Reduced Renal Mass Rats Research Articles

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.

Enhanced M1 and Impaired M2 Macrophage Polarization and Reduced Mitochondrial Biogenesis via Inhibition of AMP Kinase in Chronic Kidney Disease

Background: Macrophage polarization plays a pivotal role in the process of inflammation which is common in chronic kidney disease (CKD). Macrophages polarization under the condition of CKD remains poorly understood. Here we tested the hypothesis that CKD promotes macrophage M1 polarization. Methods: A rat model of CKD was established by reduced renal mass (RRM). Polarization of macrophages was induced in ex vivo macrophages from RRM rats and cultured ones under the condition of uremic serum. The markers were evaluated by RT-PCR, western blot, and flow cytometer. Results: Our data showed that macrophages from RRM rats displayed enhanced M1 and impaired M2 polarization as revealed by increased M1 markers (tumor necrosis factor α, IL-6, IL-12p40, nitric oxide) and decreased M2 markers (IL-10, CD206, arginase activity) in response to LPS and IL-4 induction, respectively. Treatment with uremic sera in peritoneal and bone marrow derived macrophages from normal rats led to similar results. Moreover, macrophages from RRM rats and cultured under the condition of uremic sera had reduced mitochondrial biogenesis. The disturbed macrophage polarization and mitochondrial biogenesis were accompanied by reduced activity of adenosine monophosphate-activated protein (AMP)-activated kinase (AMPK). Enhancing activation of AMPK restored mitochondrial biogenesis and M2 macrophage polarization. Conclusion: These observations suggest that CKD disturbs macrophage polarization and mitochondrial biogenesis through inhibition of AMPK. This might provide a novel therapeutic strategy for intervention of chronic inflammation in CKD.

The effect of inhibition of endoplasmic reticulum stress on lipolysis in white adipose tissue in a rat model of chronic kidney disease

Lipolysis in fat tissue plays an important role in the development of metabolic disturbances, a characteristic feature of chronic kidney disease (CKD). In the present study, we tested the hypothesis that the inhibition of endoplasmic reticulum (ER) stress could alleviate lipolysis in white adipose tissue in a rat model of CKD. A rat model of CKD was established by a method of reduced renal mass (RRM). Lipolysis was measured as the release of glycerol in ex vivo fat pads and cultured primary adipocytes. The activity of lipases and markers of ER stress were measured by Western blotting and immunoprecipitation. Our data showed that lipolysis in visceral white adipose tissue was increased in RRM rats compared with control rats. In addition, increased phosphorylation of hormone-sensitive lipase (HSL) and binding of adipose triglyceride lipase (ATGL) to comparative gene identification-58 (CGI-58) protein were observed in the RRM rats. The phosphorylation of ER stress markers, including IRE1α, PERK, and eukaryotic initiation factor (eIF) 2α, and the expression of ER stress marker 78 kDa glucose-regulated protein (GRP78) were significantly increased in RRM rats. Treatment with an inhibitor of ER stress partially but significantly alleviated lipolysis, and this alleviation was accompanied by reduced binding of ATGL to CGI-58. Our results showed that enhanced lipolysis and ER stress occurred in visceral white adipose tissue in a rat model of CKD. Moreover, inhibition of ER stress significantly alleviated lipolysis. These findings suggest that ER stress is a potential therapeutic target for the metabolic disturbances associated with CKD.

Vascular insulin resistance related to endoplasmic reticulum stress in aortas from a rat model of chronic kidney disease

Metabolic insulin resistance has been demonstrated in patients with nondiabetic chronic kidney disease (CKD), yet their vascular insulin signaling remains poorly understood. Here we tested the hypothesis that vascular insulin signaling was impaired and related with endoplasmic reticulum (ER) stress in aortas from the reduced renal mass (RRM) model of CKD. The activity of insulin signaling and markers of ER were determined in aortas from rats with RRM and cultured human umbilical vein endothelial cells. Tyrosine phosphorylation of insulin receptor-β and insulin receptor substrate (IRS)-1 and phosphorylation of protein kinase B and endothelial nitric oxide synthase were all decreased in aorta from RRM rats, whereas serine phosphorylation of IRS-1, a marker of insulin resistance, was increased. In addition, nitric oxide generation and insulin-mediated vasorelaxation were decreased in aortas from RRM rats. Insulin signaling in cultured vascular endothelial cells was impaired by induction of ER stress and was restored in aortas of RRM rats by inhibition of ER stress. Taken together, rats with RRM had vascular insulin resistance that was linked to ER stress. This identified vascular insulin resistance and ER stress as a potential therapeutic target for cardiovascular complications in patients with CKD.

