Impact of maternal protein restriction on Hypoxia-Inducible Factor (HIF) expression in male fetal kidney development.

BACKGROUND: Kidney developmental studies have demonstrated molecular pathway changes that may be related to decreased nephron numbers in the male 17 gestational days (17GD) low protein (LP) intake offspring compared to normal protein intake (NP) progeny. Here, we evaluated the HIF-1 and components of its pathway in the kidneys of 17-GD LP offspring to elucidate the molecular modulations during nephrogenesis. METHODS: Pregnant Wistar rats were allocated into two groups: NP (regular protein diet-17%) or LP (Low protein diet-6%). Taking into account miRNA transcriptome sequencing previous study (miRNA-Seq) in 17GD male offspring kidneys investigated predicted target genes and proteins related to the HIF-1 pathway by RT-qPCR and immunohistochemistry. RESULTS: In the present study, in male 17-GD LP offspring, an increased elF4, HSP90, p53, p300, NFκß, and AT2 gene encoding compared to the NP progeny. Higher labeling of HIF-1α CAP cells in 17-DG LP offspring was associated with reduced elF4 and phosphorylated elF4 immunoreactivity in LP progeny CAP cells. In 17DG LP, the NFκß and HSP90 immunoreactivity was enhanced, particularly in the CAP area. DISCUSSION AND CONCLUSION: The current study supported that the programmed reduced nephron number in the 17-DG LP offspring may be related to changes in the HIF-1α signaling pathway. Factors that facilitate the transposition of HIF-1α to progenitor renal cell nuclei, such as increased NOS, Ep300, and HSP90 expression, may have a crucial role in this regulatory system. Also, HIF-1α changes could be associated with reduced transcription of elF-4 and its respective signaling path.

Kidney safety during surgical pneumoperitoneum: an experimental study in rats.

BACKGROUND: Elevations of intraabdominal pressure during laparoscopic procedures may lead to oliguria or anuria in mammals. Despite this, previous research has not been able to confirm an associated kidney injury. This study aimed to investigate the occurrence of an early kidney lesion secondary to surgical pneumoperitoneum in a rat model using the expression of neutrophil gelatinase-associated lipocalin (N-GAL) as a biomarker for early kidney injury. METHODS: In this study, 20 male Sprague-Dawley rats under general anesthesia and mechanically ventilated were allocated to one of five experimental time-dependent groups: group 1 (1-h control), group 2 (1-h pneumoperitoneum), group 3 (2-h control), group 4 (2-h pneumoperitoneum), and group 5 (positive kidney injury group induced by intravenous administration of cisplatin 7.5 mg/kg). To evaluate the renal expression of N-GAL 24 h after the procedure, all the rats underwent a 2-h urine output evaluation as well as laparotomy and bilateral nephrectomy performed sequentially to investigate the presence of renal injury using immunofluorescence qualification and western blotting. RESULTS: Urine output was reduced and N-GAL expression was increased in the animals from the cisplatin group. The animals undergoing 1- or 2-h pneumoperitoneum displayed urine output and N-GAL expression similar to that of the animals from the matching control groups. CONCLUSIONS: Under the experimental conditions of this study, the animals with normal preoperative renal function did not show any type of acute kidney injury associated with the presence of a stabilized surgical pneumoperitoneum.

Transcriptome and morphological analysis on the heart in gestational protein-restricted aging male rat offspring.

Background: Adverse factors that influence embryo/fetal development are correlated with increased risk of cardiovascular disease (CVD), type-2 diabetes, arterial hypertension, obesity, insulin resistance, impaired kidney development, psychiatric disorders, and enhanced susceptibility to oxidative stress and inflammatory processes in adulthood. Human and experimental studies have demonstrated a reciprocal relationship between birthweight and cardiovascular diseases, implying intrauterine adverse events in the onset of these abnormalities. In this way, it is plausible that confirmed functional and morphological heart changes caused by gestational protein restriction could be related to epigenetic effects anticipating cardiovascular disorders and reducing the survival time of these animals. Methods: Wistar rats were divided into two groups according to the protein diet content offered during the pregnancy: a normal protein diet (NP, 17%) or a Low-protein diet (LP, 6%). The arterial pressure was measured, and the cardiac mass, cardiomyocytes area, gene expression, collagen content, and immunostaining of proteins were performed in the cardiac tissue of male 62-weeks old NP compared to LP offspring. Results: In the current study, we showed a low birthweight followed by catch-up growth phenomena associated with high blood pressure development, increased heart collagen content, and cardiomyocyte area in 62-week-old LP offspring. mRNA sequencing analysis identified changes in the expression level of 137 genes, considering genes with a p-value < 0.05. No gene was. Significantly changed according to the adj-p-value. After gene-to-gene biological evaluation and relevance, the study demonstrated significant differences in genes linked to inflammatory activity, oxidative stress, apoptosis process, autophagy, hypertrophy, and fibrosis pathways resulting in heart function disorders. Conclusion: The present study suggests that gestational protein restriction leads to early cardiac diseases in the LP progeny. It is hypothesized that heart dysfunction is associated with fibrosis, myocyte hypertrophy, and multiple abnormal gene expression. Considering the above findings, it may suppose a close link between maternal protein restriction, specific gene expression, and progressive heart failure.

