Acidotic Rats Research Articles

Renal Pathophysiological Changes During Chronic Metabolic Acidosis Associated Increased Blood Pressure

Activation of renal angiotensin II works synergistically with renal superoxides to promote an increase in blood pressure during chronic acidotic conditions has been previously delineated. Uncontrolled high blood pressure instigates renal pathophysiological function and eventual end organ damage is evident. This study aims to investigate the renal consequences of increased blood pressure provoked during chronic metabolic acidosis (CMA). It was a challenging prospect, as both hypertension and chronic metabolic acidosis are two different pathological conditions and can exist independent of each other. Renal functions in rats grouped in 3 groups; i) Control (nonacidotic and normotensive), ii) CMA (acidotic and hypertensive), iii) CMA+captopril+tempol (acidotic and normotensive) were critically assessed by measuring Glomerular Filtration Rate (GFR), Fractional excretion of sodium (FENa), urine flow rate, weight of the kidneys and albuminuria. Picro‐sirius staining of the cortical sections was performed to examine the collagen content as an index of fibrosis. The results demonstrated significant attenuation of GFR in the CMA group compared to the control group. However, GFR in captopril+tempol treated acidotic rats is significantly increased compared to the CMA groups, which suggested that the reduced GFR in CMA group is because the rats are hypertensive. Conversely, the FENa was significantly increased in the CMA group compared to the control but the captopril+tempol treated group showed a significant decrease in FENa compared to the CMA group, this implied that hypertension but not acidosis is the cause of increased FENa which increases blood flow to the kidney potentially leading to the pressure natriuresis and could potentially lead to kidney damage. The urinary albumin excretion was also significantly elevated in acidotic rats as compared to control but the captopril+tempol treated group showed a significant reduction in albuminuria, which revealed the acidosis‐induced hypertension is contributing for impaired renal infiltration and augmenting the albuminuria. The morphological analysis of renal changes using Picro‐sirius staining revealed significant structural damage in the glomeruli of chronically acidotic rats. However, when these rats were treated with captopril and tempol combined, the glomeruli were intact and a reduction in fibrosis was observed. Overall, our results suggested the renal consequences of acidosis induced hypertension can lead to eventual end organ damage which is independent to the function of acidosis. Hence, intrarenal Ang II and superoxide release in the kidney could be a potential target to reduce the blood pressure, albuminuria, and protection of renal function and delayed the onset of end‐stage renal disease associated with acidosis evoked hypertension.

Chronic Metabolic Acidosis Elicits Hypertension via Upregulation of Intrarenal Angiotensin II and Induction of Oxidative Stress

Chronic metabolic acidosis (CMA) can be a consequence of persistent hypertension but could potentially play a role in invoking hypertension. Currently, there is a scarcity of studies examining the outcome of induced chronic acidosis on blood pressure regulation. This study investigates CMA as a cause of hypertension. Chronic acidosis was induced in Sprague Dawley rats (100–150 g) by providing a weak acid solution of 0.28 M ammonium chloride (NH4Cl) in tap water for 8 weeks. To determine whether the rats were acidotic, blood pH was measured, while blood pressure (BP) was monitored by tail-cuff plethysmography weekly. Rats were divided into five groups: control, CMA, CMA ± spironolactone, captopril, and tempol. Serum sodium and potassium; renal interstitial fluid (for Angiotensin II concentration); and kidney proximal tubules (for Na+/K+ ATPase- α1 concentration) were analyzed. Reactive oxygen species (ROS) were detected in renal cortical homogenates using electron paramagnetic resonance (EPR). In the CMA rats, a sustained elevation in mean arterial pressure (MAP) associated with a significant decrease in blood pH was observed compared to that of control over the 8 weeks. A significant decrease in MAP was observed in acidotic rats treated with captopril/tempol, whereas spironolactone treatment caused no decrease in MAP as compared to that of the CMA group. The interstitial angiotensin II was increased in the CMA group but decreased in the CMA with captopril and tempol groups. In addition, the urinary sodium was decreased, and the serum sodium levels increased significantly in the CMA groups as compared to that of control. However, the acidotic groups with captopril and tempol showed reduced levels of serum sodium and an elevation in urinary sodium as compared to that of the CMA group. In addition, there was a significant increase in plasma renin and no change in plasma aldosterone in the CMA group with no significant differences in plasma renin or aldosterone observed during spironolactone, captopril, or tempol treatments. The increased expression of Na+/K+ ATPase-α1 in the CMA group suggests that active transport of Na+ to the blood could be causative of the observed hypertension. Furthermore, the EPR analysis confirmed an elevation in superoxide (O2-) radical levels in the CMA group, but the tempol/captopril treated acidotic groups showed less (O2-) compared to that of either the CMA group or control. Taken together, our data suggest that induction of CMA could potentially be causative of hypertension, while the mechanisms underlying the increased BP could be through the activation of intrarenal Ang II and induction of oxidative stress.