Susceptibility to renal injury is increased in a rat model of intrauterine growth restriction.

Several studies report an association between intrauterine growth restriction (IUGR) and increased risk to develop disease. Our laboratory has developed a unique rat model of IUGR induced‐hypertension in which testosterone plays a critical role in blood pressure regulation. Whether IUGR is a factor that increases renal disease is unclear. Thus, to test the hypothesis that IUGR exhibit a greater susceptibility to renal injury following renal insult, rats were exposed to 1 week high salt diet (HSD) prior to 3 weeks reduced renal mass (RRM) by 5/6 nephrectomy. Intact IUGR RRM rats exhibited a significant increase in mean arterial pressure (MAP), proteinuria, and glomerulosclerosis (GS) compared with intact control RRM; CTX ameliorated these effects (Table). Thus, these results suggest that IUGR increases susceptibility to renal injury induced by high salt diet and a reduction in renal mass; and that testosterone plays an important role in mediating the enhanced susceptibility to a renal insult in male IUGR rats.

Increased Dietary Potassium and Magnesium Attenuate Experimental Volume Dependent Hypertension Possibly Through Endogenous Sodium‐Potassium Pump Inhibitor

We and others have shown that inhibition of cardiovascular muscle (CVM) cell Na+,K‐ATPase activity (NKPTA) due to increased level of endogenous sodium potassium pump inhibitor (SPI) is involved in the mechanism of volume expanded (VE) experimental and human essential hypertension (HT). Since diets fortified with very high potassium (K) or very high magnesium (Mg) decrease blood pressure (BP), we have examined the effect of a moderate increase in dietary K alone and a moderate increase in dietary K and Mg on plasma levels of SPI, CVM cell NKPTA, and BP in reduced renal mass (RRM)‐salt HT rats, a classical model of VE HT. Seventy Percent‐RRM rats were divided in four dietary groups, (1) Na free and normal K and Mg (0Na–K–Mg); (2) normal Na, K and Mg (Na–K–Mg); (3) normal Na and high K (2 ×normal), and normal Mg (Na–2K–Mg); and (4) normal Na and high K (2 ×normal), and high Mg (2 ×normal) (Na–2K–2Mg). As expected, compared to control 0Na–K–Mg rats, Na–K–Mg rats developed HT. Blood pressure increased significantly less in Na–2K–Mg rats whereas, BP did not increase in Na–2K–2Mg rats. Hypertension in NA–K–Mg rats was associated with an increase in plasma SPI and digitalis like factor (DIF) and a decrease in renal and myocardial NKPTA. However, doubling the Mg along with K in the diet (Na–2K–2Mg) normalized SPI and DIF and increased myocardial and renal NKPTA, compared to control 0Na–K–Mg rats. Also, compared to 0Na–K–Mg rats, water consumption, urine excretion, urinary sodium excretion urinary potassium excretion (UNaV), and (UKV) increased in the other three groups, more so in Na–2K–2Mg rats. These data show that K and Mg have additive effects in preventing an increase in SPI, thus probably preventing the BP increase in RRM rats.

MECHANISM OF ANTIHYPERTENSIVE EFFECT OF DIETARY POTASSIUM IN EXPERIMENTAL VOLUME EXPANDED HYPERTENSION IN RATS

Dietary potassium supplementation lowers blood pressure (BP) and attenuates complications in hypertensive subjects, particularly those with the low renin volume expanded (LRVE) variety. We and others have shown that the plasma level of a digitalis like substance (DLS) is elevated in this type of hypertension. We therefore, examined the effect of increases in dietary potassium on the plasma level of endogenous DLS, myocardial and renal Na+, K+-ATPase (NKA) activities, BP, and renal excretory function in reduced renal mass (RRM)-salt hypertension in the rat, a classical model of LRVE hypertension. 70% RRM rats were divided in 4 groups, namely those consuming: 1) a sodium free and normal potassium (1.3% as KCl) diet (RRM-0 Na), 2) a normal sodium and normal potassium diet (RRM-NaK), 3) a normal sodium and high potassium (2 X normal) diet (RRM-Na2K), and 4) a normal sodium and 4 times normal potassium diet (RRM-Na4K). At the end of 4 weeks of dietary treatment, direct BP was recorded, plasma level of DLS determined by bioassay and with a radioimmunoassay for digoxin (DIF) and myocardial and renal NKA activities were measured. As expected, compared to RRM-0Na rats, RRM-NaK rats developed hypertension. BP increased significantly less in RRM-Na2K, whereas BP did not increase in RRM-Na4K rats. Hypertension in RRM-NaK rats was associated with an increase in plasma DLS and DIF and decrease in renal and myocardial NKA activities. DLS was increased (DIF was not changed) and myocardial NKA also decreased in rats consuming double potassium. However, quadrupling potassium in the diet (RRM-Na4K) normalized DLS and DIF and increased myocardial and renal NKA activities, compared to RRM-0Na rats. Also compared to RRM-0Na, water consumption, urinary volume excretion, sodium, and potassium increased in the other 3 groups, more so in RRM-Na4K rats. These data show that quadrupling the potassium in the diet prevents the BP increase in RRM rats and this is associated with diuresis/natriuresis and normalization of DLS, perhaps because the diuresis/natriuresis normalizes blood volume.

