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Glucocorticoids stimulate muscle atrophy through a cascade of signals that includes activation of FoxO transcription factors which then upregulate multiple genes to promote degradation of myofibrillar and other muscle proteins and inhibit protein synthesis. Our previous finding that glucocorticoids upregulate mRNA levels for FoxO1 in skeletal muscle led us to hypothesize that the FoxO1 gene contains one or more glucocorticoid response elements (GREs). Here we show that upregulation of FoxO1 expression by glucocorticoids requires the glucocorticoid receptor (GR) and binding of hormones to it. In cultured C2C12 myoblasts dexamethasone did not alter FoxO1 mRNA stability. Computational analysis predicted that the proximal promoter of the FoxO1 gene contained a cluster of eight GRE half sites and one highly conserved near-consensus SRE; the cluster is found between -800 and -2000bp upstream of the first codon of the FoxO1 gene. A reporter gene constructed using the first 2kb of the FoxO1 promoter was stimulated by dexamethasone. Removal of a 5' domain containing half of the GREs reduced reporter gene activity and removal of all GREs in this region ablated activation by dexamethasone. Restriction fragments of the cluster of 8 upstream GREs bound recombinant GR in gel shift assays. Collectively, the data demonstrate that the proximal promoter of the FoxO1 gene contains multiple functional GREs, indicating that upregulation of FoxO1 expression by glucocorticoids through GREs represents an additional mechanism by which the GR drives glucocorticoid-mediated muscle atrophy. These findings are also relevant to other physiological roles of FoxO1 such as regulation of hepatic metabolism.

In rapidly growing neonatal rats, the intestine is insensitive to vitamin D, and Ca absorption is solely mediated through a non-energy-dependent process. Changes in Ca absorption associated with pregnancy and lactation are qualitatively similar in vitamin D-replete and vitamin D-deplete rats. Moreover, in vivo studies in man and the rat have demonstrated that the bulk of Ca absorption is accomplished in the ileum, a segment with limited capacity for active Ca absorption and is relatively insensitive to the action of 1,25-dihydroxyvitamin D. In patients with intestinal bypass operations the degree of Ca malabsorption and bone mineral loss is proportional to the length of ileum, not duodenum or proximal intestine, removed. Bile salts and lactose are examples of agents which can augment vitamin D-independent ileal Ca absorption through the intercellular pathway.

Summary: Partial correction of uraemic anaemia restores the baseline hyperkinetic haemodynamics toward normal, reduces left ventricular size and mass and improves exercise tolerance in dialysis patients. These effects may, in large part, be attributed to increases in haematocrit. Information on whether erythropoietin also acts directly on the heart, as it does on the peripheral vasculature, is not available. Also remaining unanswered is the question as to whether the effects of erythropoietin on the heart reduce cardiovascular mortality in end-stage renal disease (ESRD) patients. In addition, we do not know whether complete (vs partial) correction of uraemic anaemia will further enhance the beneficial effect of erythropoietin, or whether such gains could be counterbalanced by side effects of erythropoietin, such as the development or exacerbation of hypertension. Studies in the peripheral vasculature suggest erythropoietin also acts directly on the vascular smooth muscle cells, exerting both vasopressive and growth effects. Better understanding of the mechanism and control of such non-erythropoietic actions of erythropoietin can optimize the beneficial effect and minimize the side effect of this hormone on the cardiovascular system.

Parathyroid hormone (PTH) and PTH-related peptide (PTH-rP) bind to a common receptor and initiate second-messenger cascades that stimulate bone turnover and hypercalcemia. However, PTH is more potent than PTH-rP in inducing bone resorption and coupled bone metabolism in intact tissue, suggesting that these proteins elicit dissimilar postreceptor responses. We compared the effects of PTH and PTH-rP on osteoblastic retraction, an early event that must occur before the osteoclast can achieve access to the underlying bone mineral and begin resorption. MC3T3-E1 mouse osteoblasts were incubated in vehicle or 4.8 nM PTH or PTH-rP with or without 1 mM dibutyryl cAMP (Bt2cAMP). Morphologic changes were observed from 0 to 120 min. PTH caused marked retraction within minutes, which was not enhanced by Bt2cAMP. PTH-rP or Bt2cAMP induced slower, more modest retraction than PTH. The combined effect of PTH-rP plus Bt2cAMP was greater than that of PTH-rP, but less than that of PTH. PTH-rP and PTH had similar effects on cAMP generation. Thus, compared to PTH, PTH-rP induces less osteoblastic retractile response, exposing less bone surface to osteoclastic resorption. This may account for its lower hypercalcemic potency in vivo and contribute to its relative inability to stimulate coupled bone resorption and formation.

Ileum displays little active transcellular calcium (Ca2+) absorption but is credited with the bulk of Ca2+ absorbed in vivo. We examined the effect of taurodeoxycholic acid (TDC, 2 mM), a bile salt, on mannitol (MN, a marker of intercellular solute traffic) and Ca2+ fluxes in rat ileum. In the absence of electrochemical gradients between the mucosal (M) and serosal (S) bathing media in an Ussing chamber, net flux (Jnet) was observed in the S-to-M direction for both MN and Ca2+, i.e., the unidirectional secretory S-to-M flux (Js-->m) exceeded the absorptive M-to-S flux (Jm-->s). Mucosal TDC caused simultaneous increase in transepithelial conductance and Js-->m for both MN and Ca2+. This was followed by even greater increases in MN and Ca2+ Jm-->s, so that ultimately Jm-->s equaled Js-->m in each case. In control tissue, Js-->m for Ca2+ appeared to permeate exclusively through the intercellular MN pathway while part of Jm-->s for Ca2+ appeared to traverse through a non-MN route. After the TDC-induced increase in intercellular solute permeability, both Ca2+ fluxes appeared to traverse through the aqueous MN conduit. During the postprandial state, the presence of bile salts and the relative abundance of Ca2+ in ileal lumen can cause bulk Ca2+ absorption through the intercellular pathway.

