Renal Mass Reduction Research Articles

Pre-clinical model of severe glutathione peroxidase-3 deficiency and chronic kidney disease results in coronary artery thrombosis and depressed left ventricular function.

Chronic kidney disease (CKD) patients have deficient levels of glutathione peroxidase-3 (GPx3). We hypothesized that GPx3 deficiency may lead to cardiovascular disease in the presence of chronic kidney disease due to an accumulation of reactive oxygen species and decreased microvascular perfusion of the myocardium. Methods. To isolate the exclusive effect of GPx3 deficiency in kidney disease-induced cardiac disease, we studied the GPx3 knockout mouse strain (GPx3-/-) in the setting of surgery-induced CKD. Results. Ribonucleic acid (RNA) microarray screening of non-stimulated GPx3-/- heart tissue show increased expression of genes associated with cardiomyopathy including myh7, plac9, serpine1 and cd74 compared with wild-type (WT) controls. GPx3-/- mice underwent surgically induced renal mass reduction to generate a model of CKD. GPx3-/- + CKD mice underwent echocardiography 4 weeks after injury. Fractional shortening (FS) was decreased to 32.9 ± 5.8% in GPx3-/- + CKD compared to 62.0% ± 10.3 in WT + CKD (P < 0.001). Platelet aggregates were increased in the myocardium of GPx3-/- + CKD. Asymmetric dimethylarginine (ADMA) levels were increased in both GPx3-/- + CKD and WT+ CKD. ADMA stimulated spontaneous platelet aggregation more quickly in washed platelets from GPx3-/-. In vitro platelet aggregation was enhanced in samples from GPx3-/- + CKD. Platelet aggregation in GPx3-/- + CKD samples was mitigated after in vivo administration of ebselen, a glutathione peroxidase mimetic. FS improved in GPx3-/- + CKD mice after ebselen treatment. These results suggest GPx3 deficiency is a substantive contributing factor to the development of kidney disease-induced cardiac disease.

Emodin ameliorates renal fibrosis in rats via TGF-β1/Smad signaling pathway and function study of Smurf 2.

Emodin is a natural active component extracted from Chinese herbs. In this study, we investigated the therapeutic effect of emodin on surgery-induced renal fibrosis in rat. Moreover, the function of Smad ubiquitination regulatory factor 2 (Smurf 2) was further studied in vitro. The renal fibrosis rat model was established via 5/6 renal mass reduction. The histopathological abnormalities of renal tissues in rats were tested via staining method and microscopic examination. Renal function indicators, the serum creatinine, blood urea nitrogen and total urinary protein were measured via spectrophotometric method. The renal tissues were tested via Western blotting and real-time quantitative PCR. Moreover, Smurf 2 protein expression under different conditions and its regulatory function in vitro was measured via Western blotting. Our results showed that the histopathological abnormalities, the decrease in rat body weight and the abnormal renal function caused by renal fibrosis were improved by emodin. Further, mRNA expression of TGF-β1 was decreased by emodin. Furthermore, extracellular matrix (ECM) components as well as TGF-β1/Smad signaling-related Smurf 2 were decreased by emodin both in vivo and in vitro. The protein level of mothers against decapentaplegic homolog 7 (Smad7) was up-regulated. In addition, ECM components were increased by Smurf 2 over-expression and these effects were weakened by emodin co-treatment in vitro. Emodin showed an anti-fibrosis effect in surgery-induced renal fibrotic rats. And this effect was potentially achieved via down-regulating expressions of TGF-β1 and Smurf 2 and up-regulating Smad7 expression.