Distinct mechanisms of modulation of angiotensin II type I receptor gene expression in heart and aorta.

The purpose of the present study was to test the hypothesis that hypertension induced by reduced renal mass (RRM) upregulates gene expression of the type 1 angiotensin II (Ang II) receptor (AT1) in the thoracic aorta and heart through an Ang II-dependent mechanism. Three groups of rats were given 1% NaCl water and subjected to RRM, RRM plus captopril (RRM+Cap, 30 mg/kg per day), or sham surgery. Tail-cuff systolic blood pressure was significantly elevated in RRM and RRM+Cap rats compared with sham-operated rats. The ratios of the medial wall area of the thoracic aorta and heart weight to body weight were significantly elevated in RRM and RRM+Cap rats compared with sham-operated rats. Northern blot analysis indicated that the ratio of AT1 to GAPDH mRNA in the aorta was significantly higher in RRM (1.85 +/- 0.52) compared with sham-operated (0.21 +/- 0.04) and RRM+Cap (0.55 +/- 0.20) rats. In contrast, the ratio of AT1 to GAPDH mRNA in the heart was significantly increased in both RRM (1.09 +/- 0.23) and RRM+Cap (1.00 +/- 0.09) compared with sham-operated (0.34 +/- 0.06) rats. Thus, RRM hypertension upregulates AT1 mRNA expression in both the hypertrophied aorta and heart. Captopril treatment without altering blood pressure in RRM rats prevents the increase in AT1 mRNA in the aorta but not the heart. These results suggest that different tissue-specific mechanisms of AT1 gene regulation exist; ie, in aorta, an Ang II-or kinin-dependent mechanism is operant, whereas in heart, RRM-induced upregulation of AT1 mRNA may be pressure dependent.

Mathematical analysis of type-I and type-IIb muscle fiber force generation in renal hypertension.

Previous results from our laboratory have shown that isometric tension development is significantly lower in reduced renal mass (RRM) hypertensive rats when compared to sham-operated controls. The current study was designed to mathematically analyze isometric tetanic contraction profiles and determine the relative contribution of fast-glycolytic (FG) and slow-oxidative (SO) muscle fibers produced by the isolated gastrocnemius-plantaris-soleus muscle group of RRM and sham rats. Because renal hypertension has been shown to be associated with a reduction in microvascular density, we hypothesized that renal hypertension leads to a decrease in SO muscle fiber contribution to force generation. The mathematical model determined the force contribution of two independent muscle fiber components, SO and FG, to the contraction and relaxation phase of isometric tetanic contractions. Each phase was modeled as having an exponentially rising contraction phase during the stimulus period and an exponentially decaying relaxation phase when the stimulus was removed. Each fiber type's tension was also scaled by an envelope function describing the fatigue over the contraction bout. The model, which included 10 parameters, was fit to experimental data by using a nonlinear optimization method and described certain limited characteristics of both fiber types. Results from this model suggest that renal hypertension affects skeletal muscle force generation primarily by decreasing the SO muscle fiber contribution to the total developed tension, decreasing performance and increasing muscle fatigue in RRM rats.

Regulation of ANG II receptor in hypertension: role of ANG II.

