Pulmonary ventilatory functions and obesity in Kuwait.

OBJECTIVE: To study the relationship between obesity and pulmonary ventilatory functions in Kuwaiti adults. SUBJECTS AND METHODS: A total of 200 male and 180 female Kuwaiti adults aged 20-65 years were investigated in six medical centers from April 2004 to March 2006. Parameters measured included forced expiratory volume in 1 s (FEV(1)), forced vital capacity (FVC), FEV(1) as a percentage of FVC (FEV%); body mass index (BMI in kg/m(2)) and waist-to-hip ratio (W/H). RESULTS: For the whole group, males or females, BMI (kg x m(2)) and W/H were poor individual predictors of pulmonary ventilatory functions. However, central adiposity (W/H) was associated with restrictive respiratory impairment (10.6-13.9% decrease in FEV(1) and 10-12.3% decrease in FVC), independent of sex, age or height. In obese females and males (BMI >30), increasing severity of obesity was significantly (p < 0.05, R(2) >0.6) [corrected] associated with increasing restrictive respiratory impairment (8.7-14.4% decrease in FEV(1) and 8-11.7% decrease in FVC), with no evidence of obstructive disease (FEV(1)/FVC >0.8). CONCLUSION: In adult Kuwaiti males and females, increase in body fat at BMI >30 or W/H >1 was associated with a restrictive effect on pulmonary ventilation.

Interactions of the growth-related, type IIc renal sodium/phosphate cotransporter with PDZ proteins.

Despite similar molecular structures, the growth-related sodium/phosphate cotransporter NaPiIIc is regulated differently than the main NaPiIIa phosphate transporter. Using two-hybrid systems and immunoprecipitation, we identified several proteins that interact with NaPiIIc that might account for this differential regulation. NaPiIIc interacted with the PDZ domain-containing sodium-hydrogen exchange-regulating factor (NHERF) 1 and NHERF3 through novel binding motifs in its C terminus. NaPiIIc from brush-border membranes coprecipitated with both NHERF1 and NHERF3, with more NHERF3 co-precipitated in rats fed a low-phosphorus diet. NaPiIIc colocalizes with both NHERF1 and NHERF3 in brush-border membranes of rats fed either a low- or high-phosphorus diet. When mouse NaPiIIc was transfected into opossum kidney cells, it was localized mainly in apical microvilli and the trans-Golgi. Both confocal and total internal reflection microscopy show that NaPiIIc colocalizes with NHERF1 and NHERF3 in the apical microvilli, and this was not altered by truncation of the last three amino acids of NaPiIIc. Interactions of NaPiIIc with NHERF1 and NHERF3 were modulated by the membrane-associated 17 kDa protein (MAP17) similarly to NaPiIIa, but only the MAP17-NaPiIIc-NHERF3 complexes were internalized to the trans-Golgi. Our study shows that NaPiIIc interacts with a limited number of PDZ domain proteins, and the mechanisms and consequences of such interactions differ from those of NaPiIIa.

Expression of the rat renal PiT-2 phosphate transporter.

BACKGROUND: NaPi-2a is the main sodium-dependent Pi (Na+-Pi) transporter in the apical membrane of the renal proximal tubule. Another group of Pi transporters, Glvr-1 (PiT-1) and Ram-1 (PiT-2), was identified. The PiT-2 cRNA induces Na+-dependent Pi uptake into Xenopus laevis oocytes. Prior studies have revealed the presence of the Pit-2 transporter in the kidney. OBJECTIVES: Further characterization of the PiT-2 transporter in the kidney and assessment of its developmental regulation. METHODS: Using primers specific for the PiT-2 mRNA and an antibody specific for the PiT-2 protein, we assessed the expression and developmental regulation of the renal PiT-2 mRNA and protein. RESULTS: RT-PCR analysis revealed that a 182 bp product was evident in the total kidney (TK), cortex (C), and medulla (M). Northern blots demonstrated a PiT-2 mRNA of approximately 4 kb (expected size) in the TK, C, and M. PiT-2 mRNA expression was similar in all kidney regions. RT-PCR and Northern blot analysis revealed that the PiT-2 cDNA was highly abundant in OK and MDCK culture cells. RT-PCR and Northern blot analysis revealed expected products at all ages studied. Densitometry demonstrated similar levels of expression of PiT-2 mRNA in the kidneys of older versus younger animals, and persistent expression in elderly rats. The PiT-2 protein was present in the TK, C, and M, and in OK and MDCK cells. PiT-2 protein abundance was similar at all ages studied. CONCLUSIONS: These studies further characterize the renal PiT-2 transporter and show that its expression is stable throughout development and ageing.