Long-term treatment with chaethomellic acid A reduces glomerulosclerosis and arteriolosclerosis in a rat model of chronic kidney disease

The high prevalence of end-stage renal disease emphasizes the failure to provide therapies to effectively prevent and/or reverse renal fibrosis. Therefore, the aim of this study was to evaluate the effect of long-term treatment with chaethomellic acid A (CAA), which selectively blocks Ha-Ras farnesylation, on renal mass reduction-induced renal fibrosis. Male Wistar rats were sham-operated (SO) or subjected to 5/6 renal mass reduction (RMR). One week after surgery, rats were placed in four experimental groups: SO:SO rats without treatment (n=13); SO+CAA: SO rats treated with CAA (n=13); RMR:RMR rats without treatment (n=14); and RMR+CAA:RMR rats treated with CAA (n=13). CAA was intraperitoneally administered in a dose of 0.23μg/kg three times a week for six months. Renal fibrosis was evaluated by two-dimensional ultrasonography and histopathological analysis. The kidneys of the RMR animals treated with CAA showed a significantly decrease in the medullary echogenicity (p<0.05) compared with the RMR rats that received no treatment. Glomerulosclerosis and arteriolosclerosis scores were significantly lower (p<0.001) in the RMR+CAA group when compared with the RMR group. There were no significant differences in interstitial fibrosis, interstitial inflammation and tubular dilatation scores between the RMR+CAA and RMR groups. These data suggest that CAA can be a potential future drug to attenuate the progression of chronic kidney disease.

MYH9 E1841K Mutation Augments Proteinuria and Podocyte Injury and Migration.

Intronic variants of the MYH9 gene that encodes the nonmuscle myosin heavy chain IIA are associated with diabetic nephropathy in European Americans and with sickle cell disease-associated nephropathy. However, the causal functional variants of MYH9 have remained elusive. Rare missense mutations in MYH9 cause macrothrombocytopenia and are occasionally associated with development of nephropathy. The E1841K mutation is among the common MYH9 missense mutations and has been associated with nephropathy in some carriers. To determine the contribution of the E1841K mutation in kidney disease, we studied the effects of the E1841K mutation in mice subjected to high salt or angiotensin II (Ang II) as models of hypertension and in mice subjected to renal mass reduction as a model of CKD. Despite similar levels of BP among wild-type (MYH9+/+ ) mice and mice heterozygous (MYH9+/E1841K ) and homozygous (MYH9E1841K/E1841K ) for the mutation in each model, MYH9E1841K/E1841K mice exhibited mildly increased albuminuria in response to high salt; severe albuminuria, nephrinuria, FSGS, and podocyte foot effacement in Ang II-induced hypertension; and early mortality in the renal mass reduction model. Treatment with candesartan during Ang II-induced hypertension attenuated kidney disease development in MYH9E1841K/E1841K mice. In vitro, isolated primary podocytes from MYH9E1841K/E1841K mice exhibited increased lamellipodia formation and reorganization of F-actin stress fibers. Wound healing assays revealed that MYH9+/+ podocytes had the lowest migration rate, followed by MYH9+/E1841K then MYH9E1841K/E1841K podocytes. In conclusion, the MYH9 E1841K variant alters podocyte cytoskeletal structure and renders podocytes more susceptible to injury after a damaging stimulus.

Abstract P430: Soluble Epoxide Hydrolase Inhibition Augments Ras Blockade Renoprotection in 5/6 Nephrectomized Ren-2 Transgenic Hypertensive Rats With Chronic Kidney Disease

