Aquaporin-2 and urea transporter-A1 are up-regulated in rats with type I diabetes mellitus.

AIMS/HYPOTHESIS: Although the urine flow rate is considerably higher in diabetes mellitus, water reabsorption is greatly increased to concentrate an increased amount of solutes. Our study evaluated the expression of aquaporins and urea transporters, which are essential to the urinary concentration process. METHODS: Northern blot and immunoblot were used to quantify mRNA and proteins for aquaporin-2 (AQP2) as well as urea transporters UT-A1, UT-A2 and UT-B1, in subzones of the renal medulla of rats with streptozotocin-induced diabetes. RESULTS: In these rats, glycaemia, urine flow rate and water reabsorption were respectively fourfold, nine-fold and fourfold those of control rats. The AQP2 protein isoforms were significantly up-regulated in outer and inner medulla. In the base and tip of inner medulla, UT-A1 mRNA was significantly up-regulated (three- and 1.3-fold, respectively) as well as the 117 kD protein (ten- and threefold, respectively) whereas the 97 kD protein was not changed or decreased twofold, respectively. This suggests that, in diabetes, the inner medullary collecting duct is endowed with more UT-A1, especially in its initial part. In the case of mRNA and proteins of UT-A2, located in thin descending limbs in the inner stripe of outer medulla, they were respectively not changed and down-regulated in diabetic rats. CONCLUSION/INTERPRETATION: This study shows that in diabetes, the increased expression of AQP2 and UT-A1 in medullary collecting duct is consistent with an improved concentrating activity. In addition, the underexpression of UT-A2 and the overexpression of UT-A1 in the initial medullary collecting duct are reminiscent of the changes seen after experimental reduction of urine concentration or low protein feeding.

Vasopressin and diabetes mellitus.

In diabetes mellitus (DM), the urine flow rate is increased, and the fluid turnover in the body is accelerated because of the glucose-induced osmotic diuresis. On the other hand, plasma vasopressin (VP) is elevated in both type 1 and type 2 DM. This elevation seems to be due to a resetting of the osmostat. A high VP level is beneficial in the short term because it limits to some extent the amount of water required for the excretion of a markedly enhanced load of osmoles (mainly glucose). However, in the long run, it may have adverse effects by favoring the development of diabetic nephropathy. VP has been shown in normal rats to induce kidney hypertrophy, glomerular hyperfiltration, and an increase in urinary albumin excretion (features also occurring in association in the period preceding diabetic nephropathy). Moreover, VP has been shown to participate in the progression of renal failure in rats with five-sixths reduction in renal mass. In recent studies, we have shown (1) that creatinine clearance, albuminuria and renal mass increased much less during experimental DM in Brattleboro rats unable to secrete VP than in their VP-replete Long-Evans controls, and (2) that albuminuria was prevented during experimental DM in Wistar rats when a VP nonpeptidic, highly selective V2 receptor antagonist was administered chronically for 9 weeks. Taken together, these results strongly suggest that VP plays a crucial role in the onset and aggravation of the renal complications of DM. The mechanisms by which VP exerts these adverse V2-dependent effects are not yet elucidated. They are most likely indirect and may involve several intermediate steps comprising VP-induced changes in the composition of the tubular fluid in the loop of Henle (due to solute recycling in the renal medulla associated with improved concentrating activity of the kidney), inhibition of the tubuloglomerular feedback control of glomerular function, and alterations in glomerular hemodynamics by the intrarenal renin-angiotensin system.

Vasopressin contributes to hyperfiltration, albuminuria, and renal hypertrophy in diabetes mellitus: study in vasopressin-deficient Brattleboro rats.

