Insulin binding sites in various segments of the rabbit nephron.

Insulin binds specifically to basolateral renal cortical membranes and modifies tubular electrolyte transport, but the target sites of this hormone in the nephron have not been identified. Using a microassay that permits measurement of hormone binding in discrete tubule segments we have determined the binding sites of 125I-insulin along the rabbit nephron. Assays were performed under conditions that minimize insulin degradation, and specific binding was measured as the difference between 125I-insulin bound in the presence or absence of excess (10(-5) M) unlabeled hormone. Insulin monoiodinated in position A14 was used in all assays. Specific insulin binding (attomol . cm-1 +/- SE) was highest in the distal convoluted tubule (180.5 +/- 15.0) and medullary thick ascending limb of Henle's loop (132.9 +/- 14.6), followed by the proximal convoluted and straight tubule. When expressed per milligram protein, insulin binding capacity was highest along the entire thick ascending limb (medullary and cortical portions) and the distal convoluted tubule, i.e., the "diluting segment" (congruent to 10(-13) mol . mg protein-1), and was lower (congruent to 4 X 10(-14) mol . mg protein-1), and remarkably similar, in all other nephron segments. Binding specificity was verified in competition studies with unlabeled insulin, insulin analogues (proinsulin and desoctapeptide insulin), and unrelated hormones (glucagon, 1-34 parathyroid hormone, prolactin, follicle-stimulating hormone). In addition, serum containing antiinsulin receptor antibody from two patients with type B insulin resistance syndrome markedly inhibited insulin binding to isolated tubules. Whether calculated per unit tubule length or protein content, insulin binding is highest in the thick ascending limb and the distal convoluted tubule, the same nephron sites where a regulatory role in sodium transport has been postulated for this hormone.

Mechanism of impaired water excretion in the hypothyroid rat.

The ability to excrete an oral water load and the renal diluting mechanism were studied in hypothyroid rats and in age-matched euthyroid controls. Hypothyroid animals excreted a significantly smaller fraction of a 50-ml/kg oral water load than controls, demonstrating the same limited ability to excrete free water as thyroid-deficient man. During hypotonic (0.45%) saline infusion, absolute sodium delivery to the diluting segment and free water clearance were markedly lower in hypothyroid rats. However, both fractional distal sodium delivery and fractional free water clearance were similar in hypothyroid and control animals, suggesting that the reduced absolute free water formation in hypothyroid rats was due to decreased net distal delivery. In support of this hypothesis was the observation that fractional distal sodium reabsorption was equal or higher in thyroid-deficient rats, which indicates that the sodium reabsorptive capacity of the diluting segment was preserved in these animals. The results cannot be attributed to incomplete suppression of antidiuretic hormone (ADH) since they were identical in diabetes insipidus rats, nor to different rates of non-ADH-dependent backflux of filtrate since tissue osmolality and solute concentrations in the cortex, medulla, and papilla were similar in hypothyroid and control rats of both Sprague-Dawley and Brattleboro strains. The functional integrity of the diluting segment in hypothyroid rats was further demonstrated in experiments in which distal delivery was increased by contralateral nephrectomy or by administration of carbonic anhydrase inhibitors which decrease proximal sodium reabsorption. In both studies, fractional free water clearance increased markedly reaching levels significantly greater than in euthyroid controls. These results demonstrate that the impaired ability of the hypothyroid rat to excrete a water load is not due to incomplete suppression of ADH or decreased reabsorptive capacity of the diluting segment but results from decreased filtrate delivery to this site secondary to reduced GFR.

Insulin binding and degradation by luminal and basolateral tubular membranes from rabbit kidney.