Rationale and Objective: We showed recently that increasing kidney tissue epoxyeicosatrienoic acids (EETs) by blocking soluble epoxide hydrolase (sEH) and thereby blocking EETs degradation to inactive dihydroxyeicosatrienoic acids (DHETEs) substantially attenuated the progression of chronic kidney disease (CKD) in Ren-2 transgenic hypertensive rats (TGR) subjected to 5/6 renal mass reduction (5/6 NX). In this study we examined if in this model addition of sEH inhibition to the complex (dual) renin-angiotensin system (RAS) blockade would bring additional renoprotective effects in already established CKD. Methods: TGR aged 9 weeks underwent 5/6 NX and then were left untreated for 6 weeks to develop CKD. Then dual RAS blockade : ACE inhibition (trandolapril) + angiotensin AT 1 receptor blockade (losartan) was instituted, alone or combined with sEH inhibition (c-AUCB, 3 mg/l in drinking water). During the 60 weeks’ follow-up period albuminuria and urinary creatinine excretion was repeatedly determined. The following experimental groups were investigated: ; 1) sham-operated TGR; 2) Untreated 5/6 NX TGR; 3) 5/6 NX TGR + RAS blockade ; 4) 5/6 NX TGR + RAS + sEH blockade. Sham-operated transgene-negative normotensive Hannover-Sprague Dawley (HanSD) rats served as basic controls. In separate groups renal glomerular and tubulointerstitial injury was assessed, and effects of two weeks’ treatments on systolic blood pressure (SBP, measured by telemetry) and on kidney ANG II, ANG 1-7, EETs and DHETEs levels were determined. Results: All untreated TGR died by week 14 after 5/6 NX. RAS blockade increased the final survival rate to 23%, normalized SBP (116 ± 3 vs. 198 ± 3 mmHg, p&lt;0.0015), reduced albuminuria (46 ± 5 vs. 102 ± 12 mg/24 h, p&lt;0.001) and intrarenal ANG II (27 ± 8 vs. 189 ± 14 fmol/g p&lt;0.0015) and did not alter kidney EETs/DHETEs ratio. After addition of sEH blockade kidney EETs/DHETEs ratio increased to 2.89 ± 0.42 (p&lt;0.001 vs. 5/6 NX TGR treated with RAS blockade), the final survival rate increased to 42% and indices of renal glomerular and tubulointerstial injury improved. Conclusion: Addition of sEH blockade to the RAS blockade brings additional renoprotective effects on the CKD progression in 5/6 NX TGR, even when applied in the advanced phase of the disease.

Abstract P388: Acute but Not Long-term Blood Pressure Lowering by Renal Denervation in Reduced Renal Mass, Salt Sensitive Hypertension

Chronic kidney disease is prevalent among patients with resistant hypertension and activation of the sympathetic nervous system is common in both conditions. While renal nerve ablation lowers blood pressure (BP) in some resistant hypertensive patients, the influence of kidney disease on the antihypertensive response is largely unknown. Systemic hemodynamics and sympathetically-mediated low frequency oscillations of systolic BP were determined continuously by telemetry in rats before and after unilateral nephrectomy and surgical excision of the poles of the remaining kidney to reduce renal mass (RRM) ~ 80%. Before RRM, BP was not salt sensitive. However, placing rats with RRM on a high salt diet induced sustained increases in mean arterial pressure (Figure) and cardiac output while suppressing sympathetic activity. After denervation of the remnant kidney, sympathetic activity and heart rate increased, concomitant with reductions in BP and cardiac output. However, these acute responses waned over the 2 weeks of follow up. Subsequent central sympathetic inhibition with clonidine reduced sympathetic activity, heart rate and cardiac output, but not BP as peripheral resistance increased. Finally, when the low salt diet was restored, BP decreased back to normotensive levels. These results show that renal denervation does not chronically lower BP in this model of salt-sensitive hypertension associated with substantial nephron loss, but without active parenchymal disease, thus providing insight into conditions likely to impact the antihypertensive response to renal-specific sympathoinhibition in subjects with advanced kidney disease.

Renal denervation attenuates hypertension and renal dysfunction in a model of cardiovascular and renal disease, which is associated with reduced NADPH and xanthine oxidase activity

Oxidative stress is considered a central pathophysiological event in cardiovascular disease, including hypertension. Early age reduction in renal mass is associated with hypertension and oxidative stress in later life, which is aggravated by increased salt intake. The aim of the present study was to examine if renal sympathetic denervation can exert blood pressure lowering effects in uninephrectomized (UNX) rats (3-week old) fed with high salt (HS, 4%; w/w) diet for 4 weeks. Moreover, we investigated if renal denervation is associated with changes in NADPH and xanthine oxidase-derived reactive oxygen species.Rats with UNX + HS had reduced renal function, elevated systolic and diastolic arterial pressures, which was accompanied by increased heart weight, and cardiac superoxide production compared to sham operated Controls. UNX + HS was also associated with higher expression and activity of NADPH and xanthine oxidase in the kidney. Renal denervation in rats with UNX + HS attenuated the development of hypertension and cardiac hypertrophy, but also improved glomerular filtration rate and reduced proteinuria. Mechanistically, renal denervation was associated with lower expression and activity of both NADPH oxidase and xanthine oxidase in the kidney, but also reduced superoxide production in the heart.In conclusion, our study shows for the first time that renal denervation has anti-hypertensive, cardio- and reno-protective effects in the UNX + HS model, which can be associated with decreased NADPH oxidase- and xanthine oxidase-derived reactive oxygen species (i.e., superoxide and hydrogen peroxide) in the kidney.

