Programming of Renal Development and Chronic Disease in Adult Life.

Chronic kidney disease (CKD) can have an insidious onset because there is a gradual decline in nephron number throughout life. There may be no overt symptoms of renal dysfunction until about two thirds or more of the nephrons have been destroyed and glomerular filtration rate (GFR) falls to below 25% of normal (often in mid-late life) (Martinez-Maldonaldo et al., 1992). Once End Stage Renal Disease (ESRD) has been reached, survival depends on renal replacement therapy (RRT). CKD causes hypertension and cardiovascular disease; and hypertension causes CKD. Albuminuria is also a risk factor for cardiovascular disease. The age of onset of CKD is partly determined during fetal life. This review describes the mechanisms underlying the development of CKD in adult life that results from abnormal renal development caused by an adverse intrauterine environment. The basis of this form of CKD is thought to be mainly due to a reduction in the number of nephrons formed in utero which impacts on the age dependent decline in glomerular function. Factors that affect the risk of reduced nephron formation during intrauterine life are discussed and include maternal nutrition (malnutrition and obesity, micronutrients), smoking and alcohol, use of drugs that block the maternal renin-angiotensin system, glucocorticoid excess and maternal renal dysfunction and prematurity. Since CKD, hypertension and cardiovascular disease add to the disease burden in the community we recommend that kidney size at birth should be recorded using ultrasound and those individuals who are born premature or who have small kidneys at this time should be monitored regularly by determining GFR and albumin:creatinine clearance ratio. Furthermore, public health measures aimed at limiting the prevalence of obesity and diabetes mellitus as well as providing advice on limiting the amount of protein ingested during a single meal, because they are all associated with increased glomerular hyperfiltration and subsequent glomerulosclerosis would be beneficial.

Fetal tubuloglomerular feedback in an ovine model of mild maternal renal disease.

Fetuses of pregnant ewes, which were subtotally nephrectomized prior to mating, were studied to assess whether mild maternal renal impairment would affect fetal tubuloglomerular feedback (TGF) under control conditions and after the inhibition of macula densa-derived nitric oxide (NO). Based on previous observations we hypothesized that, the TGF curve of fetuses of subtotally nephrectomized (STNx) ewes would resemble that of a volume expanded fetus with a high production rate of NO and that inhibition of neuronal nitric oxide synthase (nNOS) would increase the sensitivity of the TGF system in these fetuses. Renal function studies were performed on anaesthetized fetal sheep (133-140 days gestation; term ~150 days; Isoflurane 2-4% in oxygen). Fetuses were removed from the uterus and placed in a water bath (39.5°C) while maintaining umbilical blood flow. Glomerular filtration rate (GFR) and urine flow rate were markedly increased in fetuses of STNx ewes compared to fetuses of untreated ewes. Interestingly, and contrary to our hypothesis, the fetuses of STNx ewes exhibited no difference in TGF sensitivity in the presence or absence of 7-nitroindazole (7NI; nNOS inhibitor), compared to fetuses of untreated ewes, although sensitivity and reactivity increased in both groups after 7NI. There was however, a decrease in the stop flow pressure and net filtration pressure with an increase in the filtration coefficient (Kf). These factors suggest that maternal renal impairment drives the glomerular hypertrophy which has previously been found to be present in the neonatal period. Thus, we conclude that at ~138 days gestation, the fetal kidney has matured functionally and fetuses of STNx ewes are able to maintain fluid and electrolyte homeostasis even in the presence of increased transplacental flux.

Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits.

The beneficial effects of physical activity (PA) are well documented, yet the mechanisms by which PA prevents disease and improves health outcomes are poorly understood. To identify major gaps in knowledge and potential strategies for catalyzing progress in the field, the NIH convened a workshop in late October 2014 entitled "Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits." Presentations and discussions emphasized the challenges imposed by the integrative and intermittent nature of PA, the tremendous discovery potential of applying "-omics" technologies to understand interorgan crosstalk and biological networking systems during PA, and the need to establish an infrastructure of clinical trial sites with sufficient expertise to incorporate mechanistic outcome measures into adequately sized human PA trials. Identification of the mechanisms that underlie the link between PA and improved health holds extraordinary promise for discovery of novel therapeutic targets and development of personalized exercise medicine.