Hypoxic rise in cytosolic calcium and renal proximal tubule injury mediated by a nickel-sensitive pathway.

In the kidney, cell injury resulting from ischemia and hypoxia is thought to be due, in part, to increased cytosolic Ca(2+) levels, [Ca(2+)]i, leading to activation of lytic enzymes, cell dysfunction, and necrosis. We report evidence of a progressive and exponential increase in [Ca(2+)]i (from 245 +/- 10 to 975 +/- 100 nM at 45 mins), cell permeabilization and propidium iodide (PI) staining of the nucleus, and partial loss of cell transport functions such as Na(+)-gradient-dependent uptakes of (14)C-alpha-methylglucopyranoside and inorganic phosphate ((32)Pi) in proximal convoluted tubules of adult rabbits subjected to hypoxia. The rise in [Ca(2+)]i depended on the presence of extracellular [Ca(2+)] and could be blocked by 50 microM Ni(2+)but not by verapamil (100 microM). Presence of 50 microM Ni(2+) also reduced the hypoxia-induced morphological and functional injuries. We also used HEK 293 cells, a kidney cell line, incubated in media without glucose and exposed for 3.5 hrs to 1% O(2)-5% CO(2) and then returned to glucose-containing media for another 3.5 hrs in an air-5% CO(2) atmosphere and finally exposed for 1 min to media containing 1 microM PI. NiCl(2) (50 microM) or pentobarbital (300 microM) more than phenobarbital (1.5 mM), when present in the incubation medium during both the hypoxic and the reoxygenation periods, induced significant (P < 0.001) reductions in the number of cell nuclei stained with PI, similar to their relative potency as inhibitors of T channels. Our findings indicate that hypoxia-induced alterations in calcium level and subsequent cell injury in the proximal convoluted tubule and in HEK cells involve a nickel-sensitive and dihydropyridine insensitive pathway or channel.

Reported frequency of physical activity, fitness, and fatness in Kuwait.

We studied reported frequency of physical activity (PA), body composition, and fitness in Kuwaiti females (n = 84) and males (n = 72), urban office government employees, or students. A sedentary (SED) group reported no or occasional participation in self-directed PA of 30 minutes or more during the week for the last 6 months; group LOF (low frequency) participated in 1-2 sessions/week (walking, jogging, sports such as volleyball, soccer, or gym); group HIF (higher frequency) reported three or more sessions/week. Aerobic fitness was measured using Bruce's treadmill protocol. Body fat and fat-free mass (FFM) were estimated from seven skinfolds and body weight (BW). Grip strength and trunk flexibility were also measured. Maximal O(2) consumption (VO(2)max)/BW was proportional to reported frequency of PA in males and females, indicating reliability of the reported PA frequencies. SED males (%fat > 25), SED and LOF females (%fat > 30) were overweight. In males, LOF PA was associated with lower %fat and higher flexibility than SED, and in females with higher VO(2)max/FFM than SED. LOF females, in contrast to males, were resilient to or exercised at levels insufficient for fat loss but sufficient for aerobic conditioning of the FFM, likely related to the low VO(2)max of females in which PA would have the most effect. HIF PA (>/=3/week) was associated in females with significantly lower %fat and higher flexibility than SED females and no further aerobic conditioning of the FFM, and in males with no further differences in study variables compared with LOF males. Grip strength was higher in males than females but was unrelated to frequency of PA, likely reflecting lack of strength training in the activities of most participants. Thus, low-frequency PA was associated with health benefits both in males (lower %fat) and females (higher VO(2)max/FFM). Probably due to the generally low fitness level of females and, hence, the high aerobic effort (%VO(2)max) required for exercise sufficient to impinge on fat stores, this was only achieved in females participating in higher weekly frequency (>/=3) of PA.