Chronic lead exposure may cause hypertension in normotensive rats. This hypertensinogenic effect has been attributed to perturbations in the renin-angiotensin axis, the contractile resporase of the vascular smooth muscle, or the intracellular Ca2+ homeostasis as a consequence of the inhibition of Na+-K+-ATPase activity. In this study we examined the short-term effect of lead exposure on blood pressure, plasma renin activity, vascular contractility, and renal Na+-K+-ATPase activity and abundance in the spontaneously hypertensive rat. Our data indicate that modest lead exposure caused blood pressure elevation within two weeks in this rat strain that is genetically susceptible to the development of hypertension. This rapid blood pressure-elevating effect did not appear to depend on the mechanisms described in hypertension associated with more chronic lead exposure listed above. This acute model provides an additional approach to the study of lead-induced hypertension.

Magnesium absorption has been studied in both humans and animals under diverse experimental conditions. As a result, the data often appear confusing and conflicting. In this review we attempt to summarize information concerning Mg absorption and, where possible, to reconcile apparently conflicting observations. Most studies suggest that Mg is absorbed predominantly in the distal intestine. At usual Mg intakes, Mg absorption occurs primarily by intercellular diffusional and solvent drag mechanisms. There is evidence for a saturable component of Mg absorption in the small intestine and the descending colon that is important at low dietary Mg intakes. Pharmacological doses of vitamin D increase Mg absorption in both vitamin D-deficient and vitamin D-replete animals. A substantial amount of Mg absorption, however, occurs independent of vitamin D. In addition, vitamin D may reduce Mg retention through increases in urinary Mg excretion. Intestinal interactions between Mg and calcium or phosphate have been demonstrated in both humans and animals. The nature of these interactions cannot be readily explained by data currently available.

Transport characteristics of Ca and Mg were compared at three different levels of the absorptive process in male (120-170 g) Wistar rats. Balance studies in intact rats revealed that fractional Ca absorption decreased with increased Ca intake so that net Ca absorption remained constant. Fractional Mg absorption decreased modestly with increased Mg intake so that net Mg absorption increased proportionately with increased dietary Mg. Everted duodenal sacs demonstrated the presence of active Ca absorption with serosal-to-mucosal (S/M) ratio of 2.65 +/- 0.20 (n = 6), which was greater than unity (P less than 0.001). In contrast, the S/M for Mg did not exceed unity. Mucosal duodenal Ca uptake exhibited a large saturable (Michaelis constant of 4.80 +/- 0.34 mM, maximal velocity of 4.71 +/- 0.13 nmol.min-1.mg-1) and a small nonsaturable component (0.12 +/- 0.01 nmol.min-1.mg-1) in 5-wk-old rats (120 g). In 72-wk-old rats (600 g) the diffusional component of Ca uptake became predominant, and the slope increased significantly to 0.32 +/- 0.01 (P less than 0.05). Duodenal Mg uptake was completely concentration dependent and exhibited no age-related changes.

The epithelial tight junction (TJ) has three major functions. As a “gate,” it serves as a regulatory barrier separating and maintaining biological fluid compartments of different composition. As a “fence,” it generates and maintains the apicobasal polarity of cells that form the confluent epithelium. Finally, the TJ proteins form a trafficking and signaling platform that regulates cell growth, proliferation, differentiation, and dedifferentiation. Six examples are selected that illustrate the emerging link between TJ dysfunction and kidney disease. First, the glomerular slit diaphragm (GSD) is evolved, in part, from the TJ and, on maturation, exhibits all three functions of the TJ. GSD dysfunction leads to proteinuria and, in some instances, podocyte dedifferentiation and proliferation. Second, accumulating evidence supports epithelial-mesenchymal transformation (EMT) as a major player in renal fibrosis, the final common pathway that leads to end-stage renal failure. EMT is characterized by a loss of cell-cell contact and apicobasal polarity, which are hallmarks of TJ dysfunction. Third, in autosomal dominant polycystic kidney disease, mutations of the polycystins may disrupt their known interactions with the apical junction complex, of which the TJ is a major component. This can lead to disturbances in epithelial polarity regulation with consequent abnormal tubulogenesis and cyst formation. Fourth, evidence for epithelial barrier and polarity dysregulation in the pathogenesis of ischemic acute renal failure will be summarized. Fifth, the association between mutations of paracellin-1, the first TJ channel identified, and clinical disorders of magnesium and calcium wasting and bovine renal fibrosis will be used to highlight an integral TJ protein that can serve multiple TJ functions. Finally, the role of WNK4 protein kinase in shunting chloride across the TJ of the distal nephron will be addressed.