Fetal Undernutrition Programming, Sympathetic Nerve Activity, and Arterial Hypertension Development.

A wealth of evidence showed that low birth weight is associated with environmental disruption during gestation, triggering embryotic or fetal adaptations and increasing the susceptibility of progeny to non-communicable diseases, including metabolic and cardiovascular diseases, obesity, and arterial hypertension. In addition, dietary disturbance during pregnancy in animal models has highlighted mechanisms that involve the genesis of arterial hypertension, particularly severe maternal low-protein intake (LP). Functional studies demonstrated that maternal low-protein intake leads to the renal decrease of sodium excretion and the dysfunction of the renin-angiotensin-aldosterone system signaling of LP offspring. The antinatriuretic effect is accentuated by a reduced number of nephron units and glomerulosclerosis, which are critical in establishing arterial hypertension phenotype. Also, in this way, studies have shown that the overactivity of the central and peripheral sympathetic nervous system occurs due to reduced sensory (afferent) renal nerve activity. As a result of this reciprocal and abnormal renorenal reflex, there is an enhanced tubule sodium proximal sodium reabsorption, which, at least in part, contributes directly to arterial hypertension development in some of the programmed models. A recent study has observed that significant changes in adrenal medulla secretion could be involved in the pathophysiological process of increasing blood pressure. Thus, this review aims to compile studies that link the central and peripheral sympathetic system activity mechanisms on water and salt handle and blood pressure control in the maternal protein-restricted offspring. Besides, these pathophysiological mechanisms mainly may involve the modulation of neurokinins and catecholamines pathways.

Gestational and Breastfeeding Low-Protein Intake on Blood Pressure, Kidney Structure, and Renal Function in Male Rat Offspring in Adulthood.

Background: Our previous studies demonstrated that maternal protein-restricted (low-protein, LP) 16-week-old offspring had pronounced nephron number reduction and arterial hypertension associated with an unchanged glomerular filtration rate (GFR). An enhanced gomerular area may be related to increased glomerular filtration and overflow, which accounts for glomerular filtration barrier breakdown and early glomerulosclerosis. The effect of protein restriction during gestational and breastfeeding periods is unknown. Method: The functional e-structural kidney evaluation was obtained using lithium and creatinine clearance, kidney morphometry, immunoblotting, and immunostaining analysis in 16 and 24-week-old LP offspring compared to age-matched NP progeny. Results: Low protein rats' progeny had significantly reduced birth weight, without previous catch-up growth phenomena, in parallel with a decreased adiposity index. Transforming growth factor-beta 1 (TGF-ß1) glomerular expression was significantly enhanced in the LP group. Also, the LP offspring had a 38% lower nephron number and an increased glomerular volume. They also presented with a higher cardiac index and arterial blood pressure compared with age-matched NP offspring. The LP rats exhibited augmented Na+/K+-ATPase in the proximal segments, and NOS1 immunoreactivity in whole renal tissue was associated with sodium retention in the proximal nephron segments. We also found significantly enhanced collagen content associated with increased TGFß1 and ZEB1/2 renal immunoreactivity in LP offspring compared with NP offspring. Increased hypertrophy markers in LP podocytes were associated with an amplified IL-6/STAT3 pathway activity. Conclusion: To our knowledge, these are the first data demonstrating renal functional and structural changes in protein restriction during gestation and lactation model of fetal programming. The fetal-programmed adult offspring showed pronounced structural glomerular disorders with an accentuated and advanced fibrosis stage, without a change in the GFR. These findings suggest that the glomerular enhanced TGF-ß1 action may induce ZEB1/2 expression that may cause glomeruli epithelial-to-mesenchymal transition. Besides, decreased nephron number in the LP offspring with preserved glomerular function may be related to protective or even attenuate the activated IL-6/STAT3 pathway.

Impact of gestational low-protein intake on embryonic kidney microRNA expression and in nephron progenitor cells of the male fetus.