Phosphoenolpyruvate carboxykinase in urine exosomes reflect impairment in renal gluconeogenesis in early insulin resistance and diabetes.

Impaired insulin-induced suppression of renal gluconeogenesis could be a risk for hyperglycemia. Diabetes is associated with elevated renal gluconeogenesis; however, its regulation in early insulin resistance is unclear in humans. A noninvasive marker of renal gluconeogenesis would be helpful. Here, we show that human urine exosomes (uE) contain three gluconeogenic enzymes: phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-bisphosphatase, and glucose 6-phosphatase. Their protein levels were positively associated with whole body insulin sensitivity. PEPCK protein in uE exhibited a meal-induced suppression. However, subjects with lower insulin sensitivity had blunted meal-induced suppression. Also, uE from subjects with prediabetes and diabetic rats had higher PEPCK relative to nondiabetic controls. Moreover, uE-PEPCK was higher in drug-naïve subjects with diabetes relative to drug-treated subjects with diabetes. To determine whether increased renal gluconeogenesis is associated with hyperglycemia or PEPCK expression in uE, acidosis was induced in rats by 0.28 M NH4Cl with 0.5% sucrose in drinking water. Control rats were maintained on 0.5% sucrose. At the seventh day posttreatment, gluconeogenic enzyme activity in the kidneys, but not in the liver, was higher in acidotic rats. These rats had elevated PEPCK in their uE and a significant rise in blood glucose relative to controls. The induction of gluconeogenesis in human proximal tubule cells increased PEPCK expression in both human proximal tubules and human proximal tubule-secreted exosomes in the media. Overall, gluconeogenic enzymes are detectable in human uE. Elevated PEPCK and its blunted meal-induced suppression in human urine exosomes are associated with diabetes and early insulin resistance.

Localization and Regulation of the Disease‐Causing H+‐ATPase B1 Subunit in the Early Distal Nephron of Human and Murine Kidney

The H+‐ATPase B1 subunit is abundantly expressed in intercalated cells (IC) of the collecting system. Loss of function mutations in the gene results in distal renal tubular acidosis, due to insufficient acid secretion into urine. Curiously, mutations in the disease‐causing H+‐ATPase subunits has been reported to cause more severe forms of distal renal tubular acidosis, as compared to dominant mutations in AE1. Using 2 antibodies directed against the N‐terminal domain of the B1 subunit, we found abundant immunoreactivity in human kidney. As expected IC showed strongest signal, but significant signal was also observed in apical membrane aspects of the early distal nephron. Colocalization using known cellular markers confirmed expression of the B1 subunit in NCC positive distal convoluted tubules and NKCC2 positive cortical thick ascending limbs, but much less abundant in ENaC positive connecting tubule cells. Further immunohistochemical evaluation using an antibody directed against the other disease causing H+‐ATPase subunit A4, also showed localization to segments in the early distal nephron in human kidney. Expression was also detectable in macula densa for both subunits. In mouse and rat, B1 localization was investigated using antibodies directed against the N‐ or C‐terminal domains of the B1 subunit. Here subunit expression could be detected in apical membrane domains, beginning from the medullary thick ascending limb and stretching to the distal convoluted segment 2, in addition to the previously described IC. Using mice lacking the B1 subunit to validate the antibodies, complete loss of antigenicity was observed for two antibodies, while one showed minor staining in connecting tubules and IC, likely representing overlap with the B2 subunit epitope. To address whether the B1 subunit was subjected to regulation in the distal tubules, tissue from rats subjected to NH4Cl‐ and hypercapnia‐induced acidosis or NaHCO3‐ induced alkalosis were studied by immunohistochemistry. A mildly increased expression of the B1 subunit was noted in early distal nephron segments in kidneys of NH4Cl‐induced acidotic rats. In contrast, no change in expression could be observed in hypercapnia‐induced acidosis or NaHCO3‐ induced alkalosis. In conclusion, the B1 subunit is expressed in the early distal nephron in humans and rodents. The physiological consequences of H+‐ATPase expression in these segments remain to be delineated in detail.Support or Funding InformationThis work was funded by grants from the Novo Nordisk Foundation, the Carlsberg Foundation, the A.P. Møller Foundation, and the Lundbeck foundation.