OBJECTIVE Plasma renin is not elevated in recombinant human erythropoietin (rhEPO)-induced hypertension but angiotensin converting enzyme inhibitors reduce blood pressure in both human and animal studies. Since rhEPO elevates renin and angiotensinogen messenger RNAs in angiotensin II target tissues such as the aorta, we explored the actions of rhEPO on renin-angiotensin system-related gene transcription of cultured rat vascular smooth muscle cells. DESIGN AND METHODS To separate direct actions of rhEPO from those mediated secondarily by potential activation of the renin-angiotensin system, vascular smooth muscle cells were cultured with rhEPO and enalapril to inhibit the angiotensin converting enzyme and losartan to inhibit angiotensin II type 1 receptors. RESULTS Vascular smooth muscle cells cultured with rhEPO (6-8 units/ml) demonstrated elevations (40-120%) in messenger RNAs of the renin-angiotensin system (renin, angiotensinogen, angiotensin receptor types 1 and 2) and increased levels of several messenger RNAs known to respond to angiotensin II (transforming growth factor-beta, insulin-like growth factor-II, epidermal growth factor, c-fos and platelet-derived growth factor). In contrast, cells cultured in the presence of rhEPO and enalapril or losartan showed elevations of messenger RNA for only the two types of angiotensin II receptor. This increase was higher than that obtained when cells were cultured with rhEPO or either antagonist alone. The increase in specific binding of angiotensin II to cells cultured in the presence of rhEPO and enalapril or rhEPO and losartan paralleled the changes in receptor messenger RNA. CONCLUSIONS rhEPO exerts its primary action on vascular smooth muscle cells via an increase in angiotensin receptor messenger RNA, resulting in a parallel increase in angiotensin II receptor expression. We suggest that increased receptor expression secondarily mediates the expression of other renin-angiotensin system messenger RNAs, which leads to angiotensin II-responsive gene transcription. The elevation in angiotensin II receptors, as observed in response to rhEPO, may provide a mechanism by which other forms of renin-dependent hypertension are initiated.

Parathyroid hormone (PTH) binding to its osteoblastic receptors stimulates cytoplasmic retraction within minutes. We hypothesized that the calpains ( cal cium-activated pa pain -tike enzymes) contribute to PTH-induced osteoblastic retraction by catalyzing regulatory hydrolysis of cytoskeletal structural proteins or enzymes important in cytokinesis. N -Ac-Leu-Leunorleucinal (ALLN), a reversible calpain inhibitor, was tested for its ability to inhibit PTH-induced retraction in murine MC3T3-E1 osteoblastic cells. ALLN inhibited PTH-induced retraction for 30 minutes in cells cultured on polystyrene cultureware or gelatin-coated glass cover slips, supporting the hypothesis that PTH-induced activation of the calpains contributes to short-term changes in MC3T3-E1 cell shape. Inhibition of PTH-induced retraction occurred on two substrata, suggesting that interactions between the extracellular matrix and cell surface proteins are not the sole determinants of morphology. Intracellular events, such as hydrolysis of focal adherens junction proteins on the cytoplasmic face of the plasma membrane, may contribute to PTH-induced retraction.

In humans, blockade of the renin-angiotensin system with angiotensin converting-enzyme inhibitors (ANG CEI) prevents the rise in blood pressure associated with the administration of recombinant human erythropoietin (rhEPO). This study was conducted to determine whether rhEPO elevates blood pressure in normal Wistar rats and whether the renin-ANG system is affected. Groups of 10 rats each were given rhEPO, ANG CEI (enalapril), rhEPO + ANG CEI, or vehicle. Renin and/or renin substrate mRNA was measured in aortas, kidney, and heart; renin activity (PRA), inactive renin, and renin substrate were measured in plasma. rhEPO raised blood pressure in the normal rat without changing the plasma renin system. ANG CEI prevented this blood pressure rise. Renin-specific mRNA was increased by rhEPO in renal tissue, and renin substrate mRNA was significantly elevated in the kidney and aorta. mRNA for renin and renin substrate were not altered in the heart. In both aorta and kidney, a significant correlation was observed between renin substrate mRNA and blood pressure. The data indicate that rhEPO modulates specific tissue renin-ANG systems, which may contribute to blood pressure elevation.

Increased Na+/H+ antiport activity in the renal brush border membrane of SHR. Defect in renal salt excretion may play an important role in the pathogenesis of hypertension. We examined sodium (Na+) uptake by brush border membrane (BBM) vesicles of young (6 week old) spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) of the same age. SHR had lower urinary Na+ excretion (223.1 ± 9.3 vs. 266.3 ± 3.7 µEq/day/lOO g, N = 8, P < 0.01) and higher systolic blood pressure (98.9 ± 1.2 vs. 82.9 ± 1.8mm Hg, N = 8, P < 0.01) than WKY. BBM vesicle Na+ uptake, measured by rapid filtration technique, was higher in SHR when compared to WKY (1.44 ± 0.03 vs. 1.01 ± 0.06 nmol/mg/5 sec, N = 4, N < 0.01). This increase in Na+ influx was apparent only in the present of an outward-directed proton (H+) gradient and was abolished by 1mM amiloride. BBM permeability to H+ as assessed by acridine orange quenching was not different between SHR and WKY. Kinetic analyses of the amiloride-sensitive BBM Na+ uptake revealed a higher Vmax (2.13 ± 0.27 vs. 0.70 ± 0.30 nmol/mg/5 sec, N = 4, P < 0.01) and a higher km for Na+ (3.55 ± 0.32 vs. 1.23 ± 0.14mM, N = 4, P < 0.05) in SHR. These findings thus demonstrate an intrinsic derangement in BBM Na+ transport in young SHR which is characterized by increased Na+/H+ antiport activity. This alteration in antiport activity is not attributable to changes in membrane permeability to H+, and is characterized by higher Vmax and km. Similar reports of increased Na+/H+ antiport activity in other tissues of SHR suggest that a generalized membrane transport disorder may exist in this model of genetic hypertension.