Insulin influences certain metabolic and transport renal functions and is avidly degraded by the kidney, but the relative contribution of the luminal and basolateral tubular membranes to these events remains controversial. We studied (125)I-insulin degradation [TCA and immunoprecipitation (IP) methods] and the specific binding of the hormone by purified luminal (L) and basolateral (BL) tubular membranes. These were prepared from rabbit kidney cortical homogenates by differential and gradient centrifugation and ionic precipitation steps in sequence, which resulted in enrichment vs. homogenate of marker enzymes' activities (sodium-potassium-activated adenosine triphosphatase for BL and maltase for L) of 8- and 12-fold, respectively. Both fractions degraded insulin avidly and bound the hormone specifically without saturation even at pharmacologic concentrations (10 muM). At physiologic insulin concentrations (0.157 nM) BL membranes degraded substantial amounts of insulin (44.2+/-2.6 and 40.7+/-2.2 pg/mg protein per min by the TCA and IP methods, respectively), even though at lesser rates (P < 0.001) than the luminal fraction (67.2+/-2.3 and 75+/-6.2 pg/mg protein per min, respectively); the rate of insulin catabolism by BL membranes was significantly higher (P < 0.001) than that which could be attributed to their contamination by luminal components [12.2+/-1.9 pg/mg per min (TCA method), or 13.7+/-1.9 pg/mg per min (IP method)]. Competition experiments suggested that insulin-degrading activity in both fractions includes both specific and nonspecific components. In contrast to degradation, insulin binding by both membranes was highly specific for native insulin and was severalfold higher in BL than L membranes [17.5+/-1.3 vs. 4.5+/-0.4 fmol/mg protein (P < 0.001) at physiologic insulin concentrations]. Despite the marked difference in the binding capacity for insulin by the two membranes, the patterns of labeled insulin displacement by increasing amounts of unlabeled hormone were superimposable (50% displacement required approximately 3 nM), suggesting that their receptors' affinity for insulin was similar. These observations provide direct evidence that interaction of insulin with the kidney involves binding and degradation of the hormone at the peritubular cell membrane.

Hepatic and renal metabolism of somatostatin-like immunoreactivity. Simultaneous assessment in the dog.

The hepatic and renal metabolism of somatostatin-like immunoreactivity (SLI) was assessed simultaneously in an in vivo dog model. The hepatic extraction of this peptide was 29.4 +/- 2.3% and was similar for endogenous and infused exogenous SLI. The renal extraction was 62.3 +/- 5%. The renal clearance of SLI was significantly greater than that of inulin indicating that the peptide is handled by peritubular uptake from postglomerular blood in addition to glomerular filtration. In both organs SLI extraction was not saturable even at arterial concentrations in excess of 100 times physiological range. The overall metabolic clearance rate of SLI was 19.7 +/- 1.6 ml/kg per minute of which 32.7 +/- 4.6% was contributed by hepatic and 37 +/- 4.9% by renal uptake mechanisms. The plasma half disappearance time of exogenously infused SLI was 1.9 +/- 0.3 min. The studies indicate that in the dog, the liver and kidney are both major sites of SLI metabolism, together accounting for 70.0 +/- 8.7% of the metabolic clearance of the peptide.

Heterogeneity of plasma glucagon. Circulating components in normal subjects and patients with chronic renal failure.