Effect of salt loading on baroreflex sensitivity in reduced renal mass hypertension

ABSTRACTBackground: High dietary salt, as well as renal mass reduction, is known to decrease baroreflex sensitivity in rats. However, the effect of high salt intake on baroreflex sensitivity is unknown in reduced renal mass (RRM) hypertension; therefore, the aim of this study was to investigate the effects of salt loading on arterial baroreflex sensitivity and mean arterial pressure (MAP) in RRM hypertension.Methods: Both RRM and sham-operated control (SO) rats were loaded with 0.25 or 0.5% NaCl for five weeks. Plasma Na+, K+, and creatinine levels were measured, and baroreflex sensitivity was evaluated before and after β1 blockade. In addition, cardiac vagal tone and intrinsic heart rate (IHR) were measured. RESULTS: RRM decreased full baroreflex sensitivity of the tachycardic response under 0.5% NaCl loading and the parasympathetic bradycardic response under 0% NaCl loading. The NaCl loading did not affect the severity of RRM hypertension. Cardiac vagal tone and IHR decreased in RRM rats versus SO controls under all NaCl loading conditions. RRM decreased plasma K+ under 0% NaCl loading and increased plasma Na+ under 0.5% NaCl loading. High (0.5%) NaCl loading decreased IHR and increased plasma creatinine and left ventricular weight in RRM rats. CONCLUSIONS: RRM in combination with 0.5% NaCl loading led to a decrease in the sensitivity of full baroreflex and of the parasympathetic component of baroreflex. Changes in plasma Na+ and K+ levels, due to NaCl loading, may have contributed to the decrease in baroreflex sensitivities and IHR but had no effect upon MAP in RRM rats.

&lt;b&gt;The role of pneumoperitoneum in the glomerular filtration in an experimental model with 2/3 reduction of the renal mass

Objective: To evaluate the influence of the CO2 pneumoperitoneum in the glomerular filtration in a rat model with a 2/3 reduction of renal parenchyma. Methods: Adult Wistar male rats (n = 50) were subjected to right nephrectomy and a 2/3 ligature of the renal left vascular branch. Animals were randomly distributed as follows: GI (n = 10) - simulated, GII (n = 20) –8 mm Hg pneumoperitoneum, and GIII (n = 20) –15 mm Hg pneumoperitoneum. After two (GIIA and GIIIA) and three (GIIB and GIIIB) hours of insufflation, and one hour of disinsufflation, they were evaluated for the following aspects: mean blood pressure (MBP), microhematocrit, urinary volume and inulin clearance. Results: The microscopic aspects showed signs of glomerulosclerosis that caused proteinuria. Renal function with8 mm Hg pneumoperitoneum after two hours of disinsufflation (Δ% = 202.68) was better than after three hours (Δ% = 10.89). With15 mm Hg pneumoperitoneum, the renal function was damaged by both procedures, that is, two (Δ% = -3.57) and three hours (Δ% = -3.25). Conclusion: Inulin clearance evidenced renal insufficiency in the model with a 2/3 reduction of renal mass, and depending on both the increase of the exposure time and the pressure intensity, it can be more intensified.

Modeling the Effects of Functional Adaptation to Reduction in Renal Mass

When kidney tissue is removed or damaged, the remaining nephrons undergo compensatory growth and adaptive functional changes. The objective of this study is to assess the impact of such structural and functional adaptation. To do so, we developed computational models of solute transport along different nephron populations of a uninephrectomized and a 5/6‐nephrectomized rat kidney. The model represents detailed epithelial and paracellular transport processes along both the superficial and juxtamedullary nephrons. The loop of Henle of each model nephron extends to differing depths of the inner medulla. Each nephron segment is represented as a rigid tubule lined by a layer of epithelial cells, with apical and basolateral transporters that vary according to cell type. To simulate a uninephrectomized or 5/6‐nephrectomized kidney, we reduced the nephron population, increased single‐nephron glomerular filtration rate, and increased nephron size based on data in the literature. Model simulations were conducted to determine what tubular transport changes are necessary to preserve baseline salt excretion. Model results also revealed the differing degrees to which salt transport and oxygen consumption are changed along the different nephron segments.Support or Funding InformationThis research was supported in part by NIH grant DK‐106102.