The demands of growth are known to exacerbate the effect of phosphorus deprivation (PD). We examined whether changes associated with PD could be prevented in young rats in which growth and growth hormone (GH) were eliminated by hypophysectomy (HPX) and whether PD in normal intact rats (INT) was associated with increased secretion of GH. INT or thyroxine- and ACTH-replaced HPX rats were fed one of the three diets: 0.31% P (NP); 0.027% P (LP), and 0.31% P, pair-fed with LP-mates (NP-PF). The results indicate that HPX did not qualitatively alter several physiologic responses to PD: (a) serum and urinary phosphorus (P) decreased and urinary calcium (Ca) increased; (b) net intestinal Ca retention fell and duodenal sac uptake of 45Ca rose; and (c) external P balance was restored and duodenal sac uptake of 32P-phosphate increased. Only the hypercalcemia seen in INT, LP rats was prevented by HPX. In INT rats serum immunoassayable GH levels, measured in single samples, were not different between different dietary groups while pituitary bioassayable GH was reduced in both LP and NP-PF rats when compared to the NP rats. Thus, except for hypercalcemia, the physiologic responses associated with PD are not prevented by the elimination of growth and GH, and the development of these responses in INT rats was not associated with a consistent or specific alteration in GH secretion.

Some proposed mechanisms for the hypotensive effect of high calcium intake involve reduction in vascular responsivity. To assess the effect of dietary calcium on vascular responsivity, spontaneously hypertensive rats (SHR) were placed on normal (N-Ca; 0.4%) or high (H-Ca; 2.8%) casein-based synthetic diet for 4 wk. Intraarterial pressure, pressor response to graded intravenous infusion of norepinephrine (NE) and angiotensin II (ANG II), and in vitro vascular reactivity of tail artery segments to NE and transmural nerve stimulation (TNS) were studied. Urinary electrolyte excretion, plasma renin activity (PRA), aldosterone, NE, and epinephrine (EPI) were also determined. H-Ca SHR had a lower intraarterial systolic and diastolic pressure. However, H-Ca SHR had greater in vivo pressor response to both ANG II and NE. Maximal contractile force developed by tail artery segments in vitro in response to NE and TNS was slightly, but not significantly, higher in H-Ca SHR. In vitro dose-response curves to NE and TNS were not significantly different. Although H-Ca SHR had increased urinary excretion of sodium throughout the study period, PRA and aldosterone levels were similar in both groups. Plasma NE and EPI levels in the two groups were also not different. Despite lowered intra-arterial blood pressure, H-Ca SHR exhibited enhanced pressor response to ANG II and NE in vivo and a similar in vitro vascular reactivity to NE and TNS when compared with N-Ca SHR. Our results do not support a role for alterations in vascular reactivity to NE or ANG II in the hypotensive effect of high calcium intake in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium transport in red blood cells from dialyzed uremic patients. Studies on red blood cell (RBC) sodium (Na) transport in chronic renal failure have described abnormalities in the ouabain–sensitive Na, K pump. We now report Na transport in RBC using cation flux methodology, measuring both the ouabain–sensitive Na, K pump and the ouabain–insensitive Na, K cotransport (CoT) and Na, lithium (Li) countertransport (CTT) in 28 subjects on hemodialysis, eight subjects on chronic ambulatory peritoneal dialysis (CAPD) and 29 control subjects. Intracellular cation content and passive permeability of Na were also examined. Mean Na efflux through the ouabain–sensitive Na, K pump was not reduced in dialysis patients when compared to normal subjects, whether measured in fresh cells (1.41 ± 0.05 vs. 1.30 ± 0.03 mmole/liter RBC/hr; P < 0.05) or in Na-loaded cells (7.10 ± 0.24 vs. 6.90 ± 0.22; NS). There was, however, a marked and uniform suppression of the CoT pathway in Na-loaded cells from dialysis patients versus controls (0.14 ± 0.02 vs. 0.41 ± 0.05 mmole/liter RBC/hr; P < 0.001). Mean CTT activity, as measured by Li efflux, was not different between dialysis and normal subjects. Uremic and normal RBC had similar intracellular Na or K content as well as passive permeability for either ion. This indicates that intracellular cationic homeostasis is maintained, perhaps secondary to balanced changes in cationic flux activity through these transport pathways.

Patient 1. A 58-year-old man was hospitalized with hypertension, 168/114 mm Hg, congestive heart failure secondary to fluid retention, and a serum creatinine of 6.9 mgldl. Six years earlier he had received a cardiac transplant for end-stage idiopathic cardiomyopathy and was given cyclosporine (CsA). Prior to transplantation he was normotensive (blood pressure, 100/70 mm Hg) and had a serum creatinine concentration of 3.6 mg/dl, which was attributed to his refractory biventricular heart failure. He gave no family history for renal disease or high blood pressure. The serum creatinine decreased to 0.8 mg/dl one month after cardiac transplantation and fluctuated between 1 to 2 mg/dl for about four years before beginning an inexorably upward course. His blood pressure rose rapidly after transplantation, reaching 160/120 mm Hg by the second month. Four months before this admission, he had been evaluated for rapid deterioration in renal function and refractory hypertension; his blood pressure reached as high as 180/140 mm Hg. A percutaneous renal biopsy was obtained. An endomyocardial biopsy at the same time revealed no evidence of rejection. During this admission, acute dialysis was initiated, and thc patient's congestive heart failure improved markedly following a 10-liter fluid removal. He subsequently was maintained on chronic dialysis with continued good cardiac graft function. Patient 2. A 6-year-old girl first developed steroid-dependent nephrotic syndrome at age 26 months. A renal biopsy at 33 months showed minimal-change disease and normal juxtaglomerular apparatus (JGA, Fig. 1A). A chlorambucil-induced remission of eight weeks was followed by a relapse two months after the drug was discontinued. Her nephrotic syndrome became resistant to oral corticosteroid administration, responding only to intravenous methyiprednisolone. yclosporine (10—12 mg/kg/day), given in divided doses at eight-hour