The Sodium-Bicarbonate Cotransporter 1 (NBC1) and Monocarboxylate Transporter 1 (MCT1) in the Rat Kidney are Regulated in Response to Metabolic Acidosis

The present work was designed to study the effect of metabolic acidosis induced by ammonium chloride on the expression of sodium-bicarbonate cotransporter 1 (NBC1) and Monocarboxylate transporter 1 (MCT1) in rat kidney. Following the treatment of NH4Cl, blood pH and bicarbonate concentration were significantly decreased, while significant increase in chloride concentration was developed. Moreover, urine analysis revealed decrease in urine pH and significant increase in ammonia in acidotic rats. The protein expression of NBC1 and MCT1 was determined in the rat kidney by immunofluorescence confocal laser-scanning microscopy and Western blotting analyses. In the experimental group, the expression level of NBC1 was significantly increased in the renal proximal tubules, whereas, the expression of MCT1 was significantly decreased. Western blotting data confirmed the immunofluorescence confocal laser-scanning microscopy. In conclusion, metabolic acidosis causes adaptive increases in the basolateral NBC1 in the rat renal proximal tubule that are likely responsible for the increased ability of the proximal tubule to reabsorb filtered bicarbonates. Additionally, acidosis leads to decrease the MCT1 protein expression level in renal proximal tubule that may explain the increase in lactate excretion in the urine of acidotic rats due to the reduction in lactate co-transport.

Clinical role of dietary thiamine on regulation of renal response to metabolic acidosis in adult rats

Kidney plays a central role in maintaining the composition of body fluids by regulating water, NaCl, acid base, and solute reabsorption and excretion, respectively. The study was done to investigate the physiological role of thiamine in regulation of renal response to metabolic acidosis induced by NH 4 Cl in adult male rats. For this experiment, fifty rats were used. They were divided into five groups. Control rats received basal diet; rats fed on basal diet mixed with NH 4 Cl (4g NH 4 Cl/100g diet) to induce severe metabolic acidosis, rats fed on basal supplemented diet with thiamine (600 mg/kg diet), and rats fed on basal supplemented diet with thiamine before and after induction of metabolic acidosis by NH 4 Cl for 14 days. The results showed that the plasma levels of chloride, urea, and creatinine were significantly elevated in metabolic acidosis induced by NH 4 Cl. Thiamine supplementation at high dose before or after induction improved the chloride values. Feeding diets supplemented with thiamine modulated the plasma sodium and bicarbonate values. Supplementation with vitamin B1 as preventive agent significantly restored these changes to near control value and when used as curative agent improved plasma creatinine and urea levels. Urinary pH and potassium levels were decreased significantly in metabolic acidotic rats when compared to all experimental groups. Urinary ammonia and aldosterone levels were decreased by thiamine supplementation as protective agent. Supplementation with vitamin B1 as preventive and curative agents, restored the affected parameters and regulate the response of kidney to metabolic acidosis induced by ammonium chloride.

Acute Acidosis Attenuates Leucine Stimulated Signal Transduction and Protein Synthesis in Rat Skeletal Muscle

Background: Critical illnesses are often complicated by acute metabolic acidosis, which if persistent, adversely affects outcome. Among the harmful effects that it might cause are impaired utilization of nutrients, increased proteolysis and depressed protein synthesis, leading to muscle wasting. As the amino acid leucine stimulates protein synthesis by activating mTOR signaling, we explored whether in acidosis, impaired leucine-stimulated signaling might be a contributor to the depressed protein synthesis. Methods: Male pair-fed rats were gavaged with NH₄Cl (acidosis) or NaCl (control) for 2 days and then gavaged once with leucine and sacrificed 45 min later. Extensor digitorum longus muscles were isolated, incubated with or without leucine and protein synthesis measured. The anterior tibial muscle signaling was analysed by Western immunobloting. Results: Despite pair-feeding, acidotic rats lost body and muscle weight vs. controls. Moreover, leucine-induced protein synthesis in isolated muscle from acidotic rats was impaired. In-vivo, 45 min after an oral leucine load, anterior tibial muscle mTOR and 4E-BP1 phosphorylation increased significantly and comparably in control and acidotic rats. In contrast, leucine-stimulated phosphorylation of S6K1, a regulator of translation initiation and protein synthesis, was attenuated to approximately 56% of the control value (p < 0.05). Conclusion: This study reveals that an acute metabolic acidosis impairs leucine-stimulated protein synthesis and activation of signaling downstream of mTOR at the level of S6K1. We propose that this S6K1abnormality may account in part, for the resistance to leucine-stimulated muscle protein synthesis, and may thereby contribute to the impaired nutrient utilization and ultimately the muscle wasting that develops in acidosis.