Basolateral phosphate transport in renal proximal-tubule-like OK cells.

It is generally assumed that phosphate (Pi) effluxes from proximal tubule cells by passive diffusion across the basolateral (BL) membrane. We explored the mechanism of BL Pi efflux in proximal tubule-like OK cells grown on permeable filters and then loaded with 32P. BL efflux of 32P was significantly stimulated (P < 0.05) by exposing the BL side of the monolayer to 12.5 mM Pi, to 10 mM citrate, or by acid-loading the cells, and was inhibited by exposure to 0.05 mM Pi or 25 mM HCO3; by contrast, BL exposure to high (8.4) pH, 40 mM K+, 140 mM Na gluconate (replacing NaCl), 10 mM lactate, 10 mM succinate, or 10 mM glutamate did not affect BL 32P efflux. These data are consistent with BL Pi efflux from proximal tubule-like cells occurring, in part, via an electro-neutral sodium-sensitive anion transporter capable of exchanging two moles of intracellular acidic H2PO4- for each mole of extracellular basic HPO4= or for citrate.

Ontogeny of renal phosphate transport and the process of growth.

The kidneys of infants and children reabsorb a high fraction of the filtered phosphate (Pi), as appropriate to the needs of a growing organism. This high Pi reabsorptive rate is associated with a high capacity (Vmax) of the Na+-Pi symport system. At the molecular level this high reabsorptive capacity appears to be due to the presence of a growth-specific Na-Pi cotransporter. Several experimental findings support this assumption. Firstly, the expression of NaPi-2 mRNA is, if anything, lower in the renal cortex of young animals than of adult animals. Secondly, polyA RNA obtained from growing animals depleted of NaPi-2 by specific hybridization with an antisense 16-mer induces Na+-Pi transport in oocytes. No induction of Na+-Pi transport was observed in oocytes injected with hybridized polyA RNA obtained from adult animals. Thirdly, polyA RNA derived from young rats, depleted of NaPi-2 by subtractive hybridization with adult animal renal cortical cDNA, retains its ability to encode for Na+-Pi cotransport in oocytes. Adult animal renal cortical polyA RNA, depleted of NaPi-2 by subtractive hybridization, failed to induce Na+-Pi uptake into oocytes. Fourthly, renal cortical polyA RNA from young animals, depleted of NaPi-2, contains a region that is highly homologous (80%-92%) with the corresponding region of other modulated NaPi (type II) transporters. Fifthly, this region is also present in the polyA RNA obtained from the renal cortex of newborn rats (1st week of life), despite the fact that NaPi-2 is absent at this early age. Lastly, Npt2 (-/-) knockout mice, although hypophosphatemic and phosphaturic, filter and reabsorb Pi at rates exceeding those that can be accounted for by the expression of type I and III transporters. Based on these observations it is reasonable to surmise that the high Vmax of the Na+-Pi cotransport system observed in the young is due to a large extent to the presence of a growth-specific NaPi transporter, homologous but not identical to already cloned type II NaPi transporters.

Phosphate depletion in opossum kidney cells: apical but not basolateral or transepithelial adaptions of Pi transport.

