Frida Zaladek Gil

Functional and morphometric evaluation of offspring kidney after intrauterine undernutrition

Abstract  Pregnant rats were subjected to 50% food restriction during the first or the second half of pregnancy, or throughout pregnancy. The effects of intrauterine food restriction, on kidney function and morphometry were studied in newborn and adult (3 months) offspring. No differences in glomerular diameter were observed in newborn restricted rats compared with controls. The number of glomeruli was significantly lower both in newborn and 3-month-old restricted rats. However, glomerular diameter was increased in 3-month-old rats, which suggests that hypertrophic stimuli were present. The medulla/cortex ratio increased in adult rats submitted to food restriction during pregnancy, a finding that agrees with the preserved sodium and acid excretion, and the normal osmolar and free water clearance observed in these groups. These results show that the reduction in glomerular number is still present 3 months after birth in the progeny of mothers submitted to severe food restriction during pregnancy, suggesting impairment of glomerulogenesis even after birth. Intra utero undernutrition can be regarded as an experimental model of glomerular hypertrophy.

Long-term effects of maternal diabetes on vascular reactivity and renal function in rat male offspring.

Fetal growth impairment can occur in pregnancy complicated by diabetes. Although several studies have focused the effects of nutritional status on intrauterine development, the long-term impact of maternal diabetes on vascular and renal function in the offspring is poorly investigated. In the present study, blood pressure profiles and renal function parameters were investigated in the offspring of diabetic rats (DO). Female rats were made diabetic throughout gestation with a single dose of streptozotocyn (STZ) 10 d before mating. After weaning, the offspring had free access to food and water. Arterial pressure was evaluated every 15 d. Functional and morphometric kidney studies were performed in newborn, 3, 6 and 12-mo-old male rats in DO and in controls, C. Although maternal diabetes did not affect nephron number in the young adult rat, glomerular hypertrophy developed from 3 mo on. Glomerular Filtration Rate and Renal Plasma Flow were observed to be significantly decreased in DO when compared with C, from 3 mo on. In DO, hypertension was observed from 8 wk on and persisted elevated throughout the experimental period (12 mo). Vascular reactivity, evaluated in mesenteric arterial bed showed a decreased endothelium-dependent vasodilatation in 12-mo-old DO animals, while preserved response to sodium nitroprusside was demonstrated. Our data show that exposure to intrauterine diabetes induced by STZ does not affect nephron number in the young offspring but can cause permanent changes in Nitric Oxide (NO)-related vascular response, which, in turn may accelerate the natural age-related nephron loss.

Intrauterine undernutrition—renal and vascular origin of hypertension

OBJECTIVE A large number of clinical and experimental studies supports the hypothesis that intrauterine undernutrition is an important determinant of hypertension, coronary heart disease and non-insulin-dependent diabetes in the adult offspring. In this review, the renal and vascular repercussions of maternal undernutrition are emphasized, and the physiopatologic mechanisms discussed. The origin of hypertension is detailed based upon the findings of kidney functional parameters and endothelium function studies. A working model linking hypertension to intrauterine undernutrition is proposed.

Micronutrient prenatal supplementation prevents the development of hypertension and vascular endothelial damage induced by intrauterine malnutrition

AIMS The premise that intrauterine malnutrition plays an important role in the development of cardiovascular and renal diseases implies that these disorders can be programmed during fetal life. Here, we analyzed the hypothesis that supplementation with mixed antioxidant vitamins and essential mineral in early life could prevent later elevation of blood pressure and vascular and renal dysfunction associated with intrauterine malnutrition. MAIN METHODS For this, female Wistar rats were randomly divided into three groups on day 1 of pregnancy: control fed standard chow ad libitum; restricted group fed 50% of the ad libitum intake and a restricted plus micronutrient cocktail group treated daily with a combination of micronutrient (selenium, folate, vitamin C and vitamin E) by oral gavage. KEY FINDINGS In adult offspring, renal function and glomerular number were impaired by intrauterine malnutrition, and the prenatal micronutrient treatment did not prevent it. However, increased blood pressure and reduced endothelium-dependent vasodilation were prevented by the micronutrient prenatal treatment. Intrauterine malnutrition also led to reduced NO production associated with increased superoxide generation, and these parameters were fully normalized by this prenatal treatment. SIGNIFICANCE Our current findings indicate that programming alterations during fetal life can be prevented by interventions during the prenatal period, and that disturbance in availability of both antioxidant vitamins and mineral may play a crucial role in determining the occurrence of long-term cardiovascular injury.

