Decreased hepatic insulin-like growth factor (IGF)-I and increased IGF binding protein-1 and -2 gene expression in experimental uremia.

The imbalance between normal insulin-like growth factor-I (IGF-I) and markedly increased IGF binding protein (IGFBP) plasma levels plays a pathogenic role for growth retardation and catabolism in children with chronic renal failure. To investigate the mechanism of these alterations, experiments were performed in an experimental model of uremia in rats (5/6 nephrectomy) and in pair-fed and ad libitum-fed sham-operated controls Using a specific solution hybridization/RNase protection assay, we observed a marked reduction of hepatic IGF-I messenger RNA (mRNA) abundance at steady state in uremic animals (37 +/- 5% of control) compared both with pair-fed (65 +/- 10%) and ad libitum-fed controls (100 +/- 11%) (P < 0.001). Reduced IGF-I gene expression was clearly organ-specific; it was most pronounced in liver (significant vs., pair-fed controls) and lung and muscle tissue (significant vs., ad libitum-fed controls); no change was observed in kidney and heart tissue. To determine a potential mechanism of reduced hepatic IGF-I gene expression in uremia, the hepatic GH receptor gene expression in the same experimental animals was analyzed by specific solution hybridization/RNase protection assay. Uremic animals had a 20-30% reduction of hepatic GH receptor mRNA abundance compared with controls. Hepatic GHBP expression in uremia was decreased in parallel. Despite the reduction of hepatic IGF-I mRNA abundance, plasma IGF-I levels in uremia were not different from ad libitum-fed controls. This discrepancy is explained by an increased concentration of IGFBPs in uremic plasma. By RIA, plasma IGFBP-1 levels in uremia were increased 4-fold; by Western immunoblot, plasma IGFBP-2 levels were increased 7-fold and plasma IGFBP-4 levels were increased 2-fold compared with both control groups. Intact IGFBP-3 (M(r), approximately 48 kDa) and low molecular IGFBP-3 fragments were not significantly different among the three groups. By Northern blot analysis, hepatic IGFBP-1 mRNA levels in uremia were 2-fold higher than in controls. IGFBP-2 mRNA abundance in liver tissue was increased 4-fold, whereas in kidney there was a significant reduction of IGFBP-2 mRNA (30% of control). IGFBP-4 mRNA was increased by 50% in kidney but not in liver. Plasma insulin and corticosterone levels were not different among the groups. Our study shows that hepatic IGF-I gene expression was specifically reduced in uremia, partially as the consequence of a reduced hepatic GH receptor gene expression. One of the mechanisms contributing to increased IGFBP levels in uremia is increased hepatic gene expression of IGFBP-1 and IGFBP-2. The imbalance between reduced hepatic IGF-I production and increased hepatic IGFBP-1 and 2 production is likely to play a pathogenic role for catabolism and growth failure in CRF.

Biochemical and genetic analysis of a child with cystic fibrosis and cystinosis.

We have studied a child with cystic fibrosis (CF), nephropathic cystinosis, and manifestations of Bartter syndrome, a finding reported previously in both of these diseases (CF and cystinosis). The chance of an individual inheriting a mutant allele for both CF and cystinosis from each of his parents by independent segregation is very small. Therefore, other mechanisms of inheritance were investigated, including whether his diseases were caused by a chromosome deletion or rearrangement that caused defects in both genes, whether his phenotype was caused by a new mutation or variant of either disease, or whether both diseases were inherited together due to inheritance of 2 copies of the same chromosome from one of the parents (uniparental disomy). An investigation was made of whether having mutations for both CF and cystinosis resulted in a different phenotype for either disease and whether the child was a heterozygote rather than a homozygote for one of the mutations. The results suggest that neither disease influenced the expression of the defect in the other and that this child inherited a mutant allele for both diseases independently from each parent.

Ontogeny of blood pressure in the inbred Dahl hypertension-sensitive and -resistant rat.