Expression of genes of the cardiac and renal renin-angiotensin systems in preterm piglets: is this system a suitable target for therapeutic intervention?

OBJECTIVES: The newborn circulating, cardiac and renal renin-angiotensin systems (RASs) are essential for blood pressure control, and for cardiac and renal development. If cardiac and renal RASs are immature this may contribute to cardiovascular compromise in preterm infants. This study measured mRNA expression of cardiac and renal RAS components in preterm, glucocorticoid (GC) exposed preterm, and term piglets. METHODS: Renal and cardiac RAS mRNA levels were measured using real-time polymerase chain reaction (PCR). Genes studied were: (pro)renin receptor, renin, angiotensinogen, angiotensin converting enzyme (ACE), ACE2, angiotensin type 1 receptor (AT1R) and angiotensin type 2 receptor (AT2R). RESULTS: All the genes studied were expressed in the kidney; neither renin nor AT2R mRNA were detected in the heart. There were no gestational changes in (pro)renin receptor, renin, ACE or AT1R mRNA levels. Right ventricular angiotensinogen mRNA levels in females were lower in preterm animals than at term, and GC exposure increased levels in male piglets. Renal angiotensinogen mRNA levels in female term piglets were lower than females from both preterm groups, and lower than male term piglets. Left ventricular ACE2 mRNA expression was lower in GC treated preterm piglets. Renal AT2R mRNA abundance was highest in GC treated preterm piglets, and the AT1R/AT2R ratio was increased at term. CONCLUSIONS: Preterm cardiac and renal RAS mRNA levels were similar to term piglets, suggesting that immaturity of these RASs does not contribute to preterm cardiovascular compromise. Since preterm expression of both renal and cardiac angiotensin II-AT1R is similar to term animals, cardiovascular dysfunction in the sick preterm human neonate might be effectively treated by agents acting on their RASs.

Expression of adrenoceptor subtypes in preterm piglet heart is different to term heart.

Preterm delivery increases the risk of inadequate systemic blood flow and hypotension, and many preterm infants fail to respond to conventional inotrope treatments. If the profile of cardiac adrenoceptor subtypes in the preterm neonate is different to that at term this may contribute to these clinical problems. This study measured mRNA expression of ß1, ß2, α1A, α2A and α2B-adrenoceptor subtypes by real time PCR in term (113d), preterm (91d) and preterm piglets (91d) exposed to maternal glucocorticoid treatment. Abundance of ß-adrenoceptor binding sites in the left ventricle was measured using saturation binding assays. Relative abundance of ß1-adrenoceptor mRNA in untreated preterm hearts was ∼50% of term abundance in both left and right ventricles (P<0.001). Trends in receptor binding site density measurements supported this observation (P = 0.07). Glucocorticoid exposure increased ß1-adrenoceptor mRNA levels in the right ventricle of preterm hearts (P = 0.008) but did not alter expression in the left ventricle (P>0.1). Relative abundance of α1A-adrenoceptor mRNA was the same in preterm and term piglet hearts (P = >0.1) but was reduced by maternal glucocorticoid treatment (P<0.01); α2A-adrenoceptor mRNA abundance was higher in untreated and glucocorticoid exposed preterm piglet hearts than in term piglets (P<0.001). There was no difference between male and female piglets in mRNA abundance of any of the genes studied. In conclusion, there is reduced mRNA abundance of ß1-adrenoceptors in the preterm pig heart. If this lower expression of ß-adrenoceptors occurs in human preterm infants, it could explain their poor cardiovascular function and their frequent failure to respond to commonly used inotropes.