Monolayers of opossum kidney (OK) cells are widely used as models for the renal proximal tubule. OK cells adapt to phosphate (Pi) depletion by increasing their capacity for apical and basolateral Na+-dependent Pi uptake. Because NMR-visible cell Pi was found to be decreased in Pi-deprived kidney cells, we suggested that up-regulation of basolateral Pi efflux also occurs during adaptation to Pi deprivation [American Journal of Physiol 1994;267:C915-919]. In order to test this hypothesis, we measured the cell Pi pool, basolateral Pi efflux and transepithelial Pi fluxes in OK cells grown on permeable plastic filters, exposed overnight to solutions containing either 0.5 mM (deprived) or 2.0 mM (replete) Pi or 32Pi. Following steady state or acute loading with 32Pi, the specific activity (SA) of cell Pi, the cell Pi pool and the basolateral efflux of 32Pi were measured. In the steady state, a 2-fold increase in Pi uptake sustained the intracellular Pi pool at 85% of the control level (30 +/- 5 nmol/mg) in spite of a decrease in extracellular Pi from 2 to 0.5 mM. When the extracellular Pi was acutely (1 h) reduced to 0.1 mM, the cell Pi pool decreased (to 3 +/- 1 nmol/mg) both in cells previously adapted overnight to either 0.5 or to 2 mM Pi (p >0.3). The rates of absolute and fractional basolateral washout of cell 32Pi after 1 h loading with 0.1 mM 32Pi were similar in cells adapted to 0.5 compared to 2 mM Pi. This indicates that Pi depletion did not affect the effective permeability of the basolateral membranes to Pi. Adaptation for 16 h to 0.5 compared to 2 mM Pi did not alter the rate of net transepithelial transport of 0.1 mM Pi from the apical to the basal compartment but reduced (p < 0.05) the unidirectional fluxes of both 32Pi and 14C-mannitol. An insufficient driving force (unchanged or low Pi concentration in the transport pool, low electrical or coupled-anion gradients) and a constant effective basolateral Pi permeability must have limited basolateral Pi efflux in cells exposed to 0.1 mM Pi. Thus, in OK cells grown on plastic support there are no adaptive increases in either basolateral Pi efflux, or in transcellular and paracellular Pi transport, in response to Pi depletion. Adaptations are limited to increases in apical and basolateral sodium-dependent Pi uptakes that can maintain the cell Pi pool as long as apical Pi is not too low (> or =0.5 mM). The OK cells adapt to low Pi concentrations conserving cell Pi but not increasing basolateral Pi efflux nor transepithelial Pi transport.

Low-frequency physical activity insufficient for aerobic conditioning is associated with lower body fat than sedentary conditions.

We aimed to show the relationships between reported physical activity, fitness level, and body composition in healthy adult office-working Kuwaiti males (n = 45). Reported level of physical activity (group 0 = no routine exercise, n = 10; group 1 = routine exercise once per wk, n = 19; group 2 = routine exercise two or more times per wk, n = 16) was determined from recall questionnaires. Aerobic fitness levels were assessed from resting exercise heart rates and measurement of maximal oxygen uptake (treadmill exercise, Bruce protocol). Body composition was estimated from measurements of body weight, body height, and seven skin-folds. None of the subjects reported dieting to reduce or maintain their body weight. Despite minimal and not statistically significant differences in resting heart rates, VO(2)max, VO(2)max/kg body weight, and VO(2)max/kg lean body mass, the sedentary group (group 0) had a much higher (12--14 kg) average body weight, higher (12--13 kg) body fat, and thicker skin folds than the more physically active groups 1 and 2. Groups 1 and 2 did not differ significantly from each other with regard to fitness and body composition parameters. Thus, routine physical activities insufficient to result in aerobic conditioning were found to be associated with lower body weight and lower body fat compared to those present in subjects reporting the total absence of any routine physical activity. These data are consistent with the hypothesis that even mild levels of routine physical activity are associated with levels of energy turnover that allow less intense and/or frequent periods of positive energy balance, resulting in maintenance of lower body fat than in totally sedentary people. Frequency of participation in routine physical activities may also reflect the level of non-exercise related physical activities and thus relate to the ability to minimize weight gain.

Hormonal regulation of sodium-dependent phosphate transport in opossum kidney cells.

There are multiple regulators of renal proximal tubule sodium-dependent phosphate (Na(+)-Pi) transport, including 1,25-dihydroxyvitamin D (1,25-Vit. D), parathyroid hormone (PTH), insulin-like growth factor 1 (IGF-1), and arachidonic acid (AA) and/or its metabolites. The purpose of our studies was to determine whether the effect of these factors on Pi transport is synergistic or antagonistic. The control solution or the substances were added independently or coincidentally to opossum kidney (OK) cells before incubation for 4 h. 1,25-Vit. D (10(-8) M) had no significant effect on Pi transport ( upward arrow6.8%; p = 0.8). PTH (10(-7) M) significantly inhibited Pi transport by 39.6% (p < 0.0001). IGF-1 (10(-8) M) stimulated Pi transport by 19.6% (p < 0.0001). The AA metabolite 20-HETE (10(-7) M) had no significant impact on Pi transport ( downward arrow6.4; p = 0.3). The combined effect of 1,25-Vit. D and PTH was no different from PTH alone (p = 0.2). Likewise, addition of either 1,25-Vit. D or 20-HETE to IGF-1 failed to affect the magnitude of the increase on Pi transport induced by IGF-1 alone (p = 0.4, p = 0.6, respectively). The combination of 20-HETE and PTH was not different from that observed with PTH alone (p = 0.9). We conclude that in OK cells, PTH inhibits whereas IGF-1 stimulates Pi transport into OK cells. The effects of each of these hormones are independent and unaffected by either 1,25-Vit. D or 20-HETE.