Medullary GABAergic mechanisms contribute to electroacupuncture modulation of cardiovascular depressor responses during gastric distention in rats

Electroacupuncture (EA) at P5-P6 acupoints overlying the median nerves typically reduces sympathoexcitatory blood pressure (BP) reflex responses in eucapnic rats. Gastric distention in hypercapnic acidotic rats, by activating both vagal and sympathetic afferents, decreases heart rate (HR) and BP through actions in the rostral ventrolateral medulla (rVLM) and nucleus ambiguus (NAmb), leading to sympathetic withdrawal and parasympathetic activation, respectively. A GABAA mechanism in the rVLM mediates the decreased sympathetic outflow. The present study investigated the hypothesis that EA modulates gastric distention-induced hemodynamic depressor and bradycardia responses through nuclei that process parasympathetic and sympathetic outflow. Anesthetized hypercapnic acidotic rats manifested repeatable decreases in BP and HR with gastric distention every 10 min. Bilateral EA at P5-P6 for 30 min reversed the hypotensive response from -26 ± 3 to -6 ± 1 mmHg and the bradycardia from -35 ± 11 to -10 ± 3 beats/min for a period that lasted more than 70 min. Immunohistochemistry and in situ hybridization to detect c-Fos protein and GAD 67 mRNA expression showed that GABAergic caudal ventral lateral medulla (cVLM) neurons were activated by EA. Glutamatergic antagonism of cVLM neurons with kynurenic acid reversed the actions of EA. Gabazine used to block GABAA receptors microinjected into the rVLM or cVLM reversed EA's action on both the reflex depressor and bradycardia responses. EA modulation of the decreased HR was inhibited by microinjection of gabazine into the NAmb. Thus, EA through GABAA receptor mechanisms in the rVLM, cVLM, and NAmb modulates gastric distention-induced reflex sympathoinhibition and vagal excitation.

Response of the mitochondrial proteome of rat renal proximal convoluted tubules to chronic metabolic acidosis

Metabolic acidosis is a common clinical condition that is caused by a decrease in blood pH and bicarbonate concentration. Increased extraction and mitochondrial catabolism of plasma glutamine within the renal proximal convoluted tubule generates ammonium and bicarbonate ions that facilitate the excretion of acid and partially restore acid-base balance. Previous studies identified only a few mitochondrial proteins, including two key enzymes of glutamine metabolism, which are increased during chronic acidosis. A workflow was developed to characterize the mitochondrial proteome of the proximal convoluted tubule. Based upon the increase in specific activity of cytochrome c oxidase, the isolated mitochondria were enriched eightfold. Two-dimensional liquid chromatography coupled with mass spectrometry was utilized to compare mitochondrial-enriched samples from control and chronic acidotic rats. Proteomic analysis identified 901 proteins in the control and acidotic samples. Further analysis identified 37 peptides that contain an N-ε-acetyl-lysine; of these, 22 are novel sites. Spectral counting analysis revealed 33 proteins that are significantly altered in abundance in response to chronic metabolic acidosis. Western blot analysis was performed to validate the calculated changes in abundance. Thus the current study represents the first comprehensive analysis of the mitochondrial proteome of the rat renal proximal convoluted tubule and its response to metabolic acidosis.