The inbred S/JR rat is characterized by a genetic predisposition to NaCl-induced hypertension. Although mature S/JR but not R/JR rats develop hypertension when fed a high NaCl-containing diet, this effect has not been examined during early neonatal development. S/JR and R/JR dams were maintained on 0.15% (w/w) or 8% (w/w) NaCl diets throughout gestation and lactation. Measurements of mean abdominal aortic blood pressure (MAP) were obtained in anesthetized offspring at 5, 15, and 25 days of age. This was greater in neonatal S/JR rats than R/JR rats at 5, 15, and 25 days of age. A hypertensinogenic effect of 8% NaCl was seen in R/JR at 5 and 15 days. The results indicate that the ontogeny of MAP can be influenced by pre- and postnatal dietary NaCl. More importantly, elevated MAP in the S/JR strain is a distinguishing characteristic evident throughout the neonatal period of development.

Salt intake and renal hemodynamics in immature and mature Dahl salt-sensitive (DS/JR) and salt-resistant (DR/JR) rats.

To determine if abnormalities in the maturation of renal function in Dahl salt-sensitive rats are associated with the development of hypertension, studies were performed in anesthetized 3 week old salt-sensitive (DS/JR) and salt-resistant (DR/JR) rats whose mothers were maintained on 0.15% (low-salt) during gestation and either 0.15% or 2.0% (high-salt) NaC1 diets after parturition. Mature DS/JR and DR/JR rats were maintained on either 0.15% or 2.0% NaC1 diets after weaning and studied at 8 to 9 weeks of age. High-salt diet raised blood pressure (BP) and reduced glomerular filtration rate (GFR) and renal blood flow (RBF) in mature DS/JR rats, but had no effect on BP, GFR and RBF in mature DR/JR rats. In immature DS/JR and DR/JR animals, high-salt intake resulted in poor growth with reductions in GFR and RBF in the DS/JR group. The response to acute volume expansion, (5% body weight physiologic saline infusion) differed among the groups. Mature rats all vasodilated while immature high-salt DS/JR did not, and immature low-salt DS/JR vasoconstricted. These studies demonstrated that both mature and immature DS/JR rats evidence abnormal responses to acute and chronic salt loading. Early exposure to high-salt intake affects the maturation of renal function in the DS/JR group. An enhanced vascular sensitivity to sodium is present at critical periods of postnatal development in DS/JR rats.

A renal dopamine-1 receptor defect in two genetic models of hypertension.

Dopamine (DA), via DA-1 receptors, regulates Na+ transport in the kidneys. Dopamine is synthesized from L-DOPA in the proximal tubule and presumably secreted as an autocrine/paracrine substance to stimulate DA-1 receptors localized on proximal tubular cells. We have previously reported the presence of DA-1 receptors in renal cortical homogenates and on the isolated proximal tubule of the rat and rabbit, consistent with the dopamine autocrine/paracrine model. We have localized DA-1 receptors in the proximal straight tubule of the rabbit, and in the cortical collecting duct of the rabbit and rat, but not in the distal collecting tubule or the cortical thick ascending loop of Henle. The presence of functional DA-1 receptors has been substantiated by the coexistence of DA-1 agonist-stimulated adenylate cyclase activity in the same nephron segments in which DA-r receptors have been found. Increased concentrations of intrarenal dopamine induced by dopamine-beta-hydroxylase inhibition with SKF-102698 caused a down regulation of proximal tubular DA-1 receptors and almost complete ablation of DA-1 agonist stimulated adenylate cyclase activity. Thus, dopamine may play a role in the regulation of DA-1 receptors and their linkage with adenylate cyclase. Since alterations in the renal dopaminergic system have been measured in some forms of experimental hypertension, we studied DA-1 receptors and their coupling to adenylate cyclase in the spontaneously hypertensive rat (SHR).(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor-I restores microvascular autoregulation in experimental chronic renal failure.

Impairment of autoregulation (AR) is associated with accelerated progression of chronic renal failure (CRF). As the bioavailability of insulin-like growth factor-I (IGF-I) is low in CRF, we investigated the effects of acute luminal application of 10 nM recombinant human IGF-I on AR in juxtamedullary (JM) afferent arterioles (AA) perfused in vitro with a blood solution [(approximately 30% hematocrit (HCT)]. Studies were conducted in AA from adult male rats three to four weeks after five-sixths nephrectomy (Nx) by either surgical excision (N = 7) or infarction (N = 5) of two thirds of the remnant kidney; controls (N = 6) had sham surgery. AA from both Nx groups exhibited marked hypertrophy and impaired AR responses (60 to 140 mm Hg perfusion pressure), features more pronounced in the infarction group. Responses to abluminal acetylcholine (10 microM) were similar in sham and excision groups but were significantly blunted in the infarction group. All groups vasodilated significantly after Ca-channel blockade (10 mM MnCl2). IGF-I restored AR in AA from both Nx groups (P < 0.05, analysis of variance) while it vasodilated AA from controls. These results suggest that IGF-I may protect the glomerulus from injury by maintaining autoregulatory control of renal blood flow, thereby slowing the progression of CRF.