BACKGROUND: Here, we have demonstrated that gestational low-protein (LP) intake offspring present lower birth weight, reduced nephron numbers, renal salt excretion, arterial hypertension, and renal failure development compared to regular protein (NP) intake rats in adulthood. We evaluated the expression of various miRNAs and predicted target genes in the kidney in gestational 17-days LP (DG-17) fetal metanephros to identify molecular pathways involved in the proliferation and differentiation of renal embryonic or fetal cells. METHODS: Pregnant Wistar rats were classified into two groups based on protein supply during pregnancy: NP (regular protein diet, 17%) or LP diet (6%). Renal miRNA sequencing (miRNA-Seq) performed on the MiSeq platform, RT-qPCR of predicted target genes, immunohistochemistry, and morphological analysis of 17-DG NP and LP offspring were performed using previously described methods. RESULTS: A total of 44 miRNAs, of which 19 were up and 25 downregulated, were identified in 17-DG LP fetuses compared to age-matched NP offspring. We selected 7 miRNAs involved in proliferation, differentiation, and cellular apoptosis. Our findings revealed reduced cell number and Six-2 and c-Myc immunoreactivity in metanephros cap (CM) and ureter bud (UB) in 17-DG LP fetuses. Ki-67 immunoreactivity in CM was 48% lesser in LP compared to age-matched NP fetuses. Conversely, in LP CM and UB, ß-catenin was 154%, and 85% increased, respectively. Furthermore, mTOR immunoreactivity was higher in LP CM (139%) and UB (104%) compared to that in NP offspring. TGFß-1 positive cells in the UB increased by approximately 30% in the LP offspring. Moreover, ZEB1 metanephros-stained cells increased by 30% in the LP offspring. ZEB2 immunofluorescence, although present in the entire metanephros, was similar in both experimental groups. CONCLUSIONS: Maternal protein restriction changes the expression of miRNAs, mRNAs, and proteins involved in proliferation, differentiation, and apoptosis during renal development. Renal ontogenic dysfunction, caused by maternal protein restriction, promotes reduced reciprocal interaction between CM and UB; consequently, a programmed and expressive decrease in nephron number occurs in the fetus.

Gestational Low Protein Diet Modulation on miRNA Transcriptome and Its Target During Fetal and Breastfeeding Nephrogenesis.

BACKGROUND: The kidney ontogenesis is the most structurally affected by gestational protein restriction, reducing 28% of their functional units. The reduced nephron number is predictive of hypertension and cardiovascular dysfunctions that are generally observed in the adult age of most fetal programming models. We demonstrate miRNAs and predict molecular pathway changes associated with reduced reciprocal interaction between metanephros cap (CM) and ureter bud (UB) and a 28% decreased nephron stem cells in the 17 gestational days (17GD) low protein (LP) intake male fetal kidney. Here, we evaluated the same miRNAs and predicted targets in the kidneys of 21GD and at 7 days of life (7DL) LP offspring to elucidate the molecular modulations during nephrogenesis. METHODS: Pregnant Wistar rats were allocated into two groups: NP (regular protein diet- 17%) or LP (diet-6%). miRNA transcriptome sequencing (miRNA-Seq) was performed on the MiSeq platform from 21GD and 7DL male offspring kidneys using previously described methods. Among the top 10 dysfunctional regulated miRNAs, we validated 7 related to proliferation, differentiation, and apoptosis processes and investigated predicted target genes and proteins by RT-qPCR and immunohistochemistry. RESULTS: In 21GD, LP fetuses were identified alongside 21 differently expressed miRNAs, of which 12 were upregulated and 9 downregulated compared to age-matched NP offspring. In 7-DL LP offspring, the differentially expressed miRNAs were counted to be 74, of which 46 were upregulated and 28 downregulated. The curve from 17-GD to 7-DL shows that mTOR was fundamental in reducing the number of nephrons in fetal kidneys where the mothers were subjected to a protein restriction. IGF1 and TGFß curves also seemed to present the same mTOR pattern and were modulated by miRNAs 181a-5p, 181a-3p, and 199a-5p. The miRNA 181c-3p modulated SIX2 and Notch1 reduction in 7-DL but not in terms of the enhanced expression of both in the 21-GD, suggesting the participation of an additional regulator. We found enhanced Bax in 21-GD; it was regulated by miRNA 298-5p, and Bcl2 and Caspase-3 were controlled by miRNA (by 7a-5p and not by the predicted 181a-5p). The miRNA 144-3p regulated BCL6, which was enhanced, as well as Zeb 1 and 2 induced by BCL6. These results revealed that in 21GD, the compensatory mechanisms in LP kidneys led to the activation of UB ramification. Besides, an increase of 32% in the CM stem cells and a possible cell cycle halt of renal progenitor cells, which remaining undifferentiated, were observed. In the 7DL, much more altered miRNA expression was found in LP kidneys, and this was probably due to an increased maternal diet content. Additionally, we verified the activation of pathways related to differentiation and consumption of progenitor cells.