An angiotensin II type 1 receptor binding molecule has a critical role in hypertension in a chronic kidney disease model

Angiotensin II type 1 receptor-associated protein (ATRAP) promotes AT1R internalization along with suppression of hyperactivation of tissue AT1R signaling. Here, we provide evidence that renal ATRAP plays a critical role in suppressing hypertension in a mouse remnant kidney model of chronic kidney disease. The effect of 5/6 nephrectomy on endogenous ATRAP expression was examined in the kidney of C57BL/6 and 129/Sv mice. While 129/Sv mice with a remnant kidney showed decreased renal ATRAP expression and developed hypertension, C57BL/6 mice exhibited increased renal ATRAP expression and resistance to progressive hypertension. Consequently, we hypothesized that downregulation of renal ATRAP expression is involved in pathogenesis of hypertension in the remnant kidney model of chronic kidney disease. Interestingly, 5/6 nephrectomy in ATRAP-knockout mice on the hypertension-resistant C57BL/6 background caused hypertension with increased plasma volume. Moreover, in knockout compared to wild-type C57BL/6 mice after 5/6 nephrectomy, renal expression of the epithelial sodium channel α-subunit and tumor necrosis factor-α was significantly enhanced, concomitant with increased plasma membrane angiotensin II type 1 receptor in the kidneys. Thus, renal ATRAP downregulation is involved in the onset and progression ofblood pressure elevation caused by renal mass reduction,and implicates ATRAP as a therapeutic target forhypertension in chronic kidney disease.

The Effects of Long-Term Chaetomellic Acid A Administration on Renal Function and Oxidative Stress in a Rat Model of Renal Mass Reduction

Purpose. This study aimed to evaluate the effect of chronic treatment with chaetomellic acid A (CAA) on oxidative stress and renal function in a model of renal mass reduction. Methods. Male Wistar rats were subjected to 5/6 nephrectomy (RMR) or sham-operated (SO). One week after surgery, rats have been divided into four experimental groups: RMR: RMR rats without treatment (n = 14); RMR + CAA: RMR rats treated with CAA (n = 13); SO: SO rats without treatment (n = 13); and SO + CAA: SO rats treated with CAA (n = 13). CAA was intraperitoneally administered in a dose of 0.23 µg/Kg three times a week for six months. Results. RMR was accompanied by a significant reduction in catalase and glutathione reductase (GR) activity (p < 0.05) and a decrease in reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio. CAA administration significantly increased catalase and GR activity (p < 0.05) and increased GSH/GSSG ratio, but no significant difference between the treated and nontreated groups was found in this ratio. No significant differences were found between the RMR groups in any of the parameters of renal function. However, CAA administration slightly improves some parameters of renal function. Conclusions. These data suggest that CAA could attenuate 5/6 RMR-induced oxidative stress.

Ultrasonographic Evaluation of the Kidney in 5/6 Nephrectomized Rats: Correlation with Biochemical and Histopathological Findings.

To characterize the kidney alterations associated with renal mass reduction by two-dimensional ultrasound and assess the correlation of the ultrasonographic parameters with the histological and biochemical findings. Rats were divided into two groups: sham-operated animals (n=13), and animals which underwent renal mass reduction (RMR) through 5/6 nephrectomy (n=14). Renal length, renal thickness, cortical thickness, medullary length and echogenicity of the kidneys were evaluated by ultrasonography at 3 and 6 months after the RMR. Except for the renal length, the renal dimensions at 3 and 6 months were significantly higher in the RMR group when compared to the sham-operated group (p<0.05). Furthermore, the cortical and medullary echogenicity was significantly higher in the RMR group when compared to the sham-operated group (p<0.05). A significant correlation was observed between the plasma creatinine level and the renal length 3 months after RMR (r=-0.612, p=0.045). These data support future application of ultrasonography for monitoring the progression of renal damage in chronic studies with the 5/6 nephrectomy model.