Growth hormone treatment in growth retarded children with end stage renal failure: effect on free/dissociable IGF-I levels.

Growth retardation in children with endstage renal disease (ESRD) is associated with normal to slightly low concentrations of insulin-like growth factor (IGF)-I and increased concentrations of IGF-binding proteins (IGFBPs) in serum. Consequently, IGF-I bioactivity is reduced in serum from uremic patients presumably due to a decrease in the concentration of free IGF-I. Improvement of linear growth with growth hormone (GH) treatment of uremic children is thought to be due to increased IGF-I/IGFBP ratio, thus resulting in increased free IGF-I levels during treatment. The purpose of the present study was to determine whether free/dissociable IGF-I levels are in fact low in uremic children and whether increased growth velocity during GH treatment is associated with an increase in the free IGF-I concentration. Serum total and free/dissociable IGF-I concentrations were measured in 5 children with ESRD before and during treatment with GH, and in control children matched for age, pubertal status, and body mass index. Height velocity increased from 3.7 +/- 1.0 cm/yr to 6.5 +/- 1.2 cm/yr with an increment in height SDS at the end of the first year of GH treatment. Free/dissociable IGF-I concentrations tended to be lower in uremic children compared to control children (3.0 +/- 0.3 vs 7.3 +/- 2.1 micrograms/l, respectively). During GH treatment, free/dissociable IGF-I levels increased significantly to 8.5 +/- 1.0 micrograms/l at 3 months and 6.9 +/- 1.4 micrograms/l at 6-24 months, p < 0.05 compared to pretreatment. Total IGF-I levels were 243 +/- 18 micrograms/l in children with ESRD before treatment and these values also increased during GH treatment (740 +/- 114 micrograms/l at 3 months and 442 +/- 44 micrograms/l at 6-24 months, p < 0.05, compared to pretreatment). Total IGF-I concentration in the control group was 439 +/- 114 micrograms/l. These results support the hypothesis that growth retardation in children with chronic renal failure is associated with a reduction in the concentration of free, biologically available IGF-I, and that increased growth velocity during GH treatment of these children is associated with restoration of free IGF-I concentrations.

Ontogeny of plasma renin activity in the Dahl rat model of essential hypertension.

Plasma renin activity (PRA) is characteristically lower in the Dahl salt-sensitive (S) rat than in the salt-resistant (R) rat. To establish whether PRA differs between these strains at birth or subsequently becomes suppressed in the Dahl S rat, the ontogeny of PRA was studied in inbred Dahl hypertension-prone (S/JR) and hypertension-resistant (R/JR) rats from 5 to 51 days of age. Pregnant dams and postweaning pups were maintained on diets containing either 0.15% or 0.69% sodium chloride (w:w). Although PRA clearly distinguished the two strains in young adulthood, it was not lower in the S/JR pups at 5 and 15 days of age. However, PRA was greater in rat pups suckling dams consuming the low salt diet. These results suggest that suppressed PRA in S/JR rats is an acquired trait, perhaps occurring secondary to other physiological abnormalities and that maternal diet influences PRA in the suckling Dahl rat.

Effect of diet on renal response to salt challenge in neonatal piglets.

In piglets 14-40 days of age, blood pressure and glomerular filtration rate increased significantly with age and were lower than those reported for adult pigs. There was no effect of dietary salt on the maturation of these parameters and on tubular sodium reabsorption, although the piglets on a low salt diet had lower inulin space than piglets receiving higher dietary salt. After the completion of a 24-hour salt challenge there was little evidence of sodium retention and there was no effect of previous dietary salt intake on sodium excretion. However, salt challenge resulted in higher blood pressure and elevated serum sodium only in piglets on a low sodium diet. These results suggest that in contrast to puppies and infants, the renal response to salt challenge is physiologically adequate in piglets at 14-40 days although glomerular function is still immature. Dietary salt history appears to have little effect on the degree of sodium excretion during salt challenge in this age range.