Expression of renin-angiotensin system signalling compounds in maternal protein-restricted rats: effect on renal sodium excretion and blood pressure.

BACKGROUND: Intrauterine growth restriction due to low maternal dietary protein during pregnancy is associated with retardation of foetal growth, renal alterations and adult hypertension. The renin-angiotensin system (RAS) is a coordinated hormonal cascade in the control of cardiovascular, renal and adrenal function that governs body fluid and electrolyte balance, as well as arterial pressure. In the kidney, all the components of the renin-angiotensin system including angiotensin II type 1 (AT1) and type 2 (AT2) receptors are expressed locally during nephrogenesis. Hence, we investigated whether low protein diet intake during pregnancy altered kidney and adrenal expression of AT1(R) and AT2(R) receptors, their pathways and if the modified expression of the RAS compounds occurs associated with changes in urinary sodium and in arterial blood pressure in sixteen-week-old males' offspring of the underfed group. METHODS: The pregnancy dams were divided in two groups: with normal protein diet (pups named NP) (17% protein) or low protein diet (pups LP) (6% protein) during all pregnancy. RESULTS: The present data confirm a significant enhancement in arterial pressure in the LP group. Furthermore, the study showed a significantly decreased expression of RAS pathway protein and Ang II receptors in the kidney and an increased expression in the adrenal of LP rats. The detailed immunohistochemical analysis of RAS signalling proteins in the kidney confirm the immunoblotting results for both groups. The present investigation also showed a pronounced decrease in fractional urinary sodium excretion in maternal protein-restricted offspring, compared with the NP age-matched group. This occurred despite unchanged creatinine clearance. CONCLUSIONS: The current data led us to hypothesize that foetal undernutrition could be associated with decreased kidney expression of AT(R) resulting in the inability of renal tubules to handle the hydro-electrolyte balance, consequently causing arterial hypertension.

miRNAs, target genes expression and morphological analysis on the heart in gestational protein-restricted offspring.

Gestational protein restriction was associated with low birth weight, hypertension and higher prevalence of cardiac disorders in adults. Several mechanisms, including epigenetics, could be related with the cardiovascular phenotype on protein-restricted offspring. Thus, we investigated the morphological cardiac effects of gestational protein restriction and left ventricle miRNAs and target genes expression pattern in both 12-day and 16-week old gestational protein-restricted male offspring. Pregnant Wistar rats were allocated into two groups, according to protein supply during pregnancy: NP (normal protein diet- 17%) or LP (low protein diet-6%). Dams on the gestational protein-restricted diet had lower body weight gain and higher food intake. Gestational protein-restricted offspring had low birth weight, followed by rapidly body weight recovery, hypertension, and increased myocytes cross-sectional area and collagen fraction at 16-week old age. At 12-days old, miR-184, miR-192, miR-376c, miR-380-3p, miR-380-5p, miR-451, and miR-582-3p had increased expression, and miR-547 and miR-743a had decreased expression in the gestational protein-restricted left ventricle. At 16-week old, let-7b, miR-125a-3p, miR-142-3p, miR-182 and miR-188-5p had increased expression and let-7g, miR-107, miR-127, miR-181a, miR-181c, miR-184, miR-324-5p, miR-383, miR-423-5p and miR-484 had decreased expression in gestational protein-restricted left ventricle. Target predicted gene expression analysis showed higher expression of Dnmt3a, Oxct1, Rictor and Trps1 and lower expression of Bbs1 and Calml3 in 12-day old protein-restricted offspring. 16-week old protein-restricted offspring had higher expression of Adrbk1, Bbs1, Dnmt3a, Gpr22, Inppl1, and Oxct1 genes. In conclusion, gestational protein restriction was related to offspring low birth weight, increased systolic blood pressure and morphological heart alterations that could be related to early heart miRNA expression changes that perpetuate into adulthood and which are associated with the regulation of essential genes involved in cardiovascular development, heart morphology, function, and metabolism.

Effect of intracerebroventricular epinephrine microinjection on blood pressure and urinary sodium handling in gestational protein-restricted male adult rat offspring.

