCR and immunofluorescence for PiT and NaPi in duodenum, respectively. Grant Funding Source: Supported by FAPESP

Abstract 18065: The Upregulation of MicroRNA miR-21 Mediates Concentric Left Ventricle Remodeling in a Rat Model of Chronic Renocardiac Syndrome

Chronic kidney disease (CKD) puts patients at a greatly increased risk of cardiovascular disease in a condition termed chronic renocardiac syndrome (RCS). We have utilized the 5/6 nephrectomy (5/6 NX) model in Sprague Dawley rats to study the gradual development of cardiac dysfunction resulting from declining kidney function. Previously, we identified alterations in several microRNAs (miRNAs) in left ventricle (LV) tissue 7 weeks after 5/6 NX surgery. We hypothesized that the upregulation of miR-21 in the LV following 5/6 NX contributes to LV pathology, rather than being a consequence of it. In situ hybridization revealed that miR-21 expression in the LV of 5/6 NX animals is highest in regions of perivascular fibrosis. To study the impact of miR-21 on LV remodeling and function, we delivered locked-nucleic acid (LNA) modified anti-miR-21 or anti-scrambled-miR (Exiqon, delivered i.v. at 1 mg/kg) to 5/6 NX animals (n=6/group) in 3 sequential days at 1 week post-surgery, and again 4 weeks post-surgery. Echocardiography was performed prior to 5/6 NX surgery and bi-weekly thereafter, throughout the 7 week study, to evaluate remodeling and function (anti-miR-21 vs. anti-scramble treated for all comparisons). The average LV miR-21 expression was reduced nearly 12-fold with anti-miR-21 treatment when measured 7 weeks post-5/6 NX. Anti-miR-21 treatment preserved stroke volume and fractional shortening between weeks 3 and 5 post-5/6 NX while both functional indices declined in anti-scramble treated rats. Additionally, concentric LV remodeling (LV wall hypertrophy and reduced chamber diameter) 5 weeks post-5/6 NX was completely prevented with anti-miR-21 treatment. At 7 weeks post-5/6 NX, when LV decompensation is evident in untreated rats, anti-miR-21 treatment maintained LV wall thickness and chamber diameter while fractional shortening was significantly increased (p &lt;0.05). Anti-miR-21 treatment had no effect on blood pressure. We conclude that chronic knockdown of miR-21 in the 5/6 NX model prevented pathological concentric remodeling. Together these data suggest that the upregulation of cardiac miR-21 following 5/6 NX is an important mediator of pathological cardiac remodeling and dysfunction.

Sodium phosphate cotransporters in intestinal absorption of phosphorus in uremic rats

Hyperphosphatemia is a common disorder in patients with chronic kidney disease (CKD). We investigate the effect of diets with distinct concentrations of P in the regulation of cotransporters NaPIIb and PiT1 in the intestine of rats. 66 male Wistar rats were used, 33 of which were submitted to nephrectomy 5/6 (Nx) and the others were used as controls (C). The animals received a standard (ST) diet (20% protein and 0.54% P) for 30 days, after they were divided into 2 sets of 3 groups in accordance with the concentration of P in the diet (0.2%↓, 0.54% or 0.9%↑) and fed for 2 days. Blood samples and 24h urine were collected and the expressions of the cotransporters were analyzed by immunofluorescence and Western blot (WB). The table summarize the results (mean ± SD). NaPIIb in ileum of Nx rats. (A) Negative control (B) ↓P; (C) ST; (D) ↑P The concentrations of Cr, P, FGF23, PTH and Albu were higher in Nx animals. Pu decreased in Nx animals and some of these parameters were modified by the concentration of dietary P. The expression of NaPIIb and PiT1 and the WB analysis are being finalized.

Different administration schedules of darbepoetin alfa affect oxidized and reduced glutathione levels to a similar extent in 5/6 nephrectomized rats

The development of erythropoiesis-stimulating agents (ESAs) with extended serum half-lives has allowed marked prolongation of the administration intervals. The level of oxidative stress is increased in chronic kidney disease, and is reportedly decreased after long-term ESA treatment. However, the effect of different dosing regimens of ESAs on oxidative stress has not been elucidated. Five-sixths nephrectomized (NX) rats received either 0.4 μg/kg darbepoetin alfa (DA) weekly or 0.8 μg/kg DA fortnightly between weeks 4 and 10. NX animals receiving saline and a sham-operated (SHAM) group served as controls. The levels of oxidized and reduced glutathione (GSSG, GSH) were followed from blood samples drawn fortnightly. During the follow-up, the ratios GSSG/GSH showed similar trends in both DA groups, levels being significantly lower than those in the SHAM group at weeks 8 and 10. GSSG levels were lower than the baseline throughout the study in all groups except for NX controls. The GSH levels were increased in all three NX groups (weeks 6-10) compared with both the baseline and the SHAM group Our results suggest that the extent of oxidative stress is similar in response to different dosing regimens of DA in 5/6 NX rats when comparable hemoglobin levels are maintained. These findings remain to be confirmed in chronic kidney disease patients.