Delayed fracture healing in growth differentiation factor 5-deficient mice: a pilot study.

BACKGROUND: Growth differentiation factor-5 (GDF-5) is a key regulator of skeletogenesis and bone repair and induces bone formation in spinal fusions and nonunion applications by enhancing chondrocytic and osteocytic differentiation and stimulating angiogenesis. Elucidating the contribution of GDF-5 to fracture repair may support its clinical application in complex fractures. QUESTIONS/PURPOSE: We therefore asked whether the absence of GDF-5 during fracture repair impaired bone healing as assessed radiographically, histologically, and mechanically. METHODS: In this pilot study, we performed tibial osteotomies on 10-week-old male mice, stabilized by intramedullary and extramedullary nailing. Healing was assessed radiographically and histologically on Days 1 (n = 1 wild-type; n = 5 bp [brachopodism]), 5 (n = 3 wild-type; n = 3 bp), 10 (n = 6 wild-type; n = 3 bp), 14 (n = 6 wild-type; n = 6 bp), 21 (n = 6 wild-type; n = 6 bp), 28 (n = 7 wild-type; n = 6 bp), and 56 (n = 6 wild-type; n = 6 bp) after fracture. After 10 (n = 7 wild-type; n = 7 bp contralateral and n = 3 bp fractured tibiae), 14 (n = 6 wild-type; n = 6 bp), 21 (n = 6 wild-type; n = 6 bp), 28 (n = 6 wild-type; n = 3 bp), and 56 (n = 8 wild-type; n = 6 bp) days, the callus cross-sectional area was calculated. We characterized the mechanical integrity of the healing fracture by yield stress and Young's modulus at 28 (n = 6 wild-type; n = 3 bp) and 56 (n = 8 wild-type; n = 6 bp) days postfracture. RESULTS: The absence of GDF-5 impaired cartilaginous matrix deposition in the callus and reduced callus cross-sectional area. After 56 days, the repaired bp fracture was mechanically comparable to that of controls. CONCLUSIONS: Although GDF-5 deficiency did not compromise long-term fracture healing, a delay in cartilage formation and remodeling supports roles for GDF-5 in the early phase of bone repair. CLINICAL RELEVANCE: Local delivery of GDF-5 to clinically difficult fractures may simulate cartilage formation in the callus and support subsequent remodeling.

Programming of the renin response to haemorrhage by mild maternal renal impairment in sheep.

1. The aim of the present study was to test the hypothesis that the renin response to mechanisms activated by haemorrhage is programmed by exposure to maternal renal dysfunction. 2. In 26-27-day-old lambs born to ewes that had reduced renal function (STNxL, n=10) and lambs born to ewes with normal renal function (ConL, n=6), 1.6 mL/kg per min of blood was removed over 10 min. 3. Under basal conditions, the STNxL group had increased mean arterial pressure (P < 0.05). In response to haemorrhage, mean arterial pressure decreased in the STNxL group (P < 0.001), but there was no significant change in the ConL group. 4. Although plasma renin level increased in both groups (P < 0.05), the peak response was reduced and delayed in the STNxL group. In contrast, the rise in arginine vasopressin (AVP) level was similar in both groups and occurred over the same time course. At 24 h, both plasma renin and AVP level were the same as those measured before haemorrhage in both groups. Kidney renin level was similar in the two groups. 5. The attenuated renin response to haemorrhage in the STNxL group might explain the inability to maintain arterial pressure after haemorrhage. The results of the present study suggest that the renin response of the postnatal kidney to reductions in blood volume can be affected by the intrauterine environment. If these changes persist into adulthood, it suggests that permanent programming has occurred. Thus, the ability of an individual to respond to acute severe reductions in blood volume might be determined during intrauterine life.

Effects of intrafetal IGF-I on growth of cardiac myocytes in late-gestation fetal sheep.