In this study, we hypothesized that blunting of the natriuresis response to intracerebroventricularly (ICV) microinjected adrenergic agonists is involved in the development of hypertension in maternal low-protein intake (LP) offspring. A stainless steel cannula was stereotaxically implanted into the right lateral ventricle (LV), then we evaluated the ICV administration of adrenergic agonists at increasing concentrations, and of α1 and α2-adrenoceptor antagonists on blood pressure and urinary sodium handling in LP offspring relative to an age-matched normal-protein intake (NP) group. We confirmed that epinephrine (Epi) microinjected into the LV of conscious NP rats leads to enhanced natriuresis followed by a reduction in arterial pressure. This response is associated with increased proximal and post-proximal sodium excretion accompanied by an unchanged glomerular filtration rate. The current study showed, in both NP and LP offspring, that the natriuretic effect of Epi injection into the LV was abolished by prior local microinjection of an α1-adrenoceptor antagonist (prazosin). Conversely, LV α2-adrenoceptor antagonist (yohimbine) administration potentiated the action of Epi. The LV yohimbine pretreatment normalized urinary sodium excretion and reduced the blood pressure in LP compared with age-matched NP offspring. These are, as far as we are aware, the first results showing the role of central adrenergic receptors' interaction on hypertension pathogenesis in maternal LP fetal-programming offspring. This study also provides good evidence for the existence of central nervous system adrenergic mechanisms consisting of α1 and α2-adrenoceptors, which work reciprocally on the control of renal sodium excretion and blood pressure. Although the precise mechanism of the different natriuretic response of NP and LP rats is still uncertain, these results lead us to speculate that inappropriate neural adrenergic pathways might have significant effects on tubule sodium transport, resulting in the inability of the kidneys to control hydrosaline balance and, consequently, an increase in blood pressure.

Estimation of the kidney metabolic heat generation rate.

Thermogenesis results from the cellular metabolism and has a fundamental role for body thermoregulation in endothermic species. The motivation for this work is the analysis of the kidneys' contribution for thermoregulation. An inverse problem is solved for the estimation of the heat generation rate that results from the metabolic activities in the kidney, by using transient temperature measurements of the urine. The Markov chain Monte Carlo (MCMC) method is applied for the solution of the inverse problem, which presents inherent difficulties associated with low sensitivity of the parameters of main interest that represent the transient heat source term and strong correlation of the remaining model parameters. Such difficulties are dealt with in this work by using a version of the Metropolis-Hastings algorithm that samples the parameters in blocks. Simulated temperature measurements are used for the inverse problem solution, and the convergence of the Markov chains is verified with two different techniques.

Altered renal sodium handling in spontaneously hypertensive rats (SHR) after hypertonic saline intracerebroventricular injection: role of renal nerves.

The mechanism by which blood pressure rises in the SHR strain remains to be elucidated. Also, there is a surprising lack of experimental data on the natriuretic mechanisms induced by intracerebroventricular (ICV) injection of hyperosmotic saline (HoS) in SHR. In normotensive animals ICV injection of HoS causes coordinated responses including natriuresis and inhibition of renal sympathetic nerve activity. In the present study, we hypothesized that presumable blunting of the sympathoinhibitory response to centrally injected HoS may contribute to a lack of suppression of efferent renal nerve outflow in SHR. To test this hypothesis, the present study evaluates the influence of renal denervation after central HoS injection at increasing concentration on urinary sodium handling in SHR compared with age-matched normotensive WKy rats. The study confirmed previous data showing pronounced natriuretic response to centrally HoS stimuli but also demonstrated that the creatinine clearance (C(Cr)) and fractional sodium excretion responses diminished as graded NaCl concentrations were increased in WKy rats but not in SHR. In SHR, increased FE(Na) obtained by central administration of 0.90 M NaCl was produced by increases in proximal (FEP(Na)) and post-proximal fractional urinary sodium rejection without changes in C(Cr), indicating a direct tubular effect. Renal denervation caused significant antinatriuresis by decreased C(Cr) and increased FEP(Na) reabsorption in WKy but not in SHR. This study suggests that natriuresis observed only after higher centrally HoS stimuli with a rightward shift of dose-response curve provides evidence of a down-regulation of target organ responsiveness of periventricular areas of genetic hypertensive rats.

Expression and localization of NK(1)R, substance P and CGRP are altered in dorsal root ganglia neurons of spontaneously hypertensive rats (SHR).