P01.24. Central processing by electroacupuncture of vasodepression and bradycardia reflex responses

Purpose Electroacupuncture (EA) at P5-6 reduces sympathoexcitatory blood pressure (BP) reflex responses. Gammaaminobutyric acid (GABA) receptors in the brainstem region rostral ventral lateral medulla (rVLM) contribute to modulation of sympathoexcitatory visceral reflexes during EA. Gastric distension in hypercapnic acidotic rats, by activating both sympathetic and vagal afferents, decreases BP and heart rate (HR) through a GABAA mechanism in the rVLM. This study investigated the hypothesis that EA modulates gastric induced hemodynamic depressor responses through actions in nuclei that process both sympathetic and parasympathetic outflow. Methods Anesthetized and hypercapnic acidotic induced rats were used to examine the central processing of the actions of EA. An unstressed 2-cm diameter latex balloon attached to a polyurethane tube was inserted into the stomach through the mouth and esophagus to induce gastric distention. Acupuncture needles were placed near the wrist at P5-6 acupoints for 30-min EA (2 Hz, 0.2-0.4 mA, 0.5 ms). Results We observed repeatable decreases in BP and HR with gastric distention every 10 min. Bilateral EA at P5-6 for 30 min reversed the hypotensive response from -26±3 to -6±1 mmHg and the bradycardia from -35±11 to -10 ±3 beats/min for over 70 min. EA’s action on decreased BP and HR, respectively, was inhibited by microinjection of gabazine, a GABAA receptor antagonist, into the caudal–VLM (cVLM) or the nucleus ambiguus (NAmb). Gabazine microinjected into the rVLM reversed EA action on both depressor and bradycardia responses. Conclusion Thus, EA through GABAA receptor mechanisms modulates reflex sympathoinhibition and vagal excitation leading to cardiovascular depression through actions in the rVLM, cVLM and NAmb. These data indicate that EA can normalize elevated and depressed blood pressure.

CENTRAL PROCESSING BY ELECTROACUPUNCTURE OF CARDIOVASCULAR REFLEX VASODEPRESSION

Electroacupuncture (EA) at P5–6 reduces sympathoexcitatory blood pressure (BP) reflex responses. Gamma‐aminobutyric acid (GABA) receptors in the rostral ventral lateral medulla (rVLM) contribute to modulation of sympathoexcitatory visceral reflexes during EA. Gastric distension (GD) in hypercapnic acidotic (HA) rats, by activating both sympathetic and vagal afferents, decreases BP and heart rate (HR) through a GABAA mechanism in the rVLM. This study investigated the hypothesis that EA modulates GD‐induced hemodynamic depressor responses through actions in nuclei that process both sympathetic and parasympathetic outflow. Anesthetized and HA‐induced rats showed repeatable decreases in BP and HR with distension of the stomcah every 10 min. Bilateral EA at P5–6 for 30 min reversed the hypotensive response from − 26±3 to −6±1 mmHg and the bradycardia from −35±11 to −10±3 beats/min for over 70 min. EA's action on decreased BP and HR, respectively, was inhibited by microinjection of gabazine into the caudal–VLM (cVLM) or the nucleus ambiguus (NAmb). Gabazine microinjected into the rVLM reversed EA action on both depressor and bradycardia responses. Thus, EA through GABAA receptor mechanisms modulates reflex sympathoinhibition and vagal excitation leading to cardiovascular depression through actions in the rVLM, cVLM and NAmb. These data indicate that EA can normalize elevated and depressed blood pressure.

Proteomic profiling of the effect of metabolic acidosis on the apical membrane of the proximal convoluted tubule

The physiological response to the onset of metabolic acidosis requires pronounced changes in renal gene expression. Adaptations within the proximal convoluted tubule support the increased extraction of plasma glutamine and the increased synthesis and transport of glucose and of NH(4)(+) and HCO(3)(-) ions. Many of these adaptations involve proteins associated with the apical membrane. To quantify the temporal changes in these proteins, proteomic profiling was performed using brush-border membrane vesicles isolated from proximal convoluted tubules (BBMV(PCT)) that were purified from normal and acidotic rats. This preparation is essentially free of contaminating apical membranes from other renal cortical cells. The analysis identified 298 proteins, 26% of which contained one or more transmembrane domains. Spectral counts were used to assess changes in protein abundance. The onset of acidosis produced a twofold, but transient, increase in the Na(+)-dependent glucose transporter and a more gradual, but sustained, increase (3-fold) in the Na(+)-dependent lactate transporter. These changes were associated with the loss of glycolytic and gluconeogenic enzymes that are contained in the BBMV(PCT) isolated from normal rats. In addition, the levels of γ-glutamyltranspeptidase increased twofold, while transporters that participate in the uptake of neutral amino acids, including glutamine, were decreased. These changes could facilitate the deamidation of glutamine within the tubular lumen. Finally, pronounced increases were also observed in the levels of DAB2 (3-fold) and myosin 9 (7-fold), proteins that may participate in endocytosis of apical membrane proteins. Western blot analysis and accurate mass and time analyses were used to validate the spectral counting.