Prolonged Baroreflex Activation Abolishes Salt-Induced Hypertension After Reductions in Kidney Mass.

Chronic electric activation of the carotid baroreflex produces sustained reductions in sympathetic activity and arterial pressure and is currently being evaluated for therapy in patients with resistant hypertension. However, patients with significant impairment of renal function have been largely excluded from clinical trials. Thus, there is little information on blood pressure and renal responses to baroreflex activation in subjects with advanced chronic kidney disease, which is common in resistant hypertension. Changes in arterial pressure and glomerular filtration rate were determined in 5 dogs after combined unilateral nephrectomy and surgical excision of the poles of the remaining kidney to produce ≈70% reduction in renal mass. After control measurements, sodium intake was increased from ≈45 to 450 mol/d. While maintained on high salt, animals experienced increases in mean arterial pressure from 102±4 to 121±6 mm Hg and glomerular filtration rate from 40±2 to 45±2 mL/min. During 7 days of baroreflex activation, the hypertension induced by high salt was abolished (103±6 mm Hg) along with striking suppression of plasma norepinephrine concentration from 139±21 to 81±9 pg/mL, but despite pronounced blood pressure lowering, there were no significant changes in glomerular filtration rate (43±2 mL/min). All variables returned to prestimulation values during a recovery period. These findings indicate that after appreciable nephron loss, chronic suppression of central sympathetic outflow by baroreflex activation abolishes hypertension induced by high salt intake. The sustained antihypertensive effects of baroreflex activation occur without significantly compromising glomerular filtration rate in remnant nephrons.

Sex-linked differences in the course of chronic kidney disease and congestive heart failure: a study in 5/6 nephrectomized Ren-2 transgenic hypertensive rats with volume overload induced using aorto-caval fistula.

The role of hypertension and the renin-angiotensin system (RAS) in sex-related differences in the course of chronic kidney disease (CKD) and congestive heart failure (CHF) remain unclear, especially when the two diseases are combined. In male and female Ren-2 transgenic rats (TGR), a model of hypertension with activation of endogenous RAS, CKD was induced by 5/6 renal mass reduction (5/6 NX) and CHF was elicited by volume overload achieved by creation of an aorto-caval fistula (ACF). The primary aim of the study was to examine long-term CKD- and CHF-related mortality, especially in animals with CKD and CHF combined, with particular interest in the potential sex-related differences. The follow-up period was 23weeks after the first intervention (5/6 NX). We found, first, that TGR did not exhibit sexual dimorphism in the course of 5/6 NX-induced CKD. Second, in contrast, TGR exhibited important sex-related differences in the course of ACF-induced CHF-related mortality: intact female TGR showed higher survival rate than male TGR. This situation is reversed in the course of combined 5/6 NX-induced CKD and ACF-induced CHF-related mortality: intact female TGR exhibited poorer survival than male TGR. Third, the survival rate in animals with combined 5/6 NX-induced CKD and ACF-induced CHF was significantly worsened as compared with rat groups that were exposed to 'single organ disease'. Collectively, our present results clearly show that CKD aggravates long-term mortality of animals with CHF. In addition, TGR exhibit remarkable sexual dimorphism with respect to CKD- and CHF-related mortality, especially in animals with combined CKD and CHF.

High-salt diet induces outward remodelling of efferent arterioles in mice with reduced renal mass.