Administration of cyclosporine to allograft recipients and patients with immunologically mediated disorders is associated with a high incidence of the development of hypertension. We studied the effect of CsA on blood pressure and on the in vitro tail artery contractile response to transmural nerve stimulation (TNS) and exogenous norepinephrine (NE) in the spontaneously hypertensive rat (SHR). Fourteen days of p.o. administration of CsA 5 mg/kg/day (CsA5) or CsA 20 mg/kg/day (CsA20) resulted in significant increases in blood pressure. The effective level of TNS that resulted in a 50% of maximal response (ED50) was significantly (P less than 0.05) decreased in the CsA5 and CsA20 animals compared to controls. The values for controls (n = 7), CsA5 (n = 10), and CsA20 (n = 9) were 4.3 +/- 0.3, 3.2 +/- 0.3, and 3.1 +/- 0.4 pulses/sec, respectively. In the CsA20 group, the dose-response curve to NE was significantly shifted to the left, and the ED50 was significantly (P less than 0.01) decreased compared to controls (5.7 +/- 0.8 x 10(-5) mol/L, n = 11, vs. 8.9 +/- 0.6 x 10(-5) mol/L, n = 12). We conclude that the in vitro contractile response to nerve stimulation is augmented by CsA. Some of the increase may be related to an enhancement of NE response, but a direct effect on neurovascular function is also suggested. This effect of CsA may be important in the development of hypertension and the changes in neurovascular tone seen with the clinical administration of this immunosuppressant.

We studied the effects of cyclosporin A on the renin-aldosterone axis in Sprague-Dawley rats. Two weeks of intragastric administration of cyclosporin A (5 mg/kg/day or or 20 mg/kg/day) resulted in large increases in plasma renin concentration (23 +/- 5, 70 +/- 12, and 79 +/- 11 ng/ml/hr in control rats and rats receiving 5 mg and 20 mg of cyclosporin A, respectively), with no parallel increments in plasma aldosterone. In vitro angiotensin II (ANG II)-stimulated aldosterone secretion by zona glomerulosa cells obtained from cyclosporin A-treated rats was also reduced (4.8 +/- 0.5, 1.5 +/- 0.2, and 0.2 +/- 0.2 ng/10(5) cells in control rats and rats receiving 5 mg and 20 mg of cyclosporin A, respectively). In contrast, in vitro aldosterone response to graded increments of potassium (3.7-10.7 mmol/L) or adrenocorticotropic hormone (ACTH) (10(-11)-10(-8) M) was preserved in cyclosporin A-treated rats. When added in vitro to zona glomerulosa cells from untreated rats, cyclosporin A also attenuated ANG II-stimulated aldosterone secretion, but did not affect potassium or ACTH-mediated aldosterone production. Thus, cyclosporin A-induced hyperreninemic hypoaldosteronism in the rat depends on opposing renal and adrenal effects, with a direct or feedback stimulation of renin secretion and a specific blockade of ANG II-mediated aldosterone production.

The two major lesions of renal osteodystrophy are osteitis fibrosa cystica (OFC) and osteomalacia (OM). OFC is the characteristic bone lesion of uremic hyperparathyroidism. Although renal failure causes predictable parathyroid hyperplasia, the precise pathogenetic mechanism is still not defined. The "hyperphosphatemia-hypocalcemia-parathyroid hormone (PTH) hypersecretion" sequence of events is no longer an adequate model for the pathogenesis of uremic hyperparathyroidism. Other abnormalities associated with uremia include reduced 1,25-dihydroxyvitamin D (1,25D) synthesis, changes in intracellular phosphorus content or transcellular phosphate fluxes, or alteration in PTH metabolism, eg, change in set-point for PTH secretion. Each abnormality interacts with others and contributes to PTH hypersecretion, but none can completely account for the development and persistence of hyperparathyroidism in renal failure. The possibility that uremia may directly cause parathyroid hyperplasia remains open. It is also possible that factor(s) that initiate hyperparathyroidism may turn out to be quite different from that which sustains the hyperparathyroid state. Although both vitamin D-deficient and vitamin D-resistant OM may develop in patients with renal failure, the majority of uremic OM seen currently is "vitamin D-refractory." Although now there is persuasive evidence implicating aluminum (Al) accumulation as the major pathogenetic cause for the mineralization defect seen in this disorder, additional disturbances may play important contributory roles. Such factors would include extraskeletal effects of Al, differences in host-susceptibility to this element, the localization of Al within bone, uremia per se, and the participation of other metals and toxins. Finally, possible interactions between hyperparathyroidism and OM of uremia are speculated on.