Intrafetal insulin-like growth factor (IGF)-I promotes cardiac hypertrophy in the late-gestation fetal sheep; whether these effects are sustained is unknown. IGF-I was infused for 4 days at 80 microg/h from 121 to 125 days of gestation, and its effects at 128 days, 3 days after the infusion stopped, were determined by comparison with untreated fetal sheep. After IGF-I treatment, fetal weights were similar to those in control fetuses but kidney weights were bigger (P < 0.05), as were spleen weights of male fetuses (P < 0.05). Cardiac myocytes were larger in female than male fetal sheep (P < 0.001). IGF-I increased male (P < 0.001) but not female myocyte volumes. IGF-I did not alter the proportions of uni- or binucleated right or left ventricular myocytes. Female fetal sheep had a greater proportion of binucleated cardiac myocytes than males (P < 0.05). IGF-I-treated fetuses had a slightly greater proportion of right ventricular nuclei in cell cycle phase G(2)/M and a reduced proportion of G(0)/G(1) phase nuclei (P < 0.1). Therefore, evidence for IGF-I-stimulated cardiac cell hyperplasia in fetal sheep in late gestation was limited. In conclusion, the greater sizes and larger proportion of binucleated cardiac myocytes in female fetal sheep suggest that myocyte maturation may occur earlier in females than in males. This may explain in part the male sex-specific responsiveness of cardiac hypertrophy to IGF-I in late gestation. If IGF-I-stimulated cardiomyocyte growth is accompanied by maturation of contractile function, IGF-I may be a potential therapeutic agent for maintaining cardiac output in preterm males.

Interactions between subtotal nephrectomy and salt: effects on blood pressure and renal function in pregnant and nonpregnant ewes.

The effects of high salt intake on blood pressure and renal function were studied in nine subtotally nephrectomized pregnant ewes (STNxP) and seven intact pregnant ewes (IntP) in late gestation and in eight subtotally nephrectomized nonpregnant ewes (STNxNP) and seven intact nonpregnant ewes (IntNP). STNxP had higher mean arterial pressures (P < 0.02) and plasma creatinine levels (P < 0.001) than IntP. High salt (0.17 M NaCl as drinking water for 5 days) did not change blood pressure in either STNxP or IntP. STNxNP had higher mean arterial pressures (P = 0.03) and plasma creatinine levels (P < 0.001) than IntNP. In STNxNP, blood pressure increased with high salt intake and there was a positive relationship between diastolic pressure and sodium balance (r = 0.497, P = 0.05). This relationship was not present in IntNP, STNxP, or IntP. Because high salt intake did not cause an increase in blood pressure in STNxP, it is concluded that they were protected by pregnancy from further rises in blood pressure. The observed increase in glomerular filtration rate (P < 0.03) and depression of fractional proximal sodium reabsorption (P = 0.003) that occurred in STNxP, but not in STNxNP, in response to high salt may have contributed to this protection. As well, the increased production of vasorelaxants in pregnancy may selectively protect against the occurrence of salt-sensitive hypertension in pregnancy.

Glomerular hypertrophy in offspring of subtotally nephrectomized ewes.

We have shown that fetuses whose mothers underwent subtotal nephrectomy (STNx) before pregnancy had high urine flow rates and sodium excretions, but lower hematocrits, plasma chloride, and plasma renin levels compared with controls. To see if these functional differences in utero persist after birth and are the result of altered renal development, we studied 8 lambs born to STNx mothers (STNxL) and 10 controls (ConL) in the second week of life. These lambs were of similar body weights, nose-rump lengths and abdominal girths. Their kidney weights were not different (ConL 36.1 +/- 1.9 vs. STNxL 39.8 +/- 3.3 g), nor were kidney dimensions or glomerular number (ConL 423,520 +/- 22,194 vs. STNxL 429,530 +/- 27,471 glomeruli). However, STNxL had 30% larger glomerular volumes (both mean and total, P < 0.01) and there was a positive relationship between total glomerular volume and urinary protein excretion (P < 0.05) in STNxL. Despite this change in glomerular morphology, glomerular filtration rate, tubular function, urine flow, and sodium excretion rates were not different between STNxL and ConL, nor were plasma electrolytes, osmolality, and plasma renin levels. Thus while many of the functional differences seen in late gestation were not present at 1-2 weeks after birth, the alteration in glomerular size and its relationship to protein excretion suggests that exposure to this altered intrauterine environment may predispose offspring of mothers with renal dysfunction to renal disease in adult life.