The kidneys play a pivotal role in the pathogenesis of essential hypertension because of a primary defect in renal hemodynamics and/or tubule hydro-saline handling that results in the retention of fluid and electrolytes. Previous studies have shown that increasing the renal pelvic pressure increased ipsilateral afferent renal nerve activity (ARNA), the ipsilateral renal pelvic release of substance P (SP) and the contralateral urinary sodium excretion in Wistar--Kyoto rats (WKy). However, spontaneously hypertensive rats (SHR) present an impaired renorenal reflex activity associated, partly, with a peripheral defect at the level of the sensory receptors in the renal pelvis. Furthermore, the renal pelvic administration of SP failed to increase ARNA in most of SHR at concentrations that produced marked increases in WKy. Since we have assessed the expression and localization of NK(1) receptor (NK(1)R), SP and calcitonin gene-related peptide (CGRP) in different dorsal root ganglia (DRG) cell subtypes and renal pelvis of 7- and 14-week-old SHR. The results of this study show increased SP and CGRP expression in the dorsal ganglia root cells of SHR compared to WKy rats. Additionally, there was a progressive, significant, age-dependent, decrease in NK(1)R expression on the membrane surface in SHR DRG cells and in the renal pelvis. In conclusion, the results of the present study suggest that the impaired activation of renal sensory neurons in SHR may be related to changes in the expression of neuropeptides and/or to a decreased presence of NK(1)R in DRG cells. Such abnormalities could contribute to the enhanced sodium retention and elevation of blood pressure seen in SHR.

Importance of anesthesia for the genesis of neurogenic pulmonary edema in spinal cord injury.

There are reports describing both provocation and inhibition of neurogenic pulmonary edema by anesthetic drugs. Therefore, we compared the effect of two types of anesthesia on the formation of neurogenic pulmonary edema in rats with balloon-induced acute spinal cord injury. Animals with sham procedure (group 1) were anesthesized by intraperitoneal sodium pentobarbital. In the experimental groups, rats were submitted to acute spinal cord lesion by insufflations of a balloon in the epidural space at T8 for 1 min (group 3 under i.p. sodium pentobarbital and group 2 under i.p. xylazine-ketamine anesthesia). In rats with pentobarbital anesthesia, systolic blood pressure doubled the baseline value during compression, whereas this effect was less pronounced in the ketamine-xylazine group. The pulmonary index (100 x wet lung weight/body weight) was 0.395 (+/-0.018) in sham-operated rats, rose to 0.499 (+/-0.060) in group 2, and was maximum under pentobarbital anesthesia (0.639+/-0.14; p=0.0018). Histologic examination of the spinal cord showed parenchymal ruptures and acute hemorrhage. Comparison of the pulmonary index with histologic slides of lung parenchyma revealed that relevant intra-alveolar edema occurred only for index values above 0.55. On electron microscopy, endothelial alterations, and damage of the alveolar lining cells were found. Our study indicates that neurogenic pulmonary edema caused by spinal cord injury is less pronounced in rats under xylazine-ketamine anesthesia, when compared with pentobarbital.

Early altered renal sodium handling determined by lithium clearance in spontaneously hypertensive rats (SHR): role of renal nerves.

The mechanism by which blood pressure rises in the SHR strain remains to be elucidated. Since the long-term changes in renal sodium tubule handling associated with genetic hypertension have not been examined in detail, we hypothesized that SHR hypertension development may result from sustained renal sympathetic nerve overactivity and consequently decreased urinary sodium excretion. To test this hypothesis, we assessed renal sodium handling and cumulative sodium balance for 10 consecutive weeks in unanesthetized renal-denervated SHR, performed prior to the start of the entire 10-week metabolic studies, and their age-matched normotensive and hypertensive controls. The present investigation shows that SHR excreted less sodium than Wistar-Kyoto (WKy) rats during the initial 3-week observation period (p <0.05). This tendency was reversed when SHR were 10-wk old. Fractional urinary sodium excretion (FENa+) was significantly lower in 3 and 6-wk-old SHR when compared with the WKy age-matched group, as follows: SHR3-wk-old: 0.33 +/- 0.09% and WKy3-wk-old: 0.75 +/- 0.1% (P <0.05); SHR(6-wk-old): 0.52 +/- 0.12% and WKy6-wk-old: 0.83 +/- 0.11%. The decreased FENa+ in young SHR was accompanied by a significant increase in proximal sodium reabsorption (FEPNa+) compared with the normotensive age-matched control group (P <0.01). This increase occurred despite unchanged creatinine clearance (CCr) and fractional post-proximal sodium excretion (FEPPNa+)in all groups studied. The decreased urinary sodium excretion response in SHR up to the age of 6 weeks was significantly eradicated by bilateral renal denervation of SHR3-wk-old: 0.33 +/- 0.09% and SHR6-wk-old: 0.52 +/- 0.12% to DxSHR 3-wk-old: 1.02 +/- 0.2% and DxSHR 6-wk-old: 0.94 +/- 0.2% (P <0.01), in renal denervated rats. The current data suggest that neural pathways may play an instrumental role on renal sodium reabsorption as result of sustained sympathetic nervous system overexcitability.

Hemodynamic parameters and neurogenic pulmonary edema following spinal cord injury: an experimental model.