Growth hormone and insulin-like growth factor-I and mesangial matrix in uremic rats.

Combined growth hormone (GH) and insulin-like growth factor-I (IGF-I) therapy has been advocated for clinical use to minimize the diabetogenic effect of GH and enhance their anabolic effects. However, GH has been shown to accelerate the development of glomerular sclerosis in experimental animals and IGF-I mediates the renal effects of GH. The purpose of this study was therefore to examine morphometrically the effects of GH (1 mg intraperitoneally three times a week), IGF-I (50 micrograms/kg body weight subcutaneously twice a day), and combined GH/IGF-I treatments in vivo on mesangial matrix at 3-20 days after 5/6 nephrectomy in 140- to 150-g rats. There were no significant changes in growth and renal function after GH and/or IGF-I treatment. The effects of GH and IGF-I on glomerular size were additive, which were more prominent in juxtamedullary glomeruli. GH induced proportional increases in mesangial area (MA) and glomerular area (GA), whereas IGF-I induced a similar increase in GA without a corresponding change in MA. When compared with GH treatment alone, combined GH/ IGF-I treatment resulted in a lesser degree of mesangial expansion despite an enhanced glomerular size. While additional studies are needed to examine the long-term effects of these findings, our results suggest a potentially beneficial effect of combined GH/IGF-I therapy during uremia.

Effects of insulin-like growth factor I on the renal juxtamedullary microvasculature.

To characterize the effects on the rat renal preglomerular microvasculature of insulin-like growth factor I (IGF-I), experiments were performed using the in vitro blood-perfused juxtamedullary nephron preparation. IGF-I induced a reversible vasodilation of pre- but not postglomerular microvessels in a dose-dependent manner (10(-9)-10(-7) M). The IGF-I-induced vasodilation was similar in all preglomerular vascular segments: interlobular artery, 11.5 +/- 1.2% of control (n = 16); mid-afferent arterioles, 11.6 +/- 1.7% (n = 24); and juxtaglomerular afferent segments, 16.1 +/- 2.8% (n = 19). Renal autoregulatory capacity was not reduced by IGF-I. Pretreatment with the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (10(-4) M) completely inhibited the vasodilatory response to IGF-I. IGF-I induced a rapid increase of NO concentration in intact renal microvessels, monitored by a NO-selective voltametric microelectrode. Pretreatment with the cyclooxygenase inhibitor indomethacin (10(-5) M) not only abrogated the IGF-I-induced dilation, but, moreover, IGF-I elicited a small but significant (approximately 10%) vasoconstriction in all preglomerular vessels. These results indicate that the renal vascular effects of IGF-I involve activation of two endogenous vasodilators (NO and vasodilatory prostaglandins). In addition, IGF-I may also release an undefined vasoconstrictor.

Changes in laminin during recovery from postischemic acute renal failure in rats.

Changes in the immunoreactivity of laminin have been demonstrated in a variety of tissues undergoing injury-regeneration process and during nephrogenesis. Studies in vitro revealed that laminin is important in many cellular functions such as growth, differentiation, and cell communication. This study examined the changes of laminin in the extracellular matrix (ECM) of proximal straight tubules (PST) in the outer stripe of the outer medulla after renal ischemia, an area where most morphological damage occurred. Anesthetized male Sprague-Dawley adult rats underwent a 30-min temporary occlusion of the left renal artery. The inulin clearance (Cin) of the postischemic left kidneys was significantly decreased at 30 min, 1 day, 2 days, and 5 days after the injury, and at 7 and 10 days after the injury it rose to a level not significantly different from the sham-operated controls. Using immunogold electron microscopy, the density of immunoreactive laminin (gold-conjugated laminin grains/micron2 ECM area) in PST where flattened PST cells were present was decreased at 1 day and 2 days, and at 5 days, a time when the renal function was still depressed, it returned to a level not significantly different from that in controls. These changes were not observed in those PST or proximal convoluted tubules in which morphology was intact. Further studies are needed to determine if these changes in laminin of damaged PST cells have any significant role during their recovery from the ischemic injury.

Renal reabsorption of phosphate during development: tubular events.