Interactions between maternal subtotal nephrectomy and salt: effects on renal function and the composition of plasma in the late gestation sheep fetus.

Effects of altered maternal salt intake between 122 and 127 days gestation (term is 150 days) were studied in eight fetuses carried by ewes which had renal insufficiency caused by subtotal nephrectomy (STNxF) and seven fetuses carried by intact ewes (IntF). Plasma sodium and osmolality were increased in ewes with subtotal nephrectomy on a high-salt intake (0.17 m NaCl in place of drinking water for 5 days; P < 0.05). The STNxF had normal body weights. A high maternal salt intake did not affect fetal blood pressure or heart rate. Plasma osmolality was higher in STNxF (P < 0.001), and plasma sodium and osmolality were increased by high salt (P < 0.001 and P < 0.04, respectively). The STNxF had higher urinary osmolalities (P = 0.002), which were also increased by a high maternal salt intake (P = 0.03). Renal blood flow fell in STNxF in response to a high maternal salt intake, but increased in IntF (P = 0.003). In STNxF but not IntF, glomerular filtration rate and urinary protein excretion were positively related to fetal plasma renin levels (P < or = 0.01). It is concluded that the salt intake of pregnant ewes with renal insufficiency affects maternal and fetal osmolar balance, fetal plasma sodium and fetal renal function. Since STNxF also had altered renal haemodynamic responses to high maternal salt and evidence of renin-dependent glomerular filtration and protein excretion, we suggest that interactions between dietary salt and pre-existing maternal renal disease impair glomerular integrity and function in the fetus.

Identification and isolation of synovial dendritic cells.

In rheumatoid arthritis patients, three compartments need to be considered: peripheral blood, synovial fluid, and synovial tissue. Dendritic cells characterized from each compartment have different properties. The methods given are based on cell sorting for isolation of cells, and flow cytometry and immunohistochemical staining for analysis of cells in these compartments.

The kidney is resistant to chronic hypoglycaemia in late-gestation fetal sheep.

We imposed a sustained reduction in glucose supply to late-gestation fetal sheep to see whether the reduction in glucose and insulin levels affected renal growth, renin expression and synthesis, and renal function. Maternal glucose concentrations were lowered to 1.7-1.9 mmol/L for 12-13 days by i.v. insulin infusion (n = 9, 121 days gestation, term = 150 days). Control ewes (n = 7) received vehicle. Maternal and fetal glucose concentrations were 40% and 31% lower than in controls (p < 0.001), respectively. Fetal plasma insulin levels fell 36% +/- 7% by day 7 (p < 0.05); IGF-I levels were unchanged. Arterial PO2 and pH increased and PCO2 fell (p < 0.05). Renal function was largely unaffected. Longitudinal growth was 28% slower and spleen weights were 36% smaller (p < 0.05); body and kidney weights were not affected. Renal renin levels and renin, angiotensinogen, and angiotensin receptor mRNA levels were similar to those of controls. Plasma renin levels increased from 2.1 +/- 0.6 to 7.6 +/- 2.8 ng angiotensin I.mL-1.h-1 (p = 0.01). Thus reductions in fetal glucose and insulin levels in late gestation that were sufficient to retard skeletal growth had no effect on kidney growth or function or the renal renin-angiotensin system, possibly because IGF-I levels were not reduced. There was, however, increased activity of the circulating renin-angiotensin system similar to that seen during insulin-induced hypoglycaemia.