To investigate the role of angiotensin II (ANG II) in the development of hypertension induced by reduced renal mass (RRM) and the gene expression of ANG II type 1 (AT1) receptors in the remnant renal tissue, four groups of rats were given 1% NaCl in water and subjected to RRM, RRM+ ramipril, RRM+ losartan, or sham surgery (control). Tail-cuff systolic blood pressure was significantly higher in RRM rats than in the other three groups. Northern blot showed that AT1 gene expression was significantly decreased in RRM, RRM + ramipril, or RRM + losartan vs. control. There was no significant difference among the three RRM groups. Renal transforming growth factor-beta 1 (TGF-beta 1) mRNA levels were increased threefold (P < 0.05) in RRM, RRM+ ramipril, and RRM+ losartan vs. control. There was no significant difference among the three RRM groups. We conclude that the development of RRM hypertension is ANG II dependent but not mediated by AT1 gene expression. RRM downregulates AT1 mRNA and upregulates TGF-beta 1 mRNA in the remnant renal tissue, regardless of blood pressure or plasma levels of ANG II, suggesting that these gene responses are triggered by an effect of local injury.

Hemodynamic and microcirculatory changes during development of renal hypertension

To study the progression of vessel loss (rarefaction) during the development of hypertension, a plastic window was chronically implanted over the biceps femoralis muscle of reduced renal mass (RRM) and sham-operated control (SOC) rats on a low-salt diet (0.4%). Blood pressure was measured directly via a catheter implanted in the femoral artery, and tissue blood flow was measured by laser-Doppler flowmetry before and after local topical application of sodium nitroprusside. Measurements were made on the control day and after an increase in sodium intake (4.0%) in RRM rats on days 5, 10, 14, 21, and 28. SOC rats on a low-salt diet served as the control group. RRM rats became hypertensive (167 mmHg), and vascular resistance increased after a change in sodium intake. Resting tissue blood flow decreased by 19% by day 28 in RRM but did not change in SOC. Sodium nitroprusside administration reduced vascular resistance to the same level in both RRM and SOC during control and at day 10; however, on all other days, sodium nitroprusside was not able to fully dilate the RRM microcirculation compared with that of SOC. At the same time, microvascular density estimated by computer fluorescence microscopy was decreased by 25% in RRM rats. These studies indicate that the development of RRM hypertension is characterized by a transition from increased tone and vessel closure (functional rarefaction) to anatomic vessel loss (structural rarefaction).

Role of ouabainlike compound in rats with reduced renal mass-saline hypertension

Ouabainlike compound (OLC) has recently been identified as a likely mammalian endogenous digitalis-like factor (EDLF) from human plasma. In this study, plasma levels of OLC were determined to assess the role of OLC in a model known as volume-expanded, reduced renal mass (RRM)-saline (S) hypertension in rats with use of a newly developed radioimmunoassay for ouabain. In the first experiment, at 3 wk after subtotal nephrectomy and drinking 1% saline solution, sysolic blood pressure (SBP) of 18 rats with reduced renal mass (RRM-S rats) was significantly higher than in 17 sham-operated saline-drinking control (C-S) rats [154 +/- 4 (SE) vs. 132 +/- 2 mmHg; P < 0.01]. Plasma OLC levels were 355 +/- 68 pmol/l in RRM-S rats, sevenfold higher than in C-S rats (54 +/- 4 pmol/l; P < 0.01). In the second experiment, we measured plasma OLC levels of 10 RRM-S, 12 sham-operated control (C), and 10 subtotally nephrectomized rats drinking distilled water (RRM rats). Concomitant with a marked increase in blood pressure (203 +/- 5 mmHg), RRM-S rats showed significantly higher plasma OLC levels compared with C and RRM rats (RRM-S 114 +/- 24, C 47 +/- 11, and RRM 52 +/- 9 pmol/l; P < 0.05). In both experiments, plasma OLC levels correlated significantly with SBP (P < 0.05). These findings suggest that plasma OLC shows a similar behavior to that of EDLFs or Na(+)-K(+)-adenosinetriphosphatase inhibitors reported in previous publications and may play a role in hypertensive mechanisms in rats with RRM and excess Na intake.

Long-term ouabain administration produces hypertension in rats.