Cardio-respiratory responses to moderately heavy aerobic exercise during the Ramadan fasts.

OBJECTIVE: The aim of this study was to quantify the magnitude of the cardiovascular and respiratory changes that occur during the month of Ramadan in response to moderately heavy aerobic physical exertion. METHODS: Eighteen sedentary Kuwaiti adult males were tested under thermo-neutral conditions during a spring-like month of Ramadan and one month thereafter. RESULTS: There were no significant changes in maximal exercise capacity, treadmill walking efficiency, percentage VO2 max, in body weight and composition associated with Ramadan fasting or one month after. Cardiac (heart rate) and ventilatory responses to moderately intense bouts of sub-maximal aerobic exercise (70% of VO2 max) were actually slightly (<5%) but significantly (P<0.05) reduced, while exercise systolic but not diastolic pressure increased slightly (6%) by the end of Ramadan. CONCLUSION: Hormonal changes associated with dehydration or fasting, abstention from consumption of substances with negative inotropy and changes in circadian rhythms during Ramadan may be responsible for these mild changes in cardiorespiratory responses to exercise. Such changes had no negative effect on the physical aerobic performance of these subjects while exercising at moderately heavy intensity under thermally neutral conditions, during the month of Ramadan. Reduced ventilation during exercise may reflect a limited glycolytic capacity by the end of Ramadan.

Multiple arachidonic acid metabolites inhibit sodium-dependent phosphate transport in OK cells.

The cytochrome P450-dependent monoxygenase pathway represents a major route for the metabolism of arachidonic acid (AA) in the kidney. In turn, AA metabolites have been shown to affect renal electrolyte metabolism, including sodium transport. Specifically AA, 20-HETE and 12-HETE inhibit sodium-dependent (Na+-Pi) uptake into renal culture cells, and both 12-HETE and 14,15 EET have been shown to reduce renin release from renal cortical slices. Since the bulk of Pi transport occurs in the proximal tubule (PT), and the PT is a major site of AA metabolism, we studied the effect of AA and several of its metabolites on Na+-Pi uptake into PT-like opossum kidney (OK) cells. Incubation of OK cells in AA (10(-8) M) resulted in 17% inhibition of Pi uptake. Three metabolites of omega-hydroxylation of AA induced significant decreases in Pi uptake: 19R-HETE (10(-8) M) by 36% (P=0.008), 19S-HETE (10(-8) M) by 24% (P=0.002) and 20-COOH-AA (10(-8) M), a metabolite of 20-HETE, by 25% (P<0.0001). 14,15 EET (10(-8) M), a breakdown product of AA by the epoxygenase pathway, had the greatest effect on Pi uptake in OK cells. It decreased Pi uptake by 47% (P < 0.0001). Addition of the P450 inhibitor, 7-ER (10(-8) M), to OK cells resulted in a significant stimulation (28%) of Pi uptake (P=0.016). These results indicate that these AA metabolites have a significant inhibitory effect on Na+-Pi uptake in OK cells.

Responses to exercise, fluid, and energy balances during Ramadan in sedentary and active males.