Previous studies from our laboratory have demonstrated that 1,25-dihydroxyvitamin D3 (1,25D) stimulates separate calcium (Ca) and inorganic phosphate (P) absorptive mechanisms in rat intestine (1,2). The proportion of Ca to P “pumps” varies in different regions of the intestine. Thus, there is a preponderance of Ca pumps in the duodenum, a preponderance of P pumps in the jejunum and an equal distribution of Ca and P pumps in the ileum (3). Interestingly, the colon has only 1,25D-responsive Ca pumps (2). The active Ca absorptive mechanisms in duodenum, ileum and colon appear to share certain characteristics, all exhibiting a Km of approximately 1 mM (2,3). The expression of this 1,25D-induced Ca absorption in colon does not appear to require the presence of extracellular sodium ions (4). In the present study we examined the sodium (Na) requirement of 1,25D-stimulated P transport processes in rat jejunum.

We studied the action of a glucocorticoid (GC, dexamethasone) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on transepithelial calcium (Ca) transport in rat distal colon. GC 1.2 mg or 1,25(OH)2D3 270 ng were given daily for 4 d and Ca fluxes were measured in vitro in the absence of electrochemical gradients (Ussing technique). Results: (a) Both 1,25(OH)2D3 and GC increased Ca absorptive flux from 24 +/- 3 (SEM) to 50 +/- 1 and from 23 +/- 1 to 38 +/- 4 nmol/cm2 per h, respectively (in each case n = 9, P less than 0.01); both steroid hormones had no effect on Ca secretory flux. (b) GC, but not 1,25(OH)2D3 increased the short-circuit current Isc) from 30 +/- 5; to 111 +/- 13 microA/cm2 (P less than 0.01), reflecting stimulation of electrogenic sodium (Na) transport. Choline replacement of Na in the bathing buffer abolished both the Isc and the active Ca transport induced by GC, but has no effect on the 1,25(OH)2D3-stimulated active Ca absorption. (c) When the buffer Ca concentration ([Ca]) on both sides of the epithelium was reduced from 1.25 to 1.25 X 10(-2) mM, the GC-induced, but not the 1,25(OH)2D3-induced, stimulation in Ca absorption was abolished. This suggests that the GC-stimulated Ca absorption may require a "threshold" Ca gradient across the luminal membrane through which Ca influx occurs. Thus, contrary to the current consensus, this study demonstrates that GC stimulates active Ca transport and that this action is mediated through a mechanism dependent on the presence of Na and a critical [Ca] in the ambient medium.

Although hypertension is a frequent complication of cyclosporin A (CSA) therapy in clinical practice, little experimental information is available on the nature and the mechanism of this form of hypertension. We studied the effect of currently recommended therapeutic dosages of CSA, i.e., 5 (CSA5) and 20 (CSA20) mg.kg-1.day-1, on blood pressure and the renin-aldosterone system (RAS) in spontaneously hypertensive rats (SHR). Influence of in vivo CSA treatment on in vitro angiotensin II (ANG II)-stimulated aldosterone secretion by isolated adrenal glomerulosa cells (AGC) was also measured. CSA treatment in SHR resulted in a consistent increase in systolic blood pressure. This increase in blood pressure occurred in the absence of significant changes in creatinine clearance in CSA5 rats, whereas in CSA20 rats a significant reduction in creatinine clearance was observed. Sodium balance and serum calcium and magnesium concentrations were not different between the control group and either of the two CSA-treated groups of rats. Plasma renin concentration (PRC) and inactive renin (IR) were markedly elevated, but plasma renin substrate remained unchanged with CSA administration. Despite the presence of hyperreninemia, plasma aldosterone was not elevated, suggesting that CSA may induce relative adrenal resistance to ANG II. This possibility was tested using AGC isolated from CSA-treated rats. ANG II-stimulated aldosterone secretion in AGC was diminished by low dose and aborted by high dose CSA-treatment. Thus CSA administration in SHR induces a predictable increase in blood pressure in association with "hyperreninemic hypoaldosteronism."

1. The regulatory role of prostaglandins (PGs) E2and F2aon the zinc transport rate across the jejunal segments of rats was examined by employing the Ussing chamber technique. The Zn flux rate from mucosa to serosa across jejunal segments (Jms) was 5·24 (SE 1·54) nmol/h per cm2(n48) and that from serosa to mucosa (Jsm) was 15·16 (SE 2·38) nmol/h per cm2(n48) when both sides of the segment were bathed with Ringer's bicarbonate solution containing 0·5 mM-zinc chloride and 3 mM-L-histidine.2. When 5·0 or 50 μM of either PGE2or PGF2αwere added to the serosal side of the tissue,Jsmgenerally decreased andJmsgenerally increased, compared with controls. On the other hand, when PGE2or PGF2αwas added to the mucosal side of the tissue,Jmseither did not change or increased whileJsmhad a tendency to decrease.3. The Zn uptake capacity of tissue increased significantly when PG was added to the serosal side of the tissuebathing medium, but not when PG was added to the mucosal side. The uptake capacity of mucosal Zn by jejunal segments was approximately twice that of serosal Zn.4. When PG was included in the tissue-bathing medium, the short-circuit current, potential difference and conductance between the mucosa- and serosa-bathing media generally decreased.5. These results suggest that (a) PGs influence Zn flux rate not by chelating Zn and carrying it across the mucosal cell membrane but by interacting with the cytosolic components, (b) it is the serosal PGs which control the Zn flux rate and (c) PGs play a part in triggering a transduction mechanism in the intestinal Zn transport process.