Neurogenic pulmonary edema is a serious and always life-threatening complication following several lesions of the central nervous system. We report an experiment with 58 Wistar-Hanover adult male rats. Two groups were formed: control (n=4) and experimental (n=54). The experimental group sustained acute midthoracic spinal cord injury by Fogarty's balloon-compression technique containing 20 microL of saline for 5, 15, 30 or 60 seconds. The rats were anesthetized by intraperitoneal (i.p.) sodium pentobarbital (s.p.) 60 mg/Kg. The quantitative neurological outcome was presented at 4, 24 and 48 hours from compression to characterize the injury graduation in different groups. Poor outcome occurred with 60 seconds of compression. Six animals died suddenly with pulmonary edema. Using the procedure to investigate the pulmonary edema during 60 seconds of compression, followed by decompression and time-course of 60 seconds, 20 rats were randomly assigned to one of the following groups: control (1, n=4, anesthetized by i.p. s.p., 60 mg/Kg but without compression) and experimental (2, n=7, anesthetized by i.p. xylazine 10 mg/Kg and ketamine 75 mg/Kg) and (3, n=9, anesthetized by i.p. s.p., 60 mg/Kg). The pulmonary index (100 x wet lung weight/body weight) was 0.395 +/- 0.018 in control group, rose to 0.499 +/- 0.060 in group 2, and was 0.639 +/- 0.14 in group 3. Histologic examination of the spinal cord showed parenchymal ruptures and acute hemorrhage. Comparison of the pulmonary index with morphometric evaluation of edema fluid-filled alveoli by light microscopy showed that relevant intra-alveolar edema occurred only for index values above 0.55. The results suggest that the pulmonary edema induced by spinal compression is of neurogenic nature and that the type of anesthesia used might be important for the genesis of lung edema.

Impact of taurine supplementation on blood pressure in gestational protein-restricted offspring: Effect on the medial solitary tract nucleus cell numbers, angiotensin receptors, and renal sodium handling.

OBJECTIVE: The current study considers changes of the postnatal brainstem cell number and angiotensin receptors by maternal protein restriction (LP) and LP taurine supplementation (LPT), and its impact on arterial hypertension development in adult life. METHODS AND RESULTS: The brain tissue studies were performed by immunoblotting, immunohistochemistry, and isotropic fractionator analysis. The current study shows that elevated blood pressure associated with decreased fractional urinary sodium excretion (FENa) in adult LP offspring was reverted by diet taurine supplementation. Also, that 12-day-old LP pups present a reduction of 21% of brainstem neuron counts, and, immunohistochemistry demonstrates a decreased expression of type 1 angiotensin II receptors (AT1R) in the entire medial solitary tract nuclei (nTS) of 16-week-old LP rats compared to age-matched NP and LPT offspring. Conversely, the immunostained type 2 AngII (AT2R) receptors in 16-week-old LP nTS were unchanged. CONCLUSION: The present investigation shows a decreased FENa that occurs despite unchanged creatinine clearance. It is plausible to hypothesize an association of decreased postnatal nTS cell number, AT1R/AT2R ratio and FENa with the higher blood pressure levels found in taurine-deficient progeny (LP) compared with age-matched NP and LPT offspring.

Bothrops moojeni snake venom-induced renal glomeruli changes in rat.

The venom of Bothrops moojeni has potent proteolytic and phospholipase A2 activities. In previous work, we showed that intravenous injection of this venom in rats decreased creatinine clearance and caused tubular dysfunction and histopathological changes with no alterations in blood pressure. The current study used scanning and transmission electron microscopy to assess the ultrastructural changes caused by B. moojeni venom (0.4 mg/kg i.v.) in rat renal glomeruli and correlated these alterations with the severity of proteinuria 5 hours, 16 hours, and 48 hours after venom injection. The changes included mesangiolysis, glomerular microaneurysms, and glomerular basement membrane (GBM) abnormalities. In addition, there was a reduction in the number and width of podocyte pedicels, which caused a reduction in the number of filtration slits. Electron-dense amorphous material, which may be proteinaceous in origin, was found in the pedicels. The severity of the ultrastructural abnormalities correlated with the level of proteinuria. These morphophysiological changes were attributed to biochemical and physiological disturbances in the components of the GBM and mesangial matrix as well as in cytoskeleton-associated proteins of podocytic processes, and could account for the breakdown of optimal glomerular filtration barrier functioning. These results, together with the absence of appreciable glomerular fibrin deposits, support the hypothesis of a direct activity of B. moojeni venom on rat kidneys. Proteolytic activity of the venom on renal glomeruli could then contribute to the onset of acute renal failure, and would explain the clinical manifestations of renal injury after bites by this and other Bothrops species.

Effects of intracerebroventricular insulin microinjection on renal sodium handling in kidney-denervated rats.