The glomerular filtration rate (GFR) falls progressively in chronic kidney disease (CKD) which is caused by a reduction in the number of functional nephrons. The dysfunctional nephron exhibits a lower glomerular capillary pressure that is induced by an unbalance between afferent and efferent arteriole. Therefore, we tested the hypothesis that oxidative stress induced by CKD differentially impairs the structure or function of efferent vs. afferent arterioles. C57BL/6 mice received sham operations (sham) or 5/6 nephrectomy (RRM) and three months of normal- or high-salt diet or tempol. GFR was assessed from the plasma inulin clearance, arteriolar remodelling from media/lumen area ratio, myogenic responses from changes in luminal diameter with increases in perfusion pressure and passive wall compliance from the wall stress/strain relationships. Mice with RRM fed a high salt (vs. sham) had a lower GFR (553±25 vs. 758±36μLmin-1 g-1 kidney, P<0.01) and a larger efferent arteriolar diameter (9.6±0.8 vs. 7.4±0.7μm, P<0.05) resulting in a lower media/lumen area ratio (1.4±0.1 vs. 2.4±0.2, P<0.01). These alterations were corrected by tempol. The myogenic responses of efferent arterioles were about one-half that of afferent arterioles and were unaffected by RRM or salt. Passive wall compliance was reduced by high salt in both afferent and efferent arterioles. A reduction in renal mass with a high-salt diet induces oxidative stress that leads to an outward eutrophic remodelling in efferent arterioles and reduced wall compliance in both afferent and efferent arterioles. This may contribute to the lower GFR in this model of CKD.

Decreased renal AT1 receptor binding in rats after subtotal nephrectomy: PET study with [(18)F]FPyKYNE-losartan.

BackgroundSignificant renal mass reduction induced by 5/6 subtotal nephrectomy (Nx) is associated with a chain of events that culminates in hypertension and chronic kidney disease (CKD). Numerous studies have provided evidence for the role of angiotensin (Ang) II type 1 receptor (AT1R) in the promotion and progression of the disease; however, conflicting results were reported on intrarenal AT1R levels in CKD models.MethodsMale Sprague-Dawley rats (n = 26) underwent Nx or sham operations. Animals were scanned at 8–10 weeks post-surgery with PET using the novel AT1R radioligand [18F]FPyKYNE-losartan. Radioligand binding was quantified by kidney-to-blood ratio (KBR), standard uptake value (SUV), and distribution volume (DV). After sacrifice, plasma and kidney Ang II levels were measured. Western blot and 125I-[Sar1, Ile8]Ang II autoradiography were performed to assess AT1R expression.ResultsAt 8–10 weeks post-surgery, Nx rats developed hypertension, elevated plasma creatinine levels, left ventricle hypertrophy, increased myocardial blood flow (MBF), and reduced Ang II levels compared to shams. PET measurements displayed significant decrease in KBR (29 %), SUV (24 %), and DV (22 %) induced by Nx (p < 0.05), and these findings were confirmed by in vitro assays.ConclusionsReduced renal AT1Rs in hypertensive rats measured with [18F]FPyKYNE-losartan PET at 8–10 weeks following Nx support further use of this non-invasive approach in longitudinal studies to better understand the AT1R role in CKD progression.

Simplified Method to Measure Glomerular Filtration Rate by Iohexol Plasma Clearance in Conscious Rats

Background/Aims: Glomerular filtration rate (GFR) is the best index for evaluating renal function. We aimed to develop a simplified iohexol plasma clearance procedure for GFR measurement in rats without urine collection, animal catheterization or anesthesia, with limited sampling and requiring blood instead of plasma, to further reduce the sample volume and improve animal welfare. Methods: After iohexol injection (129.4 mg), samples were drawn according to 2-compartment kinetics and analyzed by high performance liquid chromatography. Healthy male Lewis rats were used to find a correction factor (CF) to obtain the ‘reference clearance' from the simplified 1-comparment model. This approach was validated using male or female (Lewis, Sprague-Dawley) rats and animals with renal mass reduction (RMR). In additional rats, different simplified approaches were evaluated. Results: Iohexol concentrations in blood and plasma strongly correlated (r = 0.9784, p < 0.0001). A CF of 0.90 enabled the calculation of the reference GFR. Validation results in male Lewis rats were 0.99 ± 0.27 for the reference GFR and 1.03 ± 0.29 ml/min/100 g for the simplified approach. Results in female Sprague-Dawley rats confirmed the suitability of the proposed method. In RMR rats, GFR was 0.14 ± 0.05 and 0.14 ± 0.04 ml/min/100 g for the reference and simplified model, respectively. Conclusion: The procedure we set up to measure GFR in conscious rats was proven to be reliable, required a small volume of blood at only 4 selected time points, without the need to collect urine or catheterize the animals, was applicable to rats from different strains and sexes, both healthy and with renal function impairment. Moreover, the procedure enables the monitoring of GFR changes over time in the same animal, thereby reducing the number of animals to be used.