We examined the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on intestinal 45Ca and [32P]phosphate uptake in normal and mineral- and vitamin D-replete adult rats. The results indicate that 45Ca uptake by adult rat duodenum was stimulated by "physiological" doses of 1,25(OH)2D3. With increasing dosage of 1,25(OH)2D3, 45Ca uptake also became stimulated first in the colon and then in the jejunum and ileum. The increase in duodenal and jejunal 45Ca uptake was paralleled by an increase in [32P]phosphate uptake, but this parallelism was not always seen in the ileum and was never observed in the colon. The dissociated calcium and phosphate transport response to 1,25(OH)2D3 stimulation in the colon was further confirmed by the measurement of transmural fluxes using a modified Ussing technique. These responses to 1,25(OH)2D3 are similar to those observed in younger vitamin D-deficient rats. However, supraphysiological doses of 1,25(OH)2D3 caused weight loss in normal adult rats, whereas the same metabolite, even when given in large doses, led to weight gain in vitamin D-deficient rats. We propose the normal adult rat as an additional model for evaluating the biological action of 1,25(OH)2D3.

We measured serum erythropoietin levels serially in 31 renal-transplant recipients treated with cyclosporine, using the recently developed recombinant human erythropoietin-based radioimmunoassay. The mean (+/- SEM) serum erythropoietin concentration in these patients before transplantation (14 +/- 2 U per liter) was similar to that in normal subjects who did not have anemia. A transient postoperative 9-fold increase (range, 0- to 74-fold) in the serum erythropoietin levels was followed by a smaller (3-fold) and sustained (28 +/- 3 days) second elevation. The initial increase occurred in the absence of graft function and was not accompanied by an erythropoietic response, whereas the second increase was associated with graft recovery and the complete resolution of the anemia. Serum erythropoietin levels returned to normal as the hematocrit rose above 0.32. Thereafter, the hematocrit continued to rise toward normal, while the serum erythropoietin levels remained normal. The patients in whom erythrocytosis or iron-deficiency anemia developed had persistently elevated serum erythropoietin levels. We conclude that in patients who have undergone renal transplantation, slight increases in endogenous erythropoietin levels induce erythropoiesis to the same extent as do large doses of exogenous erythropoietin in patients with uremia. Moreover, once initiated, erythropoiesis in renal-transplant recipients may be sustained by normal serum erythropoietin levels. These results suggest that the restoration of renal function improves the erythropoietic response to erythropoietin.

The transport of inorganic phosphate (P) was measured in the absence of electrochemical gradients across rat jejunum in vitro. Active P absorption was demonstrated in young, vitamin D-deficient (-D) rats, whereas active P secretion was found in normal, non-vitamin D-deficient adult rats, suggesting regulation of intestinal P transport by age-dependent but vitamin D-independent mechanisms. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] stimulated mucosal-to-serosal P flux (Jm----s) without affecting serosal-to-mucosal P flux (Js----m), causing further increases in net P absorption (Jnet) in -D rats and reduction in net P secretion in normal adult rats, confirming a previously described role of this hormone on P absorption. We then examined the effect of increasing extracellular (buffer) P concentration [P] on this 1,25(OH)2D3-stimulated active P absorption in jejunum from -D rats. At [P] of 0.024, 0.24, and 2.4 mM, 1,25(OH)2D3 consistently stimulated Jm----s without affecting Js----m, causing an increment in Jnet. At 7.5 mM [P], however, this Jm----s stimulatory effect of 1,25(OH)2D3 was no longer observed. Moreover, at this [P] the Js----m exceeded Jm----s in both -D and 1,25(OH)2D3-repleted rats, converting the active P absorption into active P secretion in both treatment groups. These observations suggest the participation of local mechanisms, such as the ambient [P] of the transporting enterocytes, in the regulation of intestinal P absorption. Finally, the influence of dietary P deprivation on P absorption was studied in -D rats, with or without either vitamin D3 or 1,25(OH)2D3 repletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased dietary calcium intake in the adult spontaneously hypertensive rat (SHR) has been reported to correct low serum ionized calcium concentration ([Ca++]) and to result in a significant amelioration of the prevailing hypertension. In the present study we examined several parameters of calcium metabolism in young (6-week-old) SHR and compared them with those observed in normotensive Wistar-Kyoto (WKY) rats fed equal amounts of a diet containing normal quantities of calcium (0.4%, wt/wt) for 4 weeks. A separate group of SHR was placed on an equal amount of a high calcium (2.8%, wt/wt) but otherwise identical diet. In SHR and WKY eating a normal calcium diet, serum total calcium concentration was not different, but [Ca++] was lower in SHR (1.58 +/- 0.06 vs 1.91 +/- 0.07 mmol/liter, p less than 0.01). Serum immunoreactive parathyroid hormone (PTH) was increased in some, but not all, SHR. No difference was noted between the two groups in the following parameters: calcium intake, serum 1,25 dihydroxycholecalciferol (1,25(OH)2D3), urinary calcium excretion, fractional stool calcium content ([stool calcium/calcium intake] X 100), and in vitro 45Ca uptake by everted gut sacs constructed from segments of duodenum, mid-jejunum, ileum, and proximal colon. A high calcium diet corrected the abnormal serum [Ca++] and PTH but did not alter the progression or severity of the hypertension in SHR. A lower net weight gain was observed in SHR on a high calcium diet when compared to SHR eating normal calcium diet (9.1 +/- 1.8 vs 27.0 +/- 2.0 g).(ABSTRACT TRUNCATED AT 250 WORDS)