Diabetic nephropathy represents a major complication of diabetes mellitus (DM), and the origin of this complication is poorly understood. Vasopressin (VP), which is elevated in type I and type II DM, has been shown to increase glomerular filtration rate in normal rats and to contribute to progression of chronic renal failure in 5/6 nephrectomized rats. The present study was thus designed to evaluate whether VP contributes to the renal disorders of DM. Renal function was compared in Brattleboro rats with diabetes insipidus (DI) lacking VP and in normal Long-Evans (LE) rats, with or without streptozotocin-induced DM. Blood and urine were collected after 2 and 4 weeks of DM, and creatinine clearance, urinary glucose and albumin excretion, and kidney weight were measured. Plasma glucose increased 3-fold in DM rats of both strains, but glucose excretion was approximately 40% lower in DI-DM than in LE-DM, suggesting less intense metabolic disorders. Creatinine clearance increased significantly in LE-DM (P < 0.01) but failed to increase in DI-DM. Urinary albumin excretion more than doubled in LE-DM but rose by only 34% in DI-DM rats (P < 0.05). Kidney hypertrophy was also less intense in DI-DM than in LE-DM (P < 0.001). These results suggest that VP plays a critical role in diabetic hyperfiltration and albuminuria induced by DM. This hormone thus seems to be an additional risk factor for diabetic nephropathy and, thus, a potential target for prevention and/or therapeutic intervention.

Vasopressin and urinary concentrating activity in diabetes mellitus.

In diabetes mellitus (DM), the high urine flow rate suggests that urinary concentrating capacity is impaired. However, several studies have shown that vasopressin is elevated in DM and the consequences of this elevation have not yet been characterized. This study reevaluated renal function and water handling in male Wistar rats with Streptozotocin-induced DM, and in control rats. During five weeks after induction of DM, urine was collected in metabolic cages and a blood sample was drawn during the third week. Control rats (CONT) were studied in parallel. On week 3, urine flow rate was tenfold higher in DM than in CONT rats and urinary osmolality was reduced by half along with a markedly higher osmolar excretion (DM/CONT = 5.87), due for a large part to glucose but also to urea (DM/CONT = 2.49). Glucose represented 52% of total osmoles (90.3 +/- 6.5 mmol/d out of 172 +/- 14 mosm/d). Free water reabsorption was markedly higher in DM rats compared to CONT (326 +/- 24 vs 81 +/- 5 ml/d). In other rats treated in the same way, urinary excretion of vasopressin was found to be markedly elevated (15.1 +/- 4.1 vs 1.44 +/- 0.23 ng/d). In DM rats, glucose concentration in urine was 17 fold higher than in plasma, and urea concentration 14 fold higher. Both urine flow rate and free water reabsorption were positively correlated with the sum of glucose and urea excretions (r = 0.967 and 0.653, respectively) thus demonstrating that the urinary concentrating activity of the kidney increased in proportion to the increased load of these two organic solutes. These results suggest that vasopressin elevation in DM contributes to increase urinary concentrating activity and thus to limit water requirements induced by the metabolic derangements of DM. The possible deleterious consequences of sustained high level of vasopressin in DM are discussed.

Plasma cAMP: a hepatorenal link influencing proximal reabsorption and renal hemodynamics?

The existence of a hepatorenal link is suggested by several pathophysiological observations (indirect actions of glucagon on the kidney, hepatorenal syndrome), but the nature of this link remains unidentified. We propose that extracellular circulating cyclic AMP could be this link. Cyclic AMP (cAMP) is the intracellular second messenger of glucagon (G) action in the liver, and this organ is known to release cAMP in the blood in relatively large amounts after G administration. On the other hand, the proximal tubule (mainly the pars recta) is known to take up cAMP through the organic acid transport system. We observed that the glucagon-induced rise in phosphate excretion, which requires supraphysiologic concentration of G, was significantly correlated with the simultaneous rise in plasma cAMP and could be mimiked by i.v. infusion of cAMP alone. Moreover, we showed that a significant hyperfiltration (similar to that induced by supraphysiologic G) can be observed if cAMP (mimicking G-induced hepatic release) is coinfused with a much lower, physiologic, amount of G. Taken together, these observations suggest that: (1) cAMP is a hepatorenal link and that plasma cAMP permanently influences the intensity of reabsorption in the pars recta of the proximal tubule; and (2) that cAMP participates, in conjunction with G, to control GFR. Insulin is known to exert an inhibitory influence on G-induced cAMP release by the liver and will thus weaken the indirect (cAMP-mediated) influence of G on renal function. This "pancreato-hepatorenal cascade" may explain the natriuretic effects of G and antinatriuretic effects of insulin, and probably contributes to disturbances observed in some pathophysiological situations such as the edema of liver cirrhosis or hyperfiltration of diabetes.