Plasma immunoreactive glucagon (IRG) concentrations were measured in 36 patients with chronic renal failure (CRF) and 32 normal subjects. In addition, the components of circulating IRG were analyzed by gel filtration in the fasting state and after physiological stimuli. Fasting IRG was elevated (P less than 0.001) in CRF patients (534 +/- 32 pg/ml) compared with the levels found in healthy subjects (113 +/- 9 pg/ml). Oral glucose suppressed plasma IRG in CRF patients from a basal level of 568 +/- 52 to a nadir of 354 +/- 57 pg/ml (120 min). This degree of suppression (38%) was comparable to that found in normal subjects (basal = 154 +/- 20 to 100 +/- 23 pg/ml) at 120 min (35%). Intravenous infusion of arginine (250 mg/kg) resulted in a 71% rise in IRG in CRF patients and a 166% increase in normal subjects. Gel filtration of fasting plasma from CRF patients showed three major peaks. The earliest (A) was found in the void volume (mol wt greater than 40,000) and constituted 16.5 +/- 4.7% of the elution profile. The middle peak (B) eluted just beyond the proinsulin marker (approximately 9,000 mol wt) and constituted the largest proportion of the elution profile (56.5 +/- 3.4%). The third peak (C) coincided with the standard glucagon and [125I]glucagon markers (3,485 mol wt) and comprised 27.0 +/- 4% of the IRG profile. In contrast, only peaks A and C were found in fasting plasma of normal subjects (53.6 +/- 10.4% in A and 46.4 +/- 10.4 in C). After oral glucose, glucagon immunoreactivity in the 3,500 mol wt peak (C) was markedly suppressed, while the B peak in patients with CRF declined to a lesser extent. The A peak in both groups was unchanged. After an arginine infusion only the C peak increased in both groups of subjects. Gel filtration of plasma in 3 M acetic acid gave similar profiles to those obtained in glycine albumin buffer. Exposure of serum to trypsin indicated that the B and C peaks were digestible, while the A peak was resistant to the action of the enzyme. In one sample, peak C increased after a 2-h exposure of serum to trypsin. We conclude that circulating IRG in normal subjects and patients with CRF is heterogenous. The hyperglucagonemia of renal failure is largely due to an increase in IRG material of approximately 9,000 mol wt, consistent with proglucagon, although the 3,500 mol wt component is also considerably elevated (threefold). The significance of circulating IRG levels should be interpreted with caution until the relative biological activity of the three components is established.

Pathogenesis and characterization of hyperglucagonemia in the uremic rat.

The pathogenesis of hyperglucagonemia and of the alterations in the pattern of circulating immunoreactive glucagon (IRG) associated with renal insufficiency was studied in rats in which a comparable degree of uremia was induced by three different methods, i.e., bilateral nephrectomy, bilateral ureteral ligation, and urine autoinfusion. Nephrectomized and ureteral-ligated rats were markedly hyperglucagonemic (575 +/- 95 pg/ml and 492 +/- 54 pg/ml, respectively), while IRG levels of urine autoinfused animals (208 +/- 35 pg/ml) were similar to those of control rats (180 +/- 26 pg/ml), indicating that uremia per se does not account for the hyperglucagonemia observed in renal failure. Similarly, plasma IRG composition in this group of animals was indistinguishable from that of controls, in which 88.2 +/- 5.9% of total IRG consisted of the 3,500-mol wt fraction. The same component was almost entirely responsible (82.6 +/- 4.1%) for the hyperglucagonemia observed in ligated rats, while it accounted for only 57.6 +/- 5.0% of the circulating IRG in nephrectomized animals. In the latter group, 36.8 +/- 6.6% of total IRG had a mol wt of approximately 9,000, consistent with a glucagon precursor. This peak was present in samples obtained as early as 2 h after renal ablation and its concentration continued to increase with time reaching maximal levels at 24 h. These results confirm that the kidney is a major site of glucagon metabolism and provide evidence that the renal handling of the various circulating IRG components may involve different mechanisms. Thus, the metabolism of the 3,500-mol wt fraction is dependent upon glomerular filtration, while the uptake of the 9,000-mol wt material can proceed in its absence, as long as renal tissue remains adequately perfused. This finding suggests that the 9,000-mol wt component may be handled by peritubular uptake.

Glucagon metabolism in the rat.