Acute and chronic acidosis influence on antioxidant equipment and transport proteins of rat jejunal enterocyte

Acidosis elicits the formation of oxidants and, in turn, ROS (reactive oxygen species)-induced intestinal diseases cause acidosis. This research investigated whether both acute and chronic acidosis influence the antioxidant enzymatic equipment of rat jejunocyte, including γ-GT activity, involved in GSH (glutathione) homoeostasis. Lipid peroxidation level and the expressions of (Na+, K+)-ATPase and GLUT2 were also investigated. The possible influence of acidosis on ROS action was tested. Isolated apical membranes, everted sac preparations and homogenates from acidotic rats were used. γ-GT activity is inhibited after incubation of isolated membranes at acidic pH, but using the whole intestinal tract this inhibition disappears, while SOD (superoxide dismutase) and GR (glutathione reductase) activities are enhanced. Also, in conditions of chronic acidosis, γ-GT activity is unaffected, but no variations of antioxidant activities are apparent. (Na+, K+)-ATPase expression increases, while GLUT2 decreases in acidotic animals. Lipid peroxidation level is unaffected by acidosis. H2O2 inhibits γ-GT activity only in isolated membranes; in the whole tissue, it enhances CAT (catalase) and SOD activities and reduces GLUT2 expression. The pattern of responses to oxidant agents is unaffected by acidosis. Although jejunum seems quite resistant to acidosis, results, suggesting specific responses to this condition, may direct further research on antioxidant supplementation.

The sodium-driven chloride/bicarbonate exchanger NDCBE in rat brain is upregulated by chronic metabolic acidosis

Acid extruders in neurons prohibit intracellular pH from falling very far below normal. Our recent report suggests that the acid-extruding sodium/bicarbonate transporter NBCn1 (Slc4a7) in rat brain is upregulated by chronic metabolic acidosis. In this study, we examined whether the Na +-driven Cl/HCO 3 exchanger NDCBE (Slc4a8) is also upregulated by similar systemic acid loads. Immunoblot revealed NDCBE protein (130 kDa) expressed in a variety of rat brain regions. In the hippocampus, NDCBE was localized to CA1–CA4 pyramidal neurons and dentate gyrus granular neurons determined by immunoperoxidase immunohistochemistry. The staining was dispersed in cell bodies and dendrites. NDCBE protein expression was then compared between rats in chronic metabolic acidosis and control rats. Immunoblot of crude plasma membrane fractions from the hippocampus showed a slight increase in NDCBE in acidotic rats ( p = 0.05). However, the expression in CA3 pyramidal neurons was significantly increased, determined by immunohistochemistry and quantitative analysis. The increase was also observed in other neurons including entorhinal cortical neurons, posterior cortical neurons, and outer stellate cells in cerebellum. The staining in choroid plexus epithelia was unaffected by chronic metabolic acidosis. These data demonstrate that the Na +-driven Cl/HCO 3 exchanger NDCBE is upregulated by chronic acid loads in a cell-specific manner.

Neuronal expression of sodium/bicarbonate cotransporter NBCn1 (SLC4A7) and its response to chronic metabolic acidosis.