This study compares the effects of the Ramadan fast (RF) on body and plasma compositions, hematology, and responses to steady state submaximal exercise in six physically active (A, 35.5+/-1.6 y) and seven sedentary (S, 37.6+/-2.3 y) Kuwaiti men. Subjects were evaluated: 1) 1 wk before RF (pre-RF); 2) 2 wk after the start of RF (mid-RF); 3) at the 4th wk of RF (end-RF). Total body weight and body fat decreased insignificantly (<1.5%, P>0.3) in both groups. At the end of Ramadan, significant increases in osmolarity (P<0.05), Na (P<0.05) and bicarbonate (P<0.05), and a decrease in serum iron were noted in sedentary but not in active subjects. Platelet count was lower in both groups by the end of RF (P<0.05). Body weight and percent fat changed little (<1.5%) in either group. During RF, submaximal exercise heart rate declined insignificantly in group S but significantly (P<0.05) in group A. The respiratory exchange ratio R during steady state submaximal exercise decreased markedly (P<0.001) by the end of RF in both groups. It is concluded that energy balance is well maintained during RF both in S and in A subjects. Metabolic adaptations during RF result in lower exercise R due to increased lipid usage. Deficits or redistribution of specific micronutrients (iron, vitamins) may account for reductions in serum iron and platelet counts, particularly in sedentary subjects that need to limit intake to maintain body weight. The decrease in submaximal exercise heart rate indicates that during RF, cardiovascular adaptation to conditioning is adequate in the more physically active group. Body fluid balance was better maintained in active than in sedentary subjects.

Gastrointestinal parasitic infection, anthropometrics, nutritional status, and physical work capacity in Colombian boys.

This article tests the hypothesis that the presence of gastrointestinal parasites in Colombian boys is negatively associated with anthropometric characteristics, physical work capacity, blood hemoglobin (Hb) levels, and nutritional status. Anthropometric, Hb, &Vdot;O(2) max, and parasite load data were collected on 1,016 boys in Cali, Colombia. The boys were classified as lower socioeconomic class (SEC) from either urban or rural environments, and upper SEC from an urban environment. Sixty-three percent of the boys were infected with gastrointestinal parasites and, of the infected boys, 80-95% had light parasite loads. Parasites found included Necator americanus, Ascaris lumbricoides, Entamoeba histolytica, Trichuris trichiura, Giardia spp., and Enterobius vermicularis. Infected boys had significantly lower weight, stature, weight-for-height (among 6-9-year-old boys), Hb levels, and &Vdot;O(2) max (ANCOVA, controlling for age and SEC). In terms of nutritional status, infected boys were 1.47 times more likely to be classified as iron deficient than noninfected boys (chi-square, P < 0.001), and 1.61 times more likely to be classified as stunted (P < 0.001). Infection was not associated with wasting in any SEC group. In conclusion, light to moderate gastrointestinal parasite loads were associated with significantly lower weight, stature, weight-for-height (in 6-9-year-old boys), Hb levels, and &Vdot;O(2) max, and a significantly higher frequency of IDA and stunting. These data suggest that comprehensive analyses of the nutritional status of populations in regions endemic for parasitic infection should include testing for the presence of infection. Am. J. Hum. Biol. 11:763-771, 1999. Copyright 1999 Wiley-Liss, Inc.

Study of renal metabolic disturbances related to renal lithiasis at school age in very-low-birth-weight children.

We studied 34 asymptomatic children who were born with a very-low-birth-weight (VLBW) and had no perinatal history of acute renal failure nor treatment with furosemide. The study was done at preschool or school age, looking for echographic changes and renal tubular disturbances which are known to predispose to renal lithiasis. The results were compared with those of a control group of 18 children who had been born at term with a body weight >2,500 g. One or more renal tubular disturbances were found in 64.70% of the VLBW children. Most frequently found were decreased ammonium excretion in response to furosemide (38.23%), enhanced N-acetylglucosaminidase excretion (35.29%), hypercalciuria (26.47%), and hypocitraturia (23.53%). Echography revealed renal cortical hyperechogenicity (17.65%) and renal lithiasis (8.82 %) in some of the VLBW children. We found a significant positive correlation (r = 0.7) between the perinatal level of plasma phosphate and the total amount of H+ excreted in response to furosemide at preschool or school age. Because these renal tubular anomalies may be precursors of future lithiasis, and the renal function and echography tests are not invasive, we suggest that renal tubular function be measured and followed up in every VLBW child, particularly when perinatal hypophosphatemia has occurred.

20-HETE mediates the effect of parathyroid hormone and protein kinase C on renal phosphate transport.