Abstract 264: Oral Activated Charcoal Adsorbent (AST-120) Inhibition of Chronic Kidney Disease-induced Atherosclerosis Involves Modulation of Intestinal Immunity

Introduction and Aims: Although patients with chronic kidney disease (CKD) are at the highest risk for atherosclerotic cardiovascular disease (CVD), current interventions (e.g. statins) that are consistently effective in other high-risk groups have been found to be insufficiently effective in CKD. Serum levels of uremic toxins including indoxyl sulphate have been implicated in cardiovascular mortality. The oral charcoal absorbent AST-120 reduces serum levels of indoxyl sulphate through adsorption of indole converted from dietary tryptophan in the gastrointestinal tract. Previously, we reported the inhibitory effects of AST-120 on progression of atherosclerosis in apoE-deficient hyperlipidemic mice. Because gut immunity can affect systemic inflammatory responses and atherogenesis, we hypothesized that the AST-120-mediated benefits in CKD-acceleration of atherosclerosis involve modulation of intestinal immune cells. Methods: Eleven week old apoE-deficient mice underwent a reduction in renal mass through subtotal nephrectomy (sNx, n=34) or sham-operation (S, n=14). Two weeks later, each of the 2 groups was further divided into AST-120-treated or untreated mice. Six weeks later, we used flow cytometry and real time PCR to examine the population of immune cells in intestinal Peyer’s patches and mesenteric lymph nodes. Results: AST-120 had no significant effect on cellular populations of Peyer’s patch or mesenteric lymph nodes in mice with intact kidneys. Interestingly, however, in sNX mice treatment with AST-120 significantly decreased numbers of Peyer’s patch cytotoxic T cells (-19.6%; P=0.008), CD8 + central memory T cells (-33.9%; P=0.014), and CD8 + naïve T cells (-42.6%; P=0.004) compared to controls. In mesenteric lymph nodes of sNX animals, AST-120 did not affect CD4 + T helper cell number, but reduced early activated CD4 + T cells (-13.1%; P=0.04). Cytokine expression in mesenteric lymph nodes of AST-treated sNX mice showed increases in TGF-β (+47.2%; P=0.022) and IL-10 (+36.5%). Conclusions: The mechanism underlying the ability of oral activated charcoal adsorbent AST-120 to inhibit the acceleration of atherosclerosis in the setting of CKD may involve modulation of immune cells in the gut mucosa.

Validation of an Experimental Model to Study Less Severe Chronic Renal Failure

ABSTRACTPurpose: The 5/6 nephrectomy, mimics the stages of human chronic renal failure (CRF), but the procedure causes severe renal functional and morphological damage that could interfere with the evaluation of therapies for slowing the progression of the disease. This study summarizes the results of renal function, histology, and immunohistochemical findings in rats undergoing a 2/3 nephrectomy. Methods: The rats were distributed in groups according to the type of nephrectomy: CRF5/6: induced by a 5/6 renal mass reduction and CRF2/3: less severe CRF. The body weight and blood pressure were monitored, and the serum creatinine (SCr), creatinine clearance (CCr), urine osmolality, and 24-h proteinuria (PT24h) were measured. CRF progression was evaluated by the rate of decline of CCr (RCCr). Histology and immunohistochemistry were performed in the remnant kidneys. Statistical analysis was done by unpaired t-test, and a P-value < 0.05 was taken as a statistical significance. Results: Compared to the CRF5/6 group, the CRF2/3 model had a lower SCr, PT24h, CCr, and variations of the SCr from baseline. The disease progression was also significantly slower. The renal histopathological findings revealed fewer chronic lesions in rats with CRF2/3. Similarly, we observed less macrophage accumulation as well as lower proliferative activity and expression of fibronectin and a-smooth muscle-actin in the CRF2/3 model. Conclusions: The CRF2/3 model presented with a pattern of less severe CRF, functionally and morphologically, compared to the classical CRF5/6 model, and the CRF2/3 model may be useful for evaluating therapeutic interventions that target the early stages of CRF.