The renal handling of the biologically active glucagon component (the 3,500-mol wt fraction of immunoreactive glucagon [IRG]) and the contribution of the kidney to its overall peripheral metabolism were studied in normal and uremic rats. The metabolic clearance rate of glucagon was 31.8 +/- 1.2 ml/min per kg in normal animals and was diminished by approximately one-third in each of three groups of rats with compromized renal function: 22.3+/-1.6 ml/min per kg in partially (70%) nephrectomized; 22.9+/-3.3 ml/min per kg in bilaterally ureteral ligated; and 23.2+/-1.2 ml/min per kg in bilaterally nephrectomized animals. In normal rats the kidney contributed 30% to the overall metabolic clearance of the hormone and the renal extraction of endogenous and exogenous glucagon was similar, averaging 22.9+/-1.6% and was independent of plasma IRG levels over a wide range of arterial concentrations. The remnant kidney of partially (70%) nephrectomized animals continued to extract substantial amounts (16.6+/-4.2%) of the hormone, but accounted for only 8% of the total peripheral catabolism of IRG. In the two groups of animals with filtering kidneys, renal glucagon uptake was linearly related to its filtered load and could be accounted for by glomerular filtration and tubular reabsorption. However, the kidneys of animals with both ureters ligated (renal extraction of inulin = 3.2+/-1.8%) and hence virtual absence of glomerular filtration, continued to extract 11.5+/-1.9% of the renal arterial glucagon, contributing by 9% to its overall metabolic clearance, indicating that IRG uptake occurs also from the post glomerular capillaries.

Heterogeneity of plasma glucagon: patterns in patients with chronic renal failure and diabetes.

Immunoreactive plasma glucagon (IRG) in normal subjects and patients with chronic renal failure, diabetic ketoacidosis and diagetic hyperosmolar syndrome circulates in several forms. In the diabetic patients most IRG eluted coincidentally with the extracted, purified pancreatic hormone (MW3500), while in normal subjects a high molecular weight component predominated. In striking contrast, the major component of plasma IRG in patients with chronic renal failure was of intermediate size (MW +/- 9000), consistent with proglucagon. The accumulation of this form of IRG suggests that the kidney plays an important role in its metabolism. If there are differences in the biological activity of the various circulating components of IRG, the significance of immunoreactive glucagon levels in some disease states will require reassessment.

First exchange neutrophilia: an index of peritonitis during chronic intermittent peritoneal dialysis.

During intermittent peritoneal dialysis (IPD) early diagnosis of peritonitis may be difficult, because of paucity in clinical signs and delays in bacteriologic studies. We examined prospectively leucocyte counts and their differential composition in initial ascites and dialysis effluent of patients on IPD and correlated these findings to the presence of subsequently bacteriologically proven clinical peritonitis. Total leucocyte counts from either ascites or first exchange effluent did not differentiate infected from noninfected patients. In contrast, first exchange effluent neutrophilia (greater than 43%) proved to be an early indicator of infection, being 100% sensitive and 94% specific for peritonitis. We conclude that in such patients peritoneal effluent neutrophilia should be considered an indication of possible infection.

Metabolism of polypeptide hormones by the normal kidney and in uremia.

Recent work from our laboratory on the mechanism of polypeptide hormone handling by the normal kidney and the pathogenesis of altered hormonal metabolism in renal failure is reviewed. The kidney extracts substantial amounts of low - and medium - molecular weight polypeptide hormones from the renal circulation by a process which probably involves both glomerular filtration plus luminal reabsorption and direct peritubular uptake, although the relative contribution of the two mechanisms under physiologic conditions is not known. The bulk of the extracted hormone is catabolized in the renal parenchyma since urinary excretion is negligible. Renal catabolism contributes an important fraction of the total metabolic clearance of polypeptide hormones, which accounts in part for their increased circulating levels in renal failure. Since certain hormones are heterogenous and a large proportion of their plasma immunoreactivity may consist of components of uncertain biologic activity, simple correlations between circulating hormone levels and endocrine abnormalities in uremia are hazardous.

Role of the kidney in metabolism of gonadotropins in rats.