The sodium-bicarbonate cotransporter NBCn1 (SLC4A7) is an acid-base transporter that normally moves Na(+) and HCO(3)(-) into the cell. This membrane protein is sensitive to cellular and systemic pH changes. We examined NBCn1 expression and localization in the brain and its response to chronic metabolic acidosis. Two new NBCn1 antibodies were generated by immunizing a rabbit and a guinea pig. The antibodies stained neurons in a variety of rat brain regions, including hippocampal pyramidal neurons, dentate gyrus granular neurons, posterior cortical neurons, and cerebellar Purkinje neurons. Choroid plexus epithelia were also stained. Double immunofluorescence labeling showed that NBCn1 and the postsynaptic density protein PSD-95 were found in the same hippocampal CA3 neurons and partially colocalized in dendrites. PSD-95 was pulled down from rat brain lysates with the GST/NBCn1 fusion protein and was also coimmunoprecipitated with NBCn1. Chronic metabolic acidosis was induced by feeding rats with normal chow or 0.4 M HCl-containing chow for 7 days. Real-time PCR and immunoblot showed upregulation of NBCn1 mRNA and protein in the hippocampus of acidotic rats. NBCn1 immunostaining was enhanced in CA3 neurons, posterior cortical neurons, and cerebellar granular cells. Intraperitoneal administration of N-methyl-d-aspartate caused neuronal death determined by caspase-3 activity, and this effect was more severe in acidotic rats. Administering N-methyl-d-aspartate also inhibited NBCn1 upregulation in acidotic rats. We conclude that NBCn1 in neurons is upregulated by chronic acid loads, and this upregulation is associated with glutamate excitotoxicity.

Effects of NH4Cl-induced systemic metabolic acidosis on kidney mitochondrial coupling and calcium transport in rats

We have previously shown that chronic metabolic acidosis, induced in rats by NH(4)Cl feeding, leads to nephron hypertrophy and to a decreased water-salt reabsorption by the kidneys. Since mitochondria are the main source of metabolic energy that drives ion transport in kidney tubules, we examined energy-linked functions (respiration, electrochemical membrane potential and coupling between respiration and ADP phosphorylation) in mitochondria isolated from rat kidney and liver at 48 h after metabolic acidosis induced by NH(4)Cl. Mitochondria isolated from the kidneys and liver of metabolic acidotic rats, induced by NH(4)Cl, was used to study of the oxygen consumption by Clark-type electrode, mitochondrial electrical transmembrane potential estimated by the safranine O method and the variations in free medium Ca(2+) concentrations examined by absorbance spectrum of Arsenazo III set at the 675-685 nm wavelength pair. Whole kidney and liver mitochondria isolated from 48 h acidotic rats presented higher resting respiration, lower respiratory control and a lower ADP/O ratio than controls. These differences in mitochondrial coupling, between respiration and oxidative phosphorylation (ATP synthesis), were totally corrected when experiments were carried out in the presence of carboxyatractyloside, GDP and BSA, indicating that mitochondrial uncoupling proteins are more active in acidotic rat kidneys. Interestingly, determination of Ca(2+) transport demonstrated a faster rate of initial Ca(2+) uptake by acidotic kidney mitochondria, which resulted in a lower concentration of extra-mitochondrial Ca(2+) under steady-state conditions (Ca(2+) set point) when compared with control mitochondria. In contrast, there were no significant differences in the rates of Na(+) or ruthenium red induced Ca(2+) efflux. We suggest that the mild uncoupling and higher Ca(2+) accumulation represents an adaptation of the mitochondria to cope with conditions of oxidative stress and high cytosolic Ca(2+), which are associated with a decreased efficiency of oxidative phosphorylation that may explain, at least in part, the striking natriuresis observed under chronic acidosis. Finally, there were no changes in Ca(2+) transport or coupling in liver mitochondria isolated from the acidotic rats.

Chronic metabolic acidosis upregulated claudin mRNA expression in the duodenal enterocytes of female rats

Previous investigations showed that chronic metabolic acidosis (CMA) increased the paracellular permeability of ion and neutral hydrophilic molecules in the duodenum of rats and small intestinal-like cell lines. Since proteins of the claudin family have been known to regulate the paracellular transport in several epithelia, an increase in the paracellular permeability during CMA may have resulted from changes in the pattern of claudin expression. The present study aimed to investigate the expression profile of 22 claudins in the duodenum of female Sprague–Dawley rats given 1.5% NH 4Cl for 21 days to induce CMA. Arterial blood gas analysis revealed plasma pH values of 7.40 in normal rats and 7.31 in acidotic rats. Blood chemistry showed increases in the total plasma calcium, free-ionized calcium and magnesium, indicating a typical adaptive response of animals to CMA. RT-PCR demonstrated mRNA expressions of claudin-1 to -12, -14, -15, -17 to -20, -22 and -23 in duodenum of normal rats. Claudin-16 was not expressed in normal duodenum, but was strongly expressed in the kidney. Claudin-13 expression was seen only in the cecum, colon, liver and kidney of mice. After 21-day CMA, mRNA expressions of claudin-2, -3, -6, -8, -11, -12, -14, -19 and -22 were significantly enhanced, whereas expressions of other claudins were not changed. Confocal laser-scanning microscopy demonstrated that duodenal enterocytes of normal rats expressed claudin-3 protein on the paracellular membrane. The distribution of claudin-3 protein along the paracellular membrane was markedly increased in CMA, especially near the apical surface. Our results, therefore, provided novel evidence that 21-day CMA markedly altered claudin profile in the duodenum of rats by upregulating specific claudin expression.