Cyclic AMP is a hepatorenal link influencing natriuresis and contributing to glucagon-induced hyperfiltration in rats.

The effects of glucagon (G) on proximal tubule reabsorption (PTR) and GFR seem to depend on a prior action of this hormone on the liver resulting in the liberation of a mediator and/or of a compound derived from amino acid metabolism. This study investigates in anesthetized rats the possible contribution of cAMP and urea, alone and in combination with a low dose of G, on phosphate excretion (known to depend mostly on PTR) and GFR. After a 60-min control period, cAMP (5 nmol/min x 100 grams of body weight [BW]) or urea (2.5 micromol/min x 100 grams BW) was infused intravenously for 200 min with or without G (1.2 ng/min x 100 grams BW, a physiological dose which, alone, does not influence PTR or GFR). cAMP increased markedly the excretion of phosphate and sodium (+303 and +221%, respectively, P < 0.01 for each) but did not alter GFR. Coinfusion of cAMP and G induced the same tubular effects but also induced a 20% rise in GFR (P < 0.05). Infusion of urea, with or without G, did not induce significant effects on PTR or GFR. After G infusion at increasing doses, the increase in fractional excretion of phosphate was correlated with a simultaneous rise in plasma cAMP concentration and reached a maximum for doubling of plasma cAMP. These results suggest that cAMP, normally released by the liver into the blood under the action of G, (a) is probably an essential hepatorenal link regulating the intensity of PTR, and (b) contributes, in conjunction with specific effects of G on the nephron, to the regulation of GFR.

Vasopressin increases glomerular filtration rate in conscious rats through its antidiuretic action.

To evaluate the possible influence of chronic alterations in urine concentrating activity (CA) on renal hemodynamics, adult male Sprague-Dawley rats were submitted for 7 days to one of three different levels of CA. CA was either reduced by increasing water intake (mixing the food with a gel bringing 1.6 mL water per g food) (Low-CA), or increased by chronic intraperitoneal infusion of 1-desamino 8-D-arginine vasopressin (200 ng/day) (High-CA). Low-CA, High-CA, and control rats were housed in metabolic cages, ate the same quantity of dry food (the amount provided being slightly lower than the spontaneous intake), and had free access to drinking water. The only difference between groups thus concerned the water intake-vasopressin axis. Radiolabeled (14C)inulin was infused chronically by osmotic minipumps. Urine was collected during Days 5, 6, and 7, and blood samples were taken for determination of plasma composition (P), absolute and fractional (FE) urinary excretion, and clearance (C) of inulin, creatinine, urea, and main electrolytes. This protocol produced mean 24-h urine osmolality (Uosm) ranging from 500 to 3500 mosmol/kg H2O without inducing any disturbance in body fluids or plasma osmolality (Posm). Results show that GFR (Cinulin) was markedly and positively correlated with Uosm (r = 0.798, P < 0.001) and free water reabsorption (r = 0.819, P < 0.001). For Uosm = 2500 mosm/kg H2O, GFR was 47% higher than for Uosm = 500 mosm/kg H2O. Ccreat underestimated GFR in High-CA and overestimated it in Low-CA. FEurea was inversely related to Uosm, as expected from the increased reabsorption known to occur at low urine flows. It is tentatively proposed that the intrarenal recycling of urea, triggered by vasopressin and essential to the urinary concentrating mechanism, might influence GFR indirectly by modifying the composition of the tubular fluid at the macula densa and thus the intensity of the tubuloglomerular feedback control of GFR. Even if this mechanism remains to be confirmed, this study unequivocally demonstrates, in normal conscious rats, that the level of urinary concentrating activity has a major influence on basal GFR.