Ouabain has recently been identified as an endogenous Na(+)-K+ pump inhibitor. We administered ouabain chronically to normotensive rats with varying degrees of reduced renal mass (RRM) and to normal two-kidney rats to see whether hypertension could be produced. Normal male Wistar rats and rats with 25%, 60%, and 70% RRM received ouabain (13.9 micrograms/kg per day IP) in normal saline for 4 weeks followed by ouabain (27.8 micrograms/kg per day IP) for 3 to 4 more weeks. Respective control animals received vehicle only. Blood pressure was recorded weekly by tail plethysmography. Animals received tap water and standard rat chow, except for 70% RRM rats, which received distilled water and sodium-free chow. After 6 to 8 weeks of treatment, with rats under thiobutabarbital anesthesia, direct blood pressure was determined. Plasma, tissue, and urinary ouabain levels were measured with a specific radioimmunoassay. Animals receiving ouabain developed significant increases in mean blood pressure compared with control animals (70% RRM, 147 +/- 4 vs 116 +/- 4 mm Hg; 60% RRM, 140 +/- 4 vs 107 +/- 3 mm Hg; 25% RRM, 131 +/- 5 vs 100 +/- 2 mm Hg; no RRM, 116 +/- 4 vs 98 +/- 5 mm Hg). Plasma ouabain levels measured 24 hours after the last ouabain dose were not different in animals receiving ouabain vs those receiving vehicle. However, kidney tissue ouabain levels were significantly greater (6.39 +/- 1.17 vs 2.36 +/- 0.52 micrograms/kg, P < .05) in animals receiving ouabain. In conclusion, ouabain, given chronically, is associated with the development of hypertension in RRM rats as well as in normal rats. Blood pressure was greater in animals with greater degrees of RRM for a given ouabain dose.

Structural alterations of microvascular smooth muscle cells in reduced renal mass hypertension.

Loss of microvessels (anatomic rarefaction) occurs in chronic reduced renal mass (RRM) hypertension and is mediated via structural degeneration of vascular smooth muscle (VSM) and endothelial cells. The purpose of the present study was to determine if structural changes occur in VSM cells of the microvessels that remain in the tissue of rats with chronic RRM hypertension. Samples of cremaster muscles were taken from normotensive control rats and rats with acute (3-7 days) and chronic (14-28 days) RRM hypertension (75% reduction in kidney mass with 4% NaCl loading). The samples were fixed in situ and processed for light and electron microscopy. Ultrastructural morphology of VSM cells in terminal arterioles of control animals was normal. Although VSM morphology in many microvessels of RRM hypertensive rats was also normal, some vessels exhibited structural changes that were not present in arterioles of the normotensive animals. The most striking change was the appearance of more extensive dense bodies anchoring the contractile filaments around the outer membrane of the cells. Extreme vasoconstriction was observed in some arterioles of RRM rats as long as 2 weeks after salt loading. Focal areas of VSM cell proliferation were evident. Many of the changes occurring in RRM were detected as early as 1 week after the onset of hypertension. These observations suggest that renal mass reduction-salt loading hypertension is associated with early structural and functional changes in the VSM cells.

Increased arterial potassium transport in reduced renal mass hypertension of the rat.

Aortic potassium turnover was studied during the development of hypertension induced by salt load in male rats after 70-75% of total renal mass was removed. Systolic blood pressure in the saline-drinking experimental reduced renal mass (RRM) rats steadily increased until the fourth week after surgery and thereafter stayed at the same level. Control RRM rats given tap water for drinking, and unilaterally nephrectomized saline-drinking control rats maintained normal blood pressure. Compared to controls, experimental RRM rats exhibited increased plasma aldosterone concentration while plasma renin activity was low in all groups with no significant difference. Aortic hypertrophy, greater 42K turnover, and elevated 42K exchange were observed with experimental RRM hypertension. Sensitivity to the effect of norepinephrine (NE) on aortic 42K turnover was increased four- to ninefold in the experimental RRM group as compared to controls. These results indicate that reduced renal mass hypertension is associated with increased potassium permeability and NE supersensitivity in vascular smooth muscle.

Prostaglandin and thromboxane formation in glomeruli from rats with reduced renal mass.

In vitro formation of prostaglandins (PG) E2, F2 alpha, 6-keto-F1 alpha, and thromboxane B2 (TxB2) by glomeruli from rats with reduced renal mass (RRM) were evaluated by radioimmunoassay. Four weeks following ablation of renal mass, PGE2, PGF2 alpha, and TxB2 production by glomeruli from RRM rats was significantly greater, when compared with glomerular PG and TxB2 production of sham-operated control (C) rats. The effect of cyclooxygenase inhibition with indomethacin and the selective inhibition of thromboxane formation with UK 38485 on glomerular filtration rate (GFR) was investigated in experiments in vivo. In RRM rats indomethacin reduced GFR from 212 +/- 17 to 138 +/- 14 microliter/100 g/body weight (p less than 0.05) without effect on C rats. Thromboxane synthesis inhibition with UK 38485, however, increased GFR significantly in RRM rats (221 +/- 26 to 303 +/- 21; p less than 0.05). The data suggest that vasodilatory PGs and TxB2 modulate GFR in rats with ablation of renal mass.