Curcumin alleviates oxidative stress, inflammation, and renal fibrosis in remnant kidney through the Nrf2–keap1 pathway

We hypothesized that curcumin, by increasing the expression of nuclear factor-erythroid-2-related factor 2 (Nrf2), could reduce oxidative stress, inflammation, and renal fibrosis in remnant kidney. Sprague-Dawley rats were subjected to 5/6 nephrectomy and randomly assigned to untreated (Nx), curcumin-treated (75 mg/kg/day, orally), and telmisartan-treated groups (10 mg/kg/day, orally; as positive control). Sham-operated rats also served as controls. Five/sixth nephrectomy caused renal dysfunction, as evidenced by elevated proteinuria, blood urea nitrogen, and plasma creatinine, and decreased creatinine clearance that were ameliorated by curcumin or telmisartan treatment. The Nx rats demonstrated reduced Nrf2 protein expression, whereas the Kelch-like ECH-associated protein 1 was upregulated and heme oxygenase-1 level was significantly diminished. Consequently, Nx animals had significantly higher kidney malondialdehyde concentration and lower glutathione peroxidase activity, which was associated with the upregulation of nicotinamide adenine dinucleotide phosphatase oxidase subunit (p67(phox) and p22(phox) ), NF-kappaB p65, TNF-α, TGF-β1, cyclooxygenase-2, and fibronectin accumulation in remnant kidney. Interestingly, all of these changes were ameliorated by curcumin or telmisartan. These findings demonstrate that, by modulating Nrf2-Keap1 pathway, the curcumin effectively attenuates oxidative stress, inflammation, and renal fibrosis, which suggest that curcumin hold promising potential for safe treatment of chronic kidney disease.

Cardiac remodeling in rats with renal failure shows interventricular differences

Chronic renal failure (CRF) is associated with an increased incidence of cardiovascular diseases. Intensive research revealed a number of alterations in the heart during CRF; however, possible interventricular differences in CRF-induced cardiac remodeling have so far not been addressed. CRF was induced by two-stage surgical 5/6 nephrectomy (NX) in male Wistar rats. Cellular hypertrophy was quantified using immunohistological morphometric analysis. Contraction force and membrane potential were recorded in left and right ventricle papillary muscles with an isometric force transducer and high-resistance glass microelectrodes. Hypertrophy was present in the left ventricle (LV) of NX animals, but not in the right ventricle (RV) of NX animals, as documented by both ventricle/body weight ratios and cellular morphometric analysis of the cross-sectional area of myocytes. The contraction force was reduced in the LV of NX animals but increased in the RV of NX animals compared with sham-operated rats. Rest potentiation of contraction force was relatively more pronounced in the LV of NX rats. Fifty percent substitution of extracellular sodium with lithium significantly increased the contraction force only in the LV of NX animals. Action potential durations were shortened in both ventricles of CRF animals. Cardiac structural and contractile remodeling in CRF shows significant interventricular differences. CRF induces hypertrophy of the LV but not of the RV. LV hypertrophy was associated with a reduction of contraction force, whereas in the RV, the contraction force was enhanced. Partial recovery of contractile function of the LV by rest potentiation or lithium substitution indicates a role of the Na(+)/Ca(2+) exchanger in this phenomenon.

Chronic VEGF Blockade Worsens Glomerular Injury in the Remnant Kidney Model

VEGF inhibition can promote renal vascular and parenchymal injury, causing proteinuria, hypertension and thrombotic microangiopathy. The mechanisms underlying these side effects are unclear. We investigated the renal effects of the administration, during 45 days, of sunitinib (Su), a VEGF receptor inhibitor, to rats with 5/6 renal ablation (Nx). Adult male Munich-Wistar rats were distributed among groups S+V, sham-operated rats receiving vehicle only; S+Su, S rats given Su, 4 mg/kg/day; Nx+V, Nx rats receiving V; and Nx+Su, Nx rats receiving Su. Su caused no change in Group S. Seven and 45 days after renal ablation, renal cortical interstitium was expanded, in association with rarefaction of peritubular capillaries. Su did not worsen hypertension, proteinuria or interstitial expansion, nor did it affect capillary rarefaction, suggesting little angiogenic activity in this model. Nx animals exhibited glomerulosclerosis (GS), which was aggravated by Su. This effect could not be explained by podocyte damage, nor could it be ascribed to tuft hypertrophy or hyperplasia. GS may have derived from organization of capillary microthrombi, frequently observed in Group Nx+Su. Treatment with Su did not reduce the fractional glomerular endothelial area, suggesting functional rather than structural cell injury. Chronic VEGF inhibition has little effect on normal rats, but can affect glomerular endothelium when renal damage is already present.