Fifteen patients with dialysis osteomalacia were treated with 24,25-dihydroxyvitamin D 3 in dosages up to 10 μg per day for two to 24 months. All had previously had no improvement during treatment with calcitriol but had been remarkably susceptible to hypercalcemia. When 24,25-dihydroxyvitamin D 3 was given with either calcitriol or dihydrotachysterol, serum calcium levels were significantly lower than during treatment with calcitriol or dihydrotachysterol alone. Eight of nine patients who received combined therapy with 24,25-dihydroxyvitamin D 3 and calcitriol for longer than two months had clinical improvement; six patients underwent repeated bone biopsy and showed evidence of improved bone mineralization. Patients who received 24,25-dihydroxyvitamin D 3 alone did not improve clinically. Since 24,25-dihydroxyvitamin D 3 appears to improve calcium homeostasis and bone mineralization in some patients with severe dialysis osteomalacia when administered with 1-hydroxylated vitamin D metabolites, further controlled studies are warranted.

1. Reduced Na+, K+ pump activity has been reported in uraemic erythrocytes, and this has been attributed to the presence of a circulating ouabain-like inhibitor. Since we have previously reported marked suppression of Na+, K+ co-transport activity in erythrocytes from dialysis patients, we have now examined the effect of incubation with uraemic plasma on Na+ efflux through the Na+, K+ pump and the co-transport pathway in normal erythrocytes from both men and women. 2. The results show that Na+ efflux via the Na+, K+ pump is not different between men and women (3.14 ± 0.14 vs 3.68 ± 0.18 mmol h−1 litre−1 of cells) and is not inhibited by incubation with uraemic plasma. 3. In contrast, co-transport-mediated Na+ efflux is lower in erythrocytes from normal women when compared with normal men (0.17 ± 0.02 vs 0.25 ± 0.05 mmol h−1 litre−1 of cells, P < 0.001). 4. Moreover, incubation with uraemic plasma causes stimulation of Na+ efflux through the co-transport pathway in erythrocytes of healthy women (0.17 ± 0.02 vs 0.27 ± 0.03 mmol h−1 litre−1 of cells, P < 0.005), but not in erythrocytes of healthy men (0.27 ± 0.03 vs 0.25 ± 0.05 mmol h−1 litre−1 of cells). 5. Our data do not support the presence of either a Na+, K+ pump or a co-transport inhibitor in plasma from dialysed uraemic patients. Thus, the suppressed co-transport activity demonstrated in uraemic erythrocytes cannot be attributed to a circulating inhibitor, and may reflect an acquired membrane or transport defect. 6. The stimulation of co-transport by uraemic plasma from women provides a further example of sex-determined characteristics in human erythrocytes, the mechanism of which remains to be elucidated.

To investigate the effect of metabolic acidosis on intestinal calcium (Ca) and phosphorus (P) absorption and vitamin D metabolism, metabolic balance studies and in vitro gut sac uptake of 45Ca and [32P]phosphate were performed in rats maintained on low-Ca and moderately low-P diet and fed NH4Cl for 3 or 9 days and pair-fed controls. Plasma 1,25(OH)2D concentration was measured in the rats fed NH4Cl for 9 days and their controls. Net Ca and P absorption was 87–92% in the acidotic rats and did not differ from control. Moreover, gut sac uptakes of 45Ca and [32P]phosphate were not different from control. Plasma 1,25(OH)2D was higher in the ammonium chloride-fed rats than in controls (213 +/- 44 vs. 110 +/- 12 pg/ml), and serum P was lower in the acidotic animals (4.6 +/- 0.7 vs. 7.6 +/- 0.3 mg/dl). These data indicate that metabolic acidosis does not depress the augmented intestinal absorption of calcium and phosphorus noted during their dietary deprivation nor reduce the plasma level of 1,25(OH)2D.

Intestinal calcium (Ca) hyperabsorption is a well-documented feature of experimental phosphorus depletion (PD). To further evaluate the effect of PD on Ca absorption we studied metabolic balance and in vitro everted duodenal sac uptake of Ca and phosphorus (P) in weanling male rats. Animals were assigned to three dietary groups: normal, 0.3% P ad libitum (NP); low, 0.03% P ad libitum (LP); and normal, 0.3% P but pair-fed with assigned LP mates (NP-PF). Results indicate that although PD led to an early but unsustained increase in 45Ca uptake by the everted duodenal sac in vitro, net intestinal Ca retention is consistently decreased in rats on the LP diet compared with rats eating either the NP or NP-PF diet. The reduction in net intestinal Ca absorption is reflected by an increase in fecal Ca, both in absolute quantities and in proportion to dietary Ca intake. The initial negative P balance after the initiation of the LP diet was promptly, albeit precariously, corrected. This was associated with a sustained increase in duodenal 32P uptake in vitro and virtual cessation of growth. Because the biosynthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its accumulation in intestinal mucosa have been reported to increase with PD, our study represents an example in which the physiological interrelationship between the activity of 1,25(OH)2D3 and intestinal Ca absorption may be dissociated.

Page G480: U. Gafter, J. A. Kraut, D. B. N. Lee, V. Silis, M. W. Walling, K. Kurokawa, M. R. Haussler, and J. W. Coburn. “Effect of metabolic acidosis on intestinal absorption of calcium and phosphorus.” Page G480: M. W. Walling deceased 30 November 1979.