Proteomic analysis of the adaptive response of rat renal proximal tubules to metabolic acidosis

Proximal tubules were isolated from control and acidotic rats by collagenase digestion and Percoll density gradient centrifugation. Western blot analysis indicated that the tubules were approximately 95% pure. The samples were analyzed by two-dimensional difference gel electrophoresis (DIGE) and DeCyder software was used to quantify the temporal changes in proteins that exhibit enhanced or reduced expression. The mass-to-charge ratios and the amino acid sequences of the recovered tryptic peptides were determined by MALDI-TOF/TOF mass spectrometry and the proteins were identified using Mascot software. This analysis confirmed the well-characterized adaptive responses in glutaminase (GA), glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxykinase (PEPCK). This approach also identified 17 previously unrecognized proteins that are increased with ratios of 1.5 to 5.6 and 16 proteins that are decreased with ratios of 0.67 to 0.03 when tubules from 7-day acidotic vs. control rats were compared. Some of these changes were confirmed by Western blot analysis. Temporal studies identified proteins that were induced either with rapid kinetics similar to PEPCK or with more gradual profiles similar to GA and GDH. All of the mRNAs that encode the latter proteins contain an AU sequence that is homologous to the pH response element found in GA mRNA. Thus selective mRNA stabilization may be a predominant mechanism by which protein expression is increased in response to acidosis.

Downregulation in the expression of the serine dehydratase in the rat liver during chronic metabolic acidosis

Blood pH controls the activity of important regulatory enzymes in the metabolism. Serine dehydratase (SerDH) transforms l-serine into pyruvate and ammonium and is involved in the regulation of gluconeogenesis from serine in the rat liver. In this work, we investigate the effect of chronic metabolic acidosis on the kinetics, specific protein level, tissue location, and mRNA levels of rat liver SerDH. Experimental acidosis was induced in rats by ingestion of 0.28 M ammonium chloride solution for 10 days. Acidosis significantly (P<0.05) decreased SerDH activity at all substrate concentrations assayed. Moreover, the Vmax value was 38.50+/-3.51 mU/mg (n=7) of mitochondrial protein in the acidotic rats and 92.49+/-6.79 mU/mg (n=7) in the control rats. Western blot analysis revealed a significant reduction (14%) in the level of SerDH protein content in the rat liver during acidosis. Immunohistochemical analysis showed that SerDH location did not change in response to chronic metabolic acidosis and confirmed previous results on SerDH protein levels. Moreover, the SerDH mRNA level, estimated by RT-PCR, was also significantly 33.8% lower than in control. These results suggest that during experimental acidosis a specific repression of rat-liver SerDH gene transcription could result, lowering the amount and activity of this enzyme. The changes found in SerDH expression are part of an overall metabolic response of liver to maintain acid-base homeostasis during acidosis.

Excess Casein in the Diet Is Not the Unique Cause of Low-Grade Metabolic Acidosis: Role of a Deficit in Potassium Citrate in a Rat Model

This study examined the effects of a dietary model of protein excess and K anion salt deficit on the occurrence of metabolic acidosis in rat. Rats were adapted to diets containing either 13 or 26% casein, together with mineral imbalance, through lowering K/increasing sodium/omitting alkalinizing anions. For each protein level, a group of rats was supplemented with K citrate. Dietary K citrate resulted in neutral urinary pH, whatever the protein level. Urea excretion was higher in rats adapted to 26% casein than 13% casein diets, but K citrate enhanced this excretion and suppressed ammonium elimination. No citraturia could be observed in acidotic rats, whereas K citrate greatly stimulated citraturia and 2-ketoglutarate excretion. In conclusion, low-grade metabolic acidosis can occur with a moderate protein level in the diet. K citrate was apparently less effective in rats adapted to the 26% casein level than in those adapted to the 13% casein level with regard to magnesium, citrate and 2-ketoglutarate concentrations in urine.