Direct and indirect cost of urea excretion.

Urea, the major end product of protein metabolism in mammals, is the most abundant solute in the urine. Urea excretion is thought to result from filtration curtailed by some passive reabsorbtion along the nephron. This reabsorption is markedly enhanced by vasopressin and slow urinary flow rate (V), the fraction of filtered urea excreted in the urine (FEurea) falling from approximately 60% at high V to only approximately 20% at low V. In concentrated urine, normal urea excretion can be maintained only if urea filtration is elevated. This can be achieved by increasing plasma urea concentration (Purea) and/or GFR. We have shown that both parameters do increase when normal rats are submitted to chronic alterations in the water intake/vasopressin axis within the normal range of physiologic regulation. This situation is very similar to that observed after alterations in protein intake. In both cases more urea needs to be filtered, either because more of it has to be excreted, or because the efficiency of its excretion is reduced. A common mechanism is proposed to explain the rise in GFR observed in the two situations. In summary, our studies demonstrate that the antidiuretic effects of vasopressin are responsible for a significant elevation of GFR. This GFR adaptation limits the rise in Purea, a favorable effect because urea is not as harmless as usually thought. However, this hyperfiltration might have deleterious consequences in diseased kidneys.

Renal hemodynamic response to intravenous and oral amino acids in animals.

Oral or parenteral application of amino acids leads to marked hyperfiltration and increased of renal plasma flow. Amino acids stimulate the release of glucagon, which increases hepatic production and release of cyclic adenosine monophosphate (cAMP). In the kidney, the combined effect of cAMP and glucagon increases glomerular filtration rate (GFR), possibly by reducing NaCl concentration at the macula densa and depression of the tubuloglomerular feedback. Vasopressin-dependent urea recycling and delivery to the thick ascending limb could similarly reduce NaCl concentration at the macula densa. Beyond that, amino acids may trigger a hepatorenal reflex or directly interfere with renal function. Mechanisms invoked include dopamine from renal nerves, prostaglandins, nitric oxide (NO), and angiotensin II. At this point, it is not clear to which extent the described mechanisms participate in, permit, or fully account for the hyperfiltrative effect of amino acids.

Influence of glucagon on GFR and on urea and electrolyte excretion: direct and indirect effects.

Clearance experiments were performed in anesthetized male Wistar rats to determine the level of peripheral glucagon concentration required to elicit changes in glomerular filtration rate (GFR) and in solute excretion. Glucagon was intravenously infused at a rate of 1.25 (group G-1, n = 8), 3.75 (group G-3, n = 7), or 12.5 (group G-10, n = 7) ng.min-1.100 g body wt-1 for 100 min. Measurements were performed before, during, and after this infusion. Group G-10 resulted in a plasma concentration of glucagon severalfold higher than usually observed in peripheral blood after a protein meal but normal for the hepatic circulation. Group G-10 simultaneously increased GFR, plasma adenosine 3',5'-cyclic monophosphate (cAMP) concentration, and the excretion of water (i.e., urinary flow rate), Na, Cl, PO4, K, and urea. Some of the effects of glucagon on electrolyte excretion were also observed with group G-1 and/or G-3 and were fully reversible, suggesting a direct renal action of glucagon. The significant and reversible increase in K excretion in group G-3 suggests that glucagon exerts a direct stimulatory influence on K secretion in the distal nephron. Increases in urinary flow rate, PO4, Na, and urea fractional excretions were seen with group G-10 only and were not reversible, suggesting an indirect action of glucagon on the proximal tubule. Because glucagon stimulates cAMP formation in hepatocytes and because this cAMP is released in the blood and secreted by proximal tubule cells, cAMP of hepatic origin could induce a parathyroid hormone-like effect in this nephron segment. In summary, these experiments suggest that glucagon influences different aspects of renal function by a combination of direct and indirect (probably liver-dependent) effects.