Studies performed in our laboratory on the isolated perfused kidney of the guinea pig have demonstrated that the rate of Pi reabsorption is substantially greater in the newborn than in the adult, when appropriate corrections are being made either for differences in glomerular filtration rate (GFR) or in renal tubular mass. In order to determine the location of this enhanced reabsorption along the nephron, micropuncture experiments were performed on euvolemic, non-fasted guinea pigs 5-14 and 42-49 days of age, maintained on standard guinea-pig chow diet (0.76% Pi). Concomitant measurements of overall kidney function were also obtained. The results confirmed that fractional reabsorption of Pi (TRPi%) across the entire kidney was significantly higher (P less than 0.01) in the newborn (89.93 +/- 2.55%) than in the adult (78.25 +/- 2.89%) animals. The difference was also significant (P less than 0.05) when TRPi was expressed in mol/ml GFR (1.87 +/- 0.14 vs 1.53 +/- 0.12, respectively). At comparable locations along the proximal tubule (TF/Pin of 1.90 +/- 0.16 in the newborn, and 1.79 +/- 0.15 in the adult, P greater than 0.70), the fraction of the filtered load of Pi reabsorbed was significantly higher (P less than 0.001) in the immature (76.66 +/- 2.74%) than in the mature (67.21 +/- 2.74%) guinea pigs. Estimates based on the differences between proximal Pi reabsorption and the urinary excretion of Pi indicate that the reabsorption of Pi in tubular segments located beyond the proximal tubule is also enhanced in the newborn when compared with the adult (15.62 +/- 2.11% vs 10.51 +/- 1.83%, respectively, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Direct visualization of renin-cell distribution in preglomerular vascular trees dissected from rat kidney.

Three methods to visualize directly the distribution of granulated renin-positive cells in vascular trees microdissected from rat kidney were developed. Kidneys were removed from anesthesized rats, hemisectioned, macerated in HCl, and soaked in distilled water for 24-48 h. Cortical preglomerular vascular trees consisting of arcuate and cortical radial arteries and afferent arterioles were microdissected with the aid of a stereomicroscope. Granulated cells can be visualized in three ways. First, under transmitted or incident light observation, granulated cells are readily distinguished from the surrounding smooth muscle cells, because of marked differences in the refractive properties of these two cell types. Second, quinacrine, a fluorescent, intravital stain selective for dense-core granules, can be administered (2 mg/kg iv) to the rat 1 h before nephrectomy. When illuminated with 440-nm light, granulated cells fluorescence strongly at 510 nm. Third, specific immunostaining for renin can be obtained with a polyclonal anti-rat renin antibody and avidin-biotin immunoperoxidase staining in vascular trees subjected to cell permeabilization with Triton. These new techniques permit the direct visualization of the distribution of granulated renin-positive cells in preglomerular vessels under conditions in which the vascular architecture is largely preserved.

Inhibition of myogenic autoregulation in cyclosporine nephrotoxicity in the rat.

The mechanisms responsible for the impairment of renal blood flow (RBF) autoregulation in cyclosporine nephrotoxicity were investigated with clearance and micropuncture studies in anesthetized rats. Early chronic cyclosporine nephrotoxicity (CCN) was induced in male rats by daily intramuscular injection of 10 mg/kg/day cyclosporine-A in olive oil for 7 days; control (CON) rats received vehicle injections. Glomerular filtration rate and RBF were both reduced by 33% in CCN when compared to CON rats. RBF autoregulation was also significantly impaired in CCN, with an autoregulation index (AI) of 0.53 +/- 0.03 vs. 0.16 +/- 0.01 in CON rats. Micropuncture studies showed that the tubuloglomerular feedback (TGF) system is not impaired in CCN. Rather, in CCN there was a slight resetting such that the maximum TGF response was greater and the onset occurred at lower rates of perfusion than in CON. In contrast, further micropuncture studies demonstrated that TGF-independent autoregulation of glomerular capillary pressure was significantly impaired in CCN, with an AI of 0.86 +/- 0.09 vs. 0.57 +/- 0.06 in CON. These results indicate that the loss of autoregulatory ability in rats with CCN results from substantial impairment of the myogenic autoregulatory mechanism that is an intrinsic property of the preglomerular vasculature of the kidney.