Parathyroid hormone (PTH) is a major inhibitor of renal proximal tubule (PT) sodium-dependent phosphate (Na+-Pi) cotransport. PTH is thought to exert its effect on Pi transport in the PT via the protein kinase A (PKA) and C (PKC) intracellular signalling pathways. PKC-dependent phosphorylation of phospholipase A2 stimulates arachidonic acid (AA) release, the latter a potent inhibitor of Pi transport. In turn, AA is metabolized to 20-hydroxyeicosatetraenoic acid (20-HETE) in the PT. In addition, 20-HETE production is stimulated by PTH. We therefore explored the possibility that 20-HETE may mediate the PTH/PKC inhibition of renal Na+-Pi cotransport. To this end, we tested the effect of 20-HETE on Na+-Pi cotransport in proximal tubule-like cells. Exposure of opossum kidney (OK) cells for 4 h to 20-HETE (10(-7) M) decreased Na+-dependent uptake of 32Pi (from 0.26 +/- 0.02 to 0.19 +/- 0.01 nmol/mg protein.min) by approximately 25% (P < 0.001). The inhibition was due to a reduction in Vmax. 20-HETE had no significant effect on either the apical amiloride-sensitive and insensitive 22Na uptakes or on basolateral ouabain-sensitive 86Rb uptake, and was specific for Pi. These results indicate that 20-HETE specifically inhibits Na+-dependent Pi transport in OK cells and that it may be a mediator of PTH action in the PT.

A putative growth-related renal Na(+)-Pi cotransporter.

The mRNA that encodes for NaPi-2, the renal Na(+)-Pi cotransporter that is upregulated by Pi depletion in the adult rat, is low in the young animal. Yet, renal Na-Pi cotransport rates are higher in rapidly growing than in fully grown rats. The aim of this study was to unravel the molecular basis of this apparent discrepancy. Poly(A) RNA obtained from the renal cortex of young animals induced higher rates of Na(+)-Pi cotransport in oocytes than equal amounts of poly(A) mRNA obtained from the renal cortex of mature rats. Moreover, poly(A) RNA obtained from renal cortex of rapidly growing animals treated with antisense NaPi-2 oligomers or depleted of NaPi-2 transcripts by subtractive hybridization with cDNA generated from the renal cortex of adult rats retained its ability to induce Na(+)-Pi cotransport in oocytes. In addition, renal poly(A) RNA of the young subjected to subtraction hybridization generated a 379-base pair reverse transcriptase-polymerase chain reaction product common to all known type II Na(+)-Pi cotransporters. These observations permit us to surmise that the high rates of Na(+)-Pi cotransport prevailing during growth are due, at least in part, to the expression of a specific mRNA that is only partially homologous to that of NaPi-2.

Renal protein degradation in streptozotocin diabetic mice.

In diabetes mellitus, the kidney and the gut--but not the liver or the muscle--undergo hypertrophy. Hypertrophy may be due to an increase in the rate of protein synthesis and/or to a decrease in proteolysis. In order to resolve this issue we assessed in vivo the rates of protein degradation by measuring the disappearance rates of 3H- and 14C-labelled valine incorporated into renal and liver proteins of control and streptozotocin-diabetic mice. The half-life of labelled valine, which is inversely related to the rate of protein degradation, was shorter and of similar magnitude both in the kidneys and livers of diabetic (70 +/- 6 h) than of control (96 +/- 8 h) animals. Yet, only the kidneys of diabetic animals underwent hypertrophy (0.33 +/- 0.01 vs. 0.29 +/- 0.01 g, P < 0.05; 2.22 +/- 0.10 vs. 1.64 +/- 0.05% of body weight, P < 0.01)). The increase in protein degradation rate affected all subcellular (mitochondrial, nuclear, microsomal and cytosolic) fractions of the kidney but not the mitochondria of liver. We surmise that an increase in the degradation of proteins is a widely generalized phenomenon in diabetes mellitus. For reasons that are still unclear, but that may relate to the hyperglycaemia, in the kidneys of diabetic animals this process is associated with a transiently larger increase in protein synthesis resulting in hypertrophy, whereas in liver, changes in protein degradation proceed 'pan passu' with those in protein synthesis resulting in the preservation of organ weight. Accelerated renal protein turnover and hypertrophy are early manifestations and perhaps harbingers of more severe renal changes in diabetes mellitus.

Age dependence of tolerance to anoxia and changes in cytosolic calcium in rabbit renal proximal tubules.