Maternal renal insufficiency alters plasma composition and renal function in the fetal sheep.

To determine the effects of chronic maternal renal insufficiency on fetal renal function, we studied nine fetuses whose mothers underwent subtotal nephrectomy at least 2 mo before mating (STNxF) and seven fetuses from intact ewes (IntF) (126-128 days of gestation, term 150 days). STNxF had lower hematocrit (P < 0.05), plasma chloride (P < 0.01), and creatinine levels (P < 0.01), and the length-to-width ratio of their kidneys was reduced (P < 0.05). They excreted twice as much urine (P < 0.05) and sodium (P < 0.01). Total (P = 0.01) and proximal fractional sodium reabsorptions (P < 0.05) were lower in STNxF; distal delivery of sodium (P < 0.05) and distal fractional sodium reabsorption (P < 0.05) were higher. They tended to have suppressed renin levels (P = 0.06). Infusions of amino acids (alanine, glycine, proline, and serine at 0.32 mmol/min for 1 h and 0.64 mmol/min for 2 h intravenously), known to stimulate renal blood flow and glomerular filtration rate in fetal sheep, did so in IntF (P < 0.01). Arterial pressure also increased (P < 0.01). These effects were not observed in STNxF. In summary, chronic maternal renal insufficiency was associated with profound alterations in fetal renal excretion of fluid and electrolytes and impaired renal hemodynamic and glomerular responses to amino acid infusion. Whether these marked changes in the renal function of fetuses carried by STNx ewes are associated with alterations in renal function in postnatal or adult life remains to be determined.

Long-term effects of a midgestational asphyxial episode in the ovine fetus.

We and others have shown previously that fetuses at midgestation can survive 30 min of complete umbilical cord occlusion, although hydrops fetalis (or gross fetal edema) results. To investigate whether this hydrops resolves by late gestation and if there are any long-term consequences of the asphyxial insult on the heart and kidneys, eight fetuses were subjected to 30 min of complete umbilical cord occlusion at 0.6 gestation (90 days; term 150 days) and were compared to a sham group (n = 10). During the occlusion period, fetuses became severely hypoxemic, hypercapnemic, and acidotic, with both blood pressure and heart rate decreasing. Most variables had returned to normal by 2-hr recovery. At 129 +/- 1 days of gestation, approximately 40 days post occlusion, some fetuses were still slightly hydropic as skin fold measurements were increased (P < 0.01), although fetal body weight was not different from the sham group. The two groups had similar heart and kidney weights, ventricular cardiac myocyte nucleation, and glomerular number. By contrast, brain weight was reduced by 37% (P < 0.001) and the cerebral lateral ventricles were grossly dilated. Lungs were 50% smaller than in sham fetuses (P < 0.001). Thus, the hydrops that develops at midgestation as a result of a severe asphyxial episode can, but does not always, fully resolve by late gestation. Also, while fetuses at midgestation can survive this asphyxial episode with no long-term impact in renal or cardiac size, nephron number, or cardiomyocyte nucleation, the brain and lungs are severely affected.

Effects of a reduction in maternal renal mass on pregnancy and cardiovascular and renal function of the pregnant ewe.