The role of the kidney in the metabolic disposal of homologous gonadotropins [renal luteinizing hormone (rLH) and renal follicle-stimulating hormone (rFSH)] was studied in rats. In analogy with other protein hormones, renal mechanisms contributed importantly to their metabolic clearance rates (MCR), which were profoundly and comparably decreased following nephrectomy (by 94 and 78% for rLH and rFSH, respectively). Absolute MCR and renal organ clearance rates of gonadotropins were, however, markedly lower and urinary clearance rates proportionally higher than those of nonglycosylated protein hormones reported previously. Nonetheless, handling of both LH and FSH by the kidney probably involves, in addition to their excretion in the urine, also intrarenal degradation because their urinary clearance rates accounted for at most a third of their respective MCR, considerably less than the striking reduction of MCR seen after acute renal ablation. Moreover, losses of LH immunoreactivity across the renal circulation were over and above those accountable for by urinary excretion alone. Thus, handling of gonadotropins by the kidney differs from that of nonglycoprotein hormones both in magnitude and in that it involves, in addition to intrarenal degradation, also substantial urinary excretion, a pattern that appears to be representative of the way the kidney disposes of glycoprotein hormones in general.

Glucagon degradation by luminal and basolateral rabbit tubular membranes.

Glucagon is avidly degraded by the kidney, but the relative contribution of the luminal and basolateral tubular membranes to this process is unknown. We studied 125I-glucagon degradation by purified luminal (L) and basolateral (BL) tubular membranes prepared from rabbit kidney cortex, which showed enrichment vs. homogenate of marker enzyme activities (Na-K-ATPase for BL and maltase for L) of 10- and 14-fold, respectively. Renal homogenates and both tubular membrane fractions degraded glucagon avidly without reaching saturation even at pharmacologic concentration (10(-5) M) of the hormone. At physiologic concentration (3 x 10(-11) M) BL membranes degraded substantial amounts of glucagon (8.1 +/- 0.9 pg . micrograms protein-1 . h-1) even though at lesser rates (P less than 0.001) than the luminal fraction (33.3 +/- 1.9 pg . micrograms protein-1 . h-1). Competition experiments suggested that glucagon-degrading activity in both fractions includes both specific and nonspecific components, and the potency of different enzyme inhibitors to decelerate glucagon degradation was strikingly similar in the two membrane preparations. Glucagon degradation differed in several important aspects from the manner in which tubular membranes catabolize insulin, including absolute degradation rates and relative degrading capacity of the membranes vs. homogenates, both being substantially higher for glucagon. These results provide direct evidence that the renal metabolism of glucagon also involves its degradation by peritubular cell membranes.

Metabolism of pure human erythropoietin in the rat.

The metabolism of pure human erythropoietin (EPO) labeled with 125I was studied in the rat. Concentrations of the labeled hormone (125I-EPO) in plasma and urine were measured by both trichloroacetic acid precipitation and gel filtration. During steady-state conditions the metabolic clearance rate of 125I-EPO was slow, averaging 256 +/- 7 microliter. min-1 X kg-1 of which only 19 +/- 2 microliter X min-1 X kg-1 (or 7.4 +/- 0.8% of the metabolic clearance rate) could be accounted for by excretion of the labeled hormone in the urine. Urinary clearance of 125I-EPO amounted to less than 0.3% of the glomerular filtration rate, and there was no detectable arteriovenous concentration difference of 125I-EPO across the kidney. After both pulse injection and constant infusion to equilibrium, disappearance of 125I-EPO from the circulation could be approximated by a single exponential function: plasma half-life was 3.5 +/- 0.2 h in normal rats and was prolonged to 4.4 +/- 0.3 h (P less than 0.05) in animals with ligated renal pedicles. Although kidney homogenates degraded 125I-EPO in vitro (optimum pH 4.5), the hormone did not accumulate in the kidney when injected intravenously. We conclude that EPO metabolism is extremely sluggish compared with that of other polypeptide hormones. Whereas kidney tissue is capable of degrading EPO in vitro, the physicochemical characteristics of this glycoprotein (molecular size, shape, and charge) probably impede its access to degrading sites and therefore account for the limited contribution of renal extraction and excretion to the metabolic clearance of the hormone.