The role of the central nervous system (CNS) in the control of hydrosaline homeostasis has been strikingly demonstrated by several studies. Growing evidence suggests that insulin may exert an influence in the modulation of many brain functions. However, there are no available data examining the CNS effect of insulin injection on renal sodium handling. Also, to examine the influence of renal nerve activity during i.c.v. administration of insulin, unanesthetized, unrestrained rats were randomly assigned to one of nine separated groups: (a) sham-operated i.c.v. 0.15 M NaCl-injected (Co, pooled data, n = 37) and sham-operated i.c.v. 0.42 ng. microl(-1) (n = 12), 4.2 ng.microl(-1) (n = 10) and 42.0 ng.microl(-1) (n = 11) insulin-injected rats (In); (b) renal-denervated i.c.v. 0.15 M NaCl (Co(Dx), n = 5), and insulin-injected rats (In(Dx), n = 5); and (c) subcutaneously insulin-injected rats (SC, n = 5). We showed that centrally administered insulin produced dose-related increased urinary output of sodium [Co: 855 +/- 85 Delta% min, 0.42 ng.microl(-1) In: 1189 +/- 308 Delta% min, 4.2 ng.microl(-1) In: 1461 +/- 594 Delta% min (p = 0.048), and 42.0 ng.microl(-1) In: 2055 +/- 411 Delta% min (p = 0.0001)], and dose-independently increased potassium excretion [Co: 460 +/- 28 Delta% min, 0.42 ng.microl(-1) In: 649 +/- 100 Delta% min (p = 0.016), 4.2 ng.microl(-1) In: 671 +/- 175 Delta% min (p = 0.003), and 42.0 ng.microl(-1) In: 669 +/- 70 Delta% min (p = 0.002)] compared to control. The urinary sodium excretion response to i.c.v. 42 ng.microl(-1) insulin injections were abolished by bilateral renal denervation. In addition, we showed that insulin-induced natriuresis occurred by increasing postproximal tubule sodium rejection (FEPP(Na)), and changed glomerular filtration rate (C(Cr)) at 42.0 ng.microl(-1) (p = 0.023) i.c.v. insulin microinjection but not at smaller insulin dose. The current data suggests that a blunted efferent insulin-sensitive nerve activity from periventricular region may contribute to the inability of renal tubules to handle the hydroelectrolyte balance.

Acute intracerebroventricular insulin microinjection after nitric oxide synthase inhibition of renal sodium handling in rats.

The role of the central nervous system (CNS) in the control of hydrosaline homeostasis has been strikingly demonstrated by several studies. Recent and growing evidence suggests that insulin or a nonapeptide-derived from the C-terminus of the insulin beta-chain may influence many brain functions. However, there is little information on the insulin-activated neural pathways regulating urinary sodium excretion. Also, we examined the influence of nitric oxide synthase activity by chronic oral administration of N(omega)-nitro-l-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, after previous i.c.v. administration of insulin to unanesthetized, unrestrained rats that were randomly assigned to one of seven separated groups: (a) i.c.v. 0.15 M NaCl-injected (n = 11) and i.c.v. 126 ng (n = 11) insulin-injected rats; (b) i.c.v. insulin-injected in systemic L-NAME-treated (n = 10) and vehicle-treated insulin-injected rats (n = 10); and (c) subcutaneously (SC) insulin-injected rats (n = 5). We showed that centrally administered insulin produced increase in the urinary output of sodium (from 0.15 M NaCl: 855.6 +/- 85.1 Delta%.min(-1) to 126 ng insulin: 2055 +/- 310.6 Delta%.min(-1)) and potassium (126 ng: from 0.15 M NaCl: 460.4 +/- 100 Delta%.min(-1) to 126 ng insulin: 669 +/- 60.8 Delta%.min(-1)). The urinary sodium excretion response to i.c.v. 126 ng insulin microinjection was significantly abolished by previous systemic treatment of animals with 15 mg/kg/day L-NAME (from vehicle + 126 ng insulin: 1935 +/- 258.3 Delta%. min(-1) to L-NAME + 126 ng insulin: 582.3 +/- 69.6 Delta%. min(-1)). In addition, we showed that insulin-induced natriuresis occurred by increasing post-proximal tubule sodium rejection (FEPP(Na)), despite an unchanged glomerular filtration rate (C(Cr)). The current data suggests the novel concept that CNS NO-dependent neural pathways may play an instrumental role on efferent insulin-sensitive nerve activity from periventricular region. Speculatively, it seems interesting to suggest that perhaps one of the efferent signals triggered by insulin in the CNS may be nitrergic in nature, and that defects in this efferent signal could result in insulin central resistance, inability of renal tubules to handle the hydro electrolyte balance and hypertension.