L-Arginine effects on blood pressure and renal function of intrauterine restricted rats

Abstract. We have previously demonstrated that 3-month-old rats submitted to 50% intrauterine food restriction showed a decreased number of nephrons with increased glomerular diameter, a fact that suggests compensatory hypertrophy. In the present study, we extended the investigation and performed serial blood pressure measurements and renal function evaluation in 8- and 12-week-old rats submitted to 50% intrauterine food restriction (groups R8 and R12) and in age-matched control rats (groups C8 and C12). After weaning, six to eight animals from each group received oral supplements of 2% L-arginine (L-arg) solution for 4 or 8 weeks (groups CA8, CA12, RA8, RA12). Our findings showed that mean blood pressure (MBP), which was significantly increased from 8 weeks on in R rats, markedly decreased after L-arg supplementation. In control animals, no alterations in MBP were observed with L-arg. Proteinuria was within normal limits in all groups studied but L-arg caused a significant decrease in this parameter in both the RA8 and RA12 groups. Glomerular filtration rate (GFR, ml/min per kg) was significantly decreased in the C8 control group (3.75±0.12) and in both restricted groups R8 and R12, (2.47±0.13 and 3.76±0.16, respectively) compared with the C12 group (6.09±0.31; P<0.05 for all comparisons). L-Arg caused an increase in GFR only in the younger groups, C8 and R8. In a separate set of experiments, acetylcholine (ACh)-induced relaxation was examined in mesenteric arteries. The R12 group showed a significant impairment of the response to ACh, which returned to normal values after L-arg supplementation. Urinary excretion of NOx (NO3– + NO2–) was significantly decreased in 8- and 12-week-old food-restricted rats relative to control rats. Our data indicate that, besides the known decrease in absolute nephron number, disturbances in the production/sensitivity to the L-arg–nitric oxide system may contribute to the early appearance of hypertension in the offspring of mothers submitted to significant food restriction.

The Influence of L-Arginine on Blood Pressure, Vascular Nitric Oxide and Renal Morphometry in the Offspring from Diabetic Mothers

The present study was designed to evaluate the effects of l-arginine (l-arg) supplementation on blood pressure, vascular nitric oxide content, and renal morphometry in the adult offspring from diabetic mothers. Diabetes mellitus was induced in female rats with a single dose of streptozotocin (50 mg/kg), before mating. The offspring was divided into four groups: group C (controls); group DO (diabetic offspring); group CA (controls receiving 2% l-arg solution dissolved in 2% sucrose in the drinking water) and group DA (DO receiving the l-arg solution). Oral supplementation began after weaning and continued until the end of the experiments. In DO, hypertension was observed, from 3 mo on. In DA, pressure levels were not different from C and CA. In 6-mo-old animals, basal NO production (assessed by DAF-2) was significantly depressed in DO in comparison to controls. The NO production was significantly increased after stimulation with Ach or BK in all groups, the increase being greater in control than in DO rats. l-Arg was able to improve the NO production and to prevent the glomerular hypertrophy in the DO. Our data suggest that the bioavailability of NO is reduced in the DO, because l-arg corrected both the hypertension and glomerular hypertrophy.

Effects of intrauterine food restriction and long-term dietary supplementation with L-arginine on age-related changes in renal function and structure of rats.

We have previously demonstrated that restricting intrauterine food by 50% in 3-mo-old rats produced lower nephron numbers and early-onset hypertension, the latter being normalized by l-arginine administration. In 18-mo-old rats, such restriction increased glomerulosclerosis. In this study, we expanded our investigation, evaluating functional, morphologic, and immunohistochemical parameters in intrauterine-food-restricted 18-mo-old rats, either receiving l-arginine (RA18) or not (R18). Age-matched, non-food-restricted controls were assigned to similar groups with l-arginine (CA18) and without (C18). After weaning, l-arginine was given daily for 17 mo. No functional or morphologic changes were observed in C18 rats. The R18 rats developed early-onset hypertension, which persisted throughout the observation period, as well as significant proteinuria from 12 mo on. In RA18 rats, l-arginine decreased both blood pressure levels and proteinuria, and glomerular diameter was significantly smaller than in R18 rats (115.63 ± 2.2 versus 134.8 ± 1.0 μm, p < 0.05). However, in RA18 rats, glomerular filtration rate remained depressed. Although l-arginine prevented glomerulosclerosis (R18 = 14%, RA18 = 4%; p < 0.05), glomerular expression of fibronectin and desmin was still greater in RA18 rats than in controls. Our data show that, although l-arginine prevented hypertension and proteinuria, glomerular injury still occurred, suggesting that intrauterine food restriction may be one of the leading causes of impaired renal function in adult life.