Maternal renal disease is associated with high maternal and fetal morbidity. To establish an animal model to study renal dysfunction in pregnancy and its potential role in programming for renal disease and hypertension in adult life, a kidney was removed from each of 16 nonpregnant ewes, and a branch of the renal artery of the remaining kidney was ligated (STNx ewes). The 16 STNx and 15 intact ewes were time mated 2.5-17 mo later and studied at 119-132 days of gestation. STNx ewes demonstrated renal hypertrophy and glomerular hyperfiltration. They had higher diastolic arterial pressures (P < 0.05) and larger left ventricles (P < 0.0005), drank more water (P < 0.01), were hypochloremic (P < 0.01) and hyperglycemic (P < 0.0005), and had higher plasma creatinine levels (P < 0.0005) than intact ewes. Effective renal plasma flows and glomerular filtration rates were lower (P < 0.01) and protein excretion was greater (P < 0.05) in STNx than in intact ewes. Glomerulotubular balance was impaired in STNx ewes. Proximal tubular Na(+) reabsorption was reduced (P < 0.05), so Na(+) excretion was increased (P < 0.05). In STNx ewes, filtered K(+) loads were reduced (P < 0.005), but K(+) excretion was the same as in intact ewes. There was net K(+) secretion in STNx ewes; in intact ewes, there was net reabsorption. Plasma renin and angiotensinogen concentrations in STNx and intact ewes were similar, so the hypertension in STNx ewes was not renin dependent. STNx fetuses grew normally, and their blood gases, blood pressure, and heart rates were normal. These alterations in maternal fluid and electrolyte balance and the potential risk of maternal salt depletion or hyperkalemia may adversely affect the fetus.

Effects of 7-day amino acid infusion on renal growth, function, and renin-angiotensin system in fetal sheep.

These experiments examined whether renal growth and the fetal renin-angiotensin system could be stimulated by infusion of amino acids and whether chronic amino acid infusions restored glomerulotubular balance, which had been disrupted during 4-h infusions. Five fetal sheep aged 122 +/- 1 days gestation received an infusion of alanine, glycine, proline and serine in 0.15 M saline at 0.22 mmol/min for 7 days. Six control fetuses were given saline at the same rate (5 ml/h). Kidney wet weights after amino acid infusion were 28% larger than control fetuses (P < 0.05), and renal angiotensinogen mRNA levels were approximately 2.6-fold higher (P < 0.005). Circulating renin levels and renal renin mRNA levels were suppressed (P < 0.05), and renal renin protein levels tended to be lower. Arterial pressure was increased, and there was a marked, sustained natriuresis and diuresis. Glomerular filtration rate and filtered sodium were approximately two-fold higher throughout infusion (P < 0.05). Fractional proximal sodium reabsorption, suppressed at 4 h (from 73.4 +/- 6.5 to 53.7 +/- 10.2%), did not return to control levels (36.1 +/- 3.4% on day 7, P < 0.05). Distal sodium reabsorption was markedly increased (from 79 +/- 25 to 261 +/- 75 mumol/min by day 7, P < 0.005), but this was not sufficient to restore glomerulotubular balance. The resultant high rates of sodium excretion led to hyponatremia and polyhydramnios. In conclusion, long-term amino acid infusions increased renal angiotensinogen gene expression, kidney weight, and distal nephron sodium reabsorptive capacity but failed to restore proximal and total glomerulotubular balance.

Rheumatoid arthritis synovium contains plasmacytoid dendritic cells.

We have previously described enrichment of antigen-presenting HLA-DR+ nuclear RelB+ dendritic cells (DCs) in rheumatoid arthritis (RA) synovium. CD123+HLA-DR+ plasmacytoid DCs (pDCs) and their precursors have been identified in human peripheral blood (PB), lymphoid tissue, and some inflamed tissues. We hypothesized recruitment of pDCs into the inflamed RA synovial environment and their contribution as antigen-presenting cells (APCs) and inflammatory cells in RA. CD11c+ myeloid DCs and CD123+ pDCs were compared in normal and RA PB, synovial fluid (SF), and synovial tissue by flow cytometry, immunohistochemistry, and electron microscopy and were sorted for functional studies. Nuclear RelB-CD123+ DCs were located in perivascular regions of RA, in a similar frequency to nuclear RelB+CD123- DCs, but not normal synovial tissue sublining. Apart from higher expression of HLA-DR, the numbers and phenotypes of SF pDCs were similar to those of normal PB pDCs. While the APC function of PB pDCs was less efficient than that of PB myeloid DCs, RA SF pDCs efficiently activated resting allogeneic PB T cells, and high levels of IFN-gamma, IL-10, and tumor necrosis factor alpha were produced in response to incubation of allogeneic T cells with either type of SF DCs. Thus, pDCs are recruited to RA synovial tissue and comprise an APC population distinct from the previously described nuclear RelB+ synovial DCs. pDCs may contribute significantly to the local inflammatory environment.