Is the process of urinary urea concentration responsible for a high glomerular filtration rate?

For subjects on a normal diet, urea is the major urinary solute and is markedly concentrated in the urine compared with in the plasma. Because urea is not known to undergo active secretion, its excretion rests on filtration lessened to a variable extent by tubular reabsorption. It is well established that the efficiency of urea excretion drops with increasing urinary concentration and decreasing urinary flow rate (from approximately 60% of filtered load, above 2 mL/min, to approximately 20% below 0.5 mL/min) because the prolonged transit time in the distal nephron favors passive urea reabsorption. Thus, a higher urinary concentration is achieved at the expense of a reduced efficiency of urea excretion. Recent experimental observations suggest that GFR could actually increase in parallel with the urinary concentrating activity, thus ensuring a normal urea excretion in the face of a high, concentration-dependent urea reabsorption, with only a moderate increase in plasma urea. A possible mechanism is proposed that could explain how the vasopressin-induced intrarenal recycling of urea (which contributes to improvement in urinary concentration), but not an exogenous urea administration, could indirectly depress the tubuloglomerular feedback and hence increase GFR. An increased concentration of an osmotically active solute in the thick ascending limb of Henle's loop (such as urea and, in some cases, glucose) could enable a lower NaCl concentration to be achieved at the macula densa by reducing the osmotically driven water leakage in this nephron segment. This mechanism could explain the hyperfiltration seen in various pathophysiologic situations such as chronic vasopressin infusion, high protein intake, severe burns, and diabetes mellitus. Whatever the mechanism, if the need to excrete relatively high amounts of urea in a concentrated urine leads to a sustained elevation of GFR, the price to pay for this water economy is higher than generally assumed. It is not limited to the energy spent in the sodium reabsorption providing the "single effect" for the urinary concentrating process. It also includes the consequences on the glomerular filter of sustained high pressure and flow and the energy spent in reabsorbing the extra load of solutes filtered. In chronic renal failure, the ability to form hypertonic urine declines but is nevertheless well preserved with respect to declining GFR, thus imposing on remnant nephrons an additional permanent stimulus for hyperfiltration.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of glucagon on glomerular filtration rate and urea and water excretion.

Clearance experiments were performed in anesthetized male Wistar rats to reevaluate the renal effects of glucagon (Gluc) on glomerular filtration rate (GFR) and solute and water excretion. After an 80-min control period, these effects were evaluated in the last 80 min of a 2-h intravenous Gluc infusion. Gluc induced significant increases in GFR (+20%), urine flow rate (+150%), free water reabsorption (+50%), urea synthesis and urea excretion (+66%), and nonurea solute excretion (+67%). In addition, fractional urea excretion (FEurea) increased by 43% (P less than 0.01). Additional experiments showed that increases in either urea excretion or urine flow rate (induced by appropriate infusion of urea or half-dilute saline), similar to those seen after Gluc, could not account for the increased FEurea. All significant effects of Gluc were also observed during infusion of antidiuretic hormone or during water diuresis. The tubular effects of Gluc could be explained by a reduction in proximal reabsorption. The dose of Gluc required to induce all the effects described above was 12 ng.min-1.100 g body wt-1, a dose producing an approximately 10-fold supraphysiological peripheral plasma concentration but a "physiological" level for the liver. Infusion of 1.2 ng induced almost no change in renal function, and infusion of 120 ng induced no greater effects than 12 ng. These results suggest 1) that Gluc, a hormone liberated after protein ingestion, exerts coordinated effects on liver and kidney to increase simultaneously urea synthesis and excretion and to promote water conservation and 2) that these effects could, at least in part, be indirect and depend on the Gluc-induced stimulation of hepatocyte metabolism.