Calcium(Ca2+)-dependent processes mediate, in part, anoxic cell injury. These may account for the difference in sensitivity to anoxia between certain immature and mature renal cells. To address this question, we studied the effects of anoxia on cytosolic free Ca2+ concentration ([Ca2+]i), cell integrity, and transport functions in microdissected proximal convoluted tubules (PCT) of < 3-week-old (newborn) and > 12-week-old (adult) rabbits. Tubules were loaded with 10 microM fura-2 AM by incubation for 60 min at 37 degrees C, and then superfused with isosmotic saline solution gassed with either 95%O2-5%CO2 (control group) or 95%N2-5%CO2 (anoxia group) for 30 min. [Ca2+]i was measured ratiometrically; cell damage was assessed by nuclear binding of propidium iodide (PI). Anoxia resulted in a fourfold increase in [Ca2+]i in adult tubules (from resting values of 245 +/- 10 to 975 +/- 100 nM, P < 0.001), whereas in newborn tubules the rise was significantly less (from resting values of 137 +/- 5 to 165 +/- 5 nM, P < 0.001 between anoxic groups). Transient exposure to 100 mM potassium chloride, which depolarizes the PCT cells, induced increases in [Ca2+]i from baseline, to 920 +/- 90 nM in tubules from adult and to 396 +/- 16 nM in those from newborn rabbits (P < 0.001 between age groups). After exposure to ligands such as parathyroid hormone (PTH) and ATP, [Ca2+]i increased in both newborn and adult tubules, but to lower levels in newborn tubules. The response to PTH and ATP was transient in both age groups, [Ca2+]i returning to baseline levels after 2 min. Following anoxia, tubules from adult animals exhibited staining of all cell nuclei by 1 min exposure to PI, indicative of gross permeabilization of the cells. Nuclei of anoxic immatures tubules did not stain with PI. The sodium-dependent uptakes of a glucose analogue (14C-alpha-methyl-glucopyranoside) and phosphate (32Pi) were preserved in agarose-filled tubules of newborns after anoxia, whereas in those of adults recovery from anoxia was associated with drastic reduction in the uptake of these solutes. Overall, our results suggest that: (1) during anoxia, cell Ca2+ rises to critical levels in PCTs of adults compared with those of < 3-week-old animals, (2) Ca2+ influx occurs via a pathway activated by exposure to high [K+]o, presumably voltage-sensitive Ca2+ channels or reversal of Na(+)-Ca2+ exchange, (3) these pathways are either less active or less abundant in proximal tubules of newborn compared with adult rabbits, and (4) secondary active transport activity and cellular integrity are well preserved after anoxia in PCT cells of newborn but not of adult rabbits.

Mechanism of renal phosphate retention during growth.

We have previously demonstrated that the retention of phosphate required for growth is due to a a high Vmax of the Na(+)-Pi cotransport system located in the brush border membrane of the proximal tubule. Because of this and other similarities between adaptation of the kidney to a high Pi demand (growth) and that to low Pi supply, we measured the levels of NaPi-2 mRNA and cDNA present in kidney cortex of 3- and > 12-week-old rats. Like in Pi depletion, Western blots revealed that a 80 to 85 kDa protein recognized by a polyclonal antibody directed against the N-terminal region of the NaPi-2 protein was 2.3-fold more abundant in renal microvilli of the young than of adult animals. However, unlike in Pi depletion, Northern blot analysis failed to reveal a significant difference between mRNA levels at the two ages. Furthermore, suppression of NaPi-2 mRNA activity by annealing with antisense oligomers, or removal of the NaPi-2 transcripts by subtractive hybridization did not affect the rate of Na(+)-Pi cotransport induced in oocytes by polyA RNA of rapidly growing animals, while abolishing the ability of the renal cortical polyA RNA of adult rats to encode for Na(+)-Pi cotransport. RT-PCR of subtracted polyA RNA using primers specific for a region conserved in NaPi type II (Pi modulated) cotransporters yielded a product that was 98% homologous with that region, despite the absence of NaPi-2 cDNA. The results of these experiments demonstrate that the polyA RNA from kidneys of young animals contains unique mRNA transcripts able to encode for a NaPi protein homologous to, but distinct from NaPi-2.