ALTERATIONS OF THE RENAL HANDLING OF H+ IN DIABETIC RATS

Renal acid excretion and proximal and distal nephron acidification were evaluated 20 days after induction of diabetes, in rats, by intraperitoneal injection of streptozotocin (45 mg/kg). Titratable acidity in urine was measured by microtitration and ammonium excretion (NH4+) by spectrophotometry. Proximal tubular acidification was evaluated by the kinetics of reabsorption of perfused HCO3-. Distal nephron acidification was evaluated by measuring urine - blood pCO2 differences under alkaline overload. The net acid excretion (titratable acidity + NH4+ - HCO3-) was higher (p < 0.001) in diabetic rats (9.82+/-0.65 micromol/min/kg, n = 26) than in the control group (6.34+/-0.14, n = 24). Proximal HCO3- reabsorption was also higher (p < 0.001) in diabetic rats (8.38+/-0.11 nmol/cm2/s, n = 12) than in the control group (2.30+/-0.10, n = 22); however, evaluation of distal nephron H+ secretion by urine - blood pCO2 methodology was similar in both groups. We concluded that in rats with induced diabetes mellitus there is an increased rate of proximal HCO3- reabsorption, possibly effected by a higher density of Na+/H+ antiporter in the luminal membrane of the proximal tubule and by an increased proton-motive force of the H+ secretory mechanism. The higher rates of H+ secretion generate lower stationary proximal luminal pH and probably maintain the blood pH within the physiological range.

Inflammatory milieu as an early marker of kidney injury in offspring rats from diabetic mothers.

The present study investigated the early presence of inflammatory response in renal tissue of young offspring from diabetic mothers. The effect of L-arginine (L-arg) supplementation was also investigated. The offspring was divided into four groups: group CO (controls); group DO (diabetic offspring); group CA (CO receiving 2% L-arg solution) and group DA (DO receiving the 2% L-arg solution). Glycemia, arterial pressure and renal function were evaluated; gene and protein expression of pro-inflammatory cytokines were also measured. Blood pressure levels were significantly increased in 2 and 6 month-old DO rats, whereas L-arg administration caused a significant decrease in the DA group, at both ages. DO rats showed a significantly blunted glycemic response to exogenous insulin. In 2 month-old DO animals, renal protein expression of pro-inflammatory molecules was significantly increased. At six months of age, we also observed an increase in gene expression of pro-inflammatory molecules, whereas L-arg supplementation prevented this increase at both ages. Our data suggest that activation of inflammatory pathways is present early in the kidney of DO rats, and that L-arg can attenuate the expression of these markers of tissue inflammation. Our results also reinforce the concept that intrauterine environmental factors are a fundamental determinant in the development of metabolic and vascular diseases later in life.

Effect of Gentamicin on Urinary Acidification in the Rat

The effect of gentamicin, an aminoglycoside antibiotic, on renal function and especially on acid excretion was studied in normal and acidotic rats. The doses used were 1 (G4) and 10 (G40) times the suggested human therapeutic dose on a weight basis. After 10 days of each treatment, the glomerular filtration rate (GFR) was unchanged in G4 but fell significantly (p less than 0.05) in G40. In the acidotic groups (AG4 and AG40) there was an accentuated reduction in GFR, renal plasma flow and urine/plasma insulin ratio. Normal rats showed a normal acid excretion even with the high-dose treatment but, in the acidotic group, there was a significant decrease in ammonia excretion. The amount of bicarbonate excretion was significantly elevated in those groups, leading to a greater urinary pH. These results indicate that acute metabolic acidosis enhanced the nephrotoxic effects of gentamicin and impaired the excretion of an acid overload.