Thyroid hormone and the kidney.

Thyroid hormones affect both renal morphology and function. They are required for kidney growth and development, and thyroid deficiency results in decreased renal plasma flow and glomerular filtration rate and in impaired urinary concentration and dilution. Thyroid hormones also influence membrane transport and electrolyte metabolism, and alterations in mineral metabolism in hyperthyroidism frequently cause calcium nephropathy which affects renal function adversely. The kidney plays an important role in the peripheral metabolism of iodine and thyroid hormones, and thyroid function is altered in certain kidney diseases, particularly chronic renal failure. The pathogenesis of these alterations is currently under active investigation.

Urinary concentration and dilution after unilateral nephrectomy in the rat.

1. The effects of unilateral nephrectomy on urinary concentration and dilution were studied in Sprague-Dawley rats. To exclude incomplete suppression of antidiuretic hormone, free water formation was also sutdied in rats with congenital diabetes insipidus (Brattleboro strain). 2. Urinary solute-free water formation was similar in Sprague-Dawley and Brattleboro rats. Uninephrectomized animals excreted a water load promptly and diluted their urine to the same degree as control rats. Maximal values for Cwater and TCwater per kidney were higher after nephrectomy, but were similar to those of control rats at comparable rates of fluid delivery to the distal nephron. Renal tissue osmolaity was similar in uninephrectomized and sham-operated animals, indicating that nonantidiuretic hormone-dependent backflux of filtrate was the same in the two groups. The only defect observed in uninephrectomized animals was a small reduction in maximal urine osmolaity. 3. These results demonstrate that free water formation and reabsorption are unaffected by unilateral nephrectomy and suggest that, in the remaining kidney, filtrate reabsorption along the entire nephron increases in proportion to the increment in glomerular filtration.

Role of the kidney in hormone metabolism and its implications in clinical medicine.

Uremia is accompanied by a variety of metabolic and endocrine disorders, due in part to impaired degradation of hormonally active peptides by the diseased kidney, and in part to the fact that the uremic environment interferes with the extrarenal degradation of certain hormones, or with their synthesis or secretion. It is not always possible to establish a direct cause and effect relationship between alterations in immunoassayable hormone levels and endocrine abnormalities because in uremia the circulating hormonal immunoreactivity frequently includes crossreacting components without biological activity and, in addition, hormonal effects on target organs are often altered. Alterations in the metabolism of pancreatic alpha and beta cell hormones and of prolactin in chronic renal failure and their effect on the metabolism of lipids and carbohydrates and on reproductive function in this condition are discussed.

Prolactin metabolism in the rat: role of the kidney in degradation of the hormone.

The contribution of impaired prolactin (PRL) degradation to the altered dynamics of this hormone in uremia was investigated in rats. Hyperprolactinemia developed after bilateral nephrectomy (BNx) or ligation of both ureters (BUL), whereas PRL levels remained normal in comparably azotemic animals undergoing urine autoinfusion in which glomerular filtration rate (GFR) was maintained. The renal organ clearance of PRL in control rats accounted for two-thirds of its metabolic clearance rate and was consistently less than GFR. Following BUL and BNx the metabolic clearance of PRL decreased predictably also by two-thirds. The importance of the renal parenchyma in the degradation of prolactin was confirmed during perfusion of isolated rat kidneys. Renal PRL handling involves mainly glomerular filtration and tubular reabsorption, although uptake from peritubular blood is also demonstrable under the high plasma flow conditions obtaining during in vitro kidney perfusion. We conclude that the hyperprolactinemia associated with acute uremia in the rat is not the consequence of the uremic state per se, but results from impaired renal degradation of the hormone.