The CMRF58 antibody recognizes a subset of CD123hi dendritic cells in allergen-challenged mucosa.

CD123(hi) CD11c(-) dendritic cells (CD123(hi) DC) are a distinct subset of human DC present in bone marrow, blood, lymphoid organs, and peripheral tissues. Pathogen stimulation, cytokine, or CD40 ligation induces CD123(hi) DC maturation, involving a shift from their innate immune to cognate antigen-presenting functions. In this study, we revealed that blood CD123(hi) DC in the presence of cytokine (granulocyte macrophage-colony stimulating factor and interleukin-3) undergo progressive, step-wise maturation through an "early" stage, delineated by expression of the antigen detected by the new monoclonal antibody CMRF58 (CD123(hi)CMRF58(+)CD40(-)CD86(-)CD83(-)) to the "late" stage with costimulatory antigen expression (CD123(hi)CMRF58(+)CD40(+)CD86(+)CD83(+/-)). In this early stage, cytokine-maintained CD123(hi) DC do not display changes in their morphology, no longer produce interferon-alpha (IFN-alpha) in response to bacteria, and develop the capacity to induce proliferation and polarization of allogeneic T cells. CD123(hi)CMRF58(+) DC, phenotypically similar to in vitro cytokine-maintained CD123(hi) DC, were not detected in tonsil but are present in allergen-challenged nasal mucosa of allergic individuals. Thus, CD123(hi) DC in certain tissue environments such as allergen-challenged nasal mucosa share a common CD123(hi)CMRF58(+) phenotype with in vitro cytokine-maintained blood CD123(hi) DC characterized by lack of IFN-alpha production.

Extracellular zinc and ATP restore chloride secretion across cystic fibrosis airway epithelia by triggering calcium entry.

Cystic fibrosis (CF) is caused by defective cyclic AMP-dependent cystic fibrosis transmembrane conductance regulator Cl(-) channels. Thus, CF epithelia fail to transport Cl(-) and water. A postulated therapeutic avenue in CF is activation of alternative Ca(2+)-dependent Cl(-) channels. We hypothesized that stimulation of Ca(2+) entry from the extracellular space could trigger a sustained Ca(2+) signal to activate Ca(2+)-dependent Cl(-) channels. Cytosolic [Ca(2+)](i) was measured in non-polarized human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Primary human CF and non-CF airway epithelial monolayers as well as Calu-3 monolayers were used to assess anion secretion. In vivo nasal potential difference measurements were performed in non-CF and two different CF mouse (DeltaF508 homozygous and bitransgenic gut-corrected but lung-null) models. Zinc and ATP induced a sustained, reversible, and reproducible increase in cytosolic Ca(2+) in CF and non-CF cells with chemistry and pharmacology most consistent with activation of P2X purinergic receptor channels. P2X purinergic receptor channel-mediated Ca(2+) entry stimulated sustained Cl(-) and HCO(3)(-) secretion in CF and non-CF epithelial monolayers. In non-CF mice, zinc and ATP induced a significant Cl(-) secretory response similar to the effects of agonists that increase intracellular cAMP levels. More importantly, in both CF mouse models, Cl(-) permeability of nasal epithelia was restored in a sustained manner by zinc and ATP. These effects were reversible and reacquirable upon removal and readdition of agonists. Our data suggest that activation of P2X calcium entry channels may have profound therapeutic benefit for CF that is independent of cystic fibrosis transmembrane conductance regulator genotype.