Interaction between alpha 2-adrenergic and angiotensin II systems in the control of glomerular hemodynamics as assessed by renal micropuncture in the rat.

The hypothesis that renal alpha 2 adrenoceptors influence nephron filtration rate (SNGFR) via interaction with angiotensin II (AII) was tested by renal micropuncture. The physical determinants of SNGFR were assessed in adult male Munich Wistar rats 5-7 d after ipsilateral surgical renal denervation (DNX). DNX was performed to isolate inhibitory central and presynaptic alpha 2 adrenoceptors from end-organ receptors within the kidney. Two experimental protocols were employed: one to test whether prior AII receptor blockade with saralasin would alter the glomerular hemodynamic response to alpha 2 adrenoceptor stimulation with the selective agonist B-HT 933 under euvolemic conditions, and the other to test whether B-HT 933 would alter the response to exogenous AII under conditions of plasma volume expansion. In euvolemic rats, B-HT 933 caused SNGFR to decline as the result of a decrease in glomerular ultrafiltration coefficient (LpA), an effect that was blocked by saralasin. After plasma volume expansion, B-HT 933 showed no primary effect on LpA but heightened the response of arterial blood pressure, glomerular transcapillary pressure gradient, and LpA to AII. The parallel results of these converse experiments suggest a complementary interaction between renal alpha 2-adrenergic and AII systems in the control of LpA.

Functional effects on glomerular hemodynamics of short-term chronic cyclosporine in male rats.

We evaluated the effects of chronic cyclosporine (CsA) administration on the determinants of nephron filtration rate (SNGFR) using micropuncture techniques (mp) in male Munich-Wistar rats. Animals received CsA (30 mg/kg SQ) in olive oil daily for 8 d before mp. Controls (PFC) were pair fed. SNGFR, glomerular capillary hydrostatic pressure gradient (delta P), nephron plasma flow (SNPF), plasma protein oncotic pressure (pi A), and glomerular ultrafiltration coefficient (LpA) were quantitated in each experiment. CsA was associated with a lower SNGFR due to decreases in SNPF and a major reduction in delta P but no decrease in LpA. Plasma volume expansion (PVE) caused SNGFR, delta P, and SNPF to increase in both CsA and PFC without eliminating the differences between CsA and PFC. CsA/PVE rats responded normally to angiotensin II (AII) infusion indicating that the low delta P associated with CsA is not due to unresponsiveness to AII. Prior renal denervation caused SNGFR and SNPF to increase in CsA-treated animals but failed to alter the reduction in glomerular capillary pressure after CsA or to eliminate the glomerular hemodynamic differences between treated animals and pair-fed controls. This constellation of glomerular hemodynamic abnormalities suggests that the renal effect of short-term chronic CsA administration is mediated primarily by a reduction in the afferent effective filtration pressure resulting from an imbalance between pre- and postglomerular vascular resistances.

Arginine feeding modifies cyclosporine nephrotoxicity in rats.

Glycine (G) infusion causes renal vasodilation mediated by nitric oxide (NO). Cyclosporine A (CsA) nephrotoxicity is characterized by preglomerular vasoconstriction and decreased efferent arteriolar tone probably related to reduced NO and angiotensin II, respectively. L-Arginine (ARG) is a precursor to NO. To test the hypothesis that chronic CsA decreases renal NO activity, we compared the glomerular hemodynamic response to glycine infusion in rats after 8 d of CsA (30 mg/kg per d s.c.), CsA and ARG (1.6 g/kg per d p.o.) (A/CsA), and in two groups of pair-fed controls (CON, A/CON). Single nephron GFR (SNGFR), single nephron plasma flow (SNPF), glomerular capillary hydrostatic pressure gradient (delta P), proximal tubular reabsorption (APR), and kidney tissue angiotensin II (AIIk) were measured before and during G. CsA was associated with baseline decrements in SNGFR, SNPF, delta P, and AIIk, and with a blunted hemodynamic response to G. In CON, ARG did not affect baseline hemodynamics or modify the response to G. In CsA, ARG decreased baseline preglomerular resistance and restored the glomerular hemodynamic response to G. G was associated with a significant increase in AIIk in both CON and CsA. These findings suggest that (a) CsA is associated with decreased AIIk, and (b) CsA may diminish NO activity within the kidney, and that this capacity may be partially restored by arginine feeding.

Ornithine decarboxylase, kidney size, and the tubular hypothesis of glomerular hyperfiltration in experimental diabetes.

In early diabetes, the kidney grows and the glomerular filtration rate (GFR) increases. This growth is linked to ornithine decarboxylase (ODC). The study of hyperfiltration has focused on microvascular abnormalities, but hyperfiltration may actually result from a prior increase in capacity for proximal reabsorption which reduces the signal for tubuloglomerular feedback (TGF). Experiments were performed in Wistar rats after 1 week of streptozotocin diabetes. Kidney weight, ODC activity, and GFR were correlated in diabetic and control rats given difluoromethylornithine (DFMO; Marion Merrell Dow, Cincinnati, Ohio, USA) to inhibit ODC. We assessed proximal reabsorption by micropuncture, using TGF as a tool for manipulating single-nephron GFR (SNGFR), then plotting proximal reabsorption versus SNGFR. ODC activity was elevated 15-fold in diabetic kidneys and normalized by DFMO, which also attenuated hyperfiltration and hypertrophy. Micropuncture data revealed an overall increase in proximal reabsorption in diabetic rats too great to be accounted for by glomerulotubular balance. DFMO prevented the overall increase in proximal reabsorption. These data confirm that ODC is required for the full effect of diabetes on kidney size and proximal reabsorption in early streptozotocin diabetes and are consistent with the hypothesis that diabetic hyperfiltration results from normal physiologic actions of TGF operating in a larger kidney, independent of any primary malfunction of the glomerular microvasculature.

Agmatine, a bioactive metabolite of arginine. Production, degradation, and functional effects in the kidney of the rat.

Until recently, conversion of arginine to agmatine by arginine decarboxylase (ADC) was considered important only in plants and bacteria. In the following, we demonstrate ADC activity in the membrane-enriched fraction of brain, liver, and kidney cortex and medulla by radiochemical assay. Diamine oxidase, an enzyme shown here to metabolize agmatine, was localized by immunohistochemistry in kidney glomeruli and other nonrenal cells. Production of labeled agmatine, citrulline, and ornithine from [3H]arginine was demonstrated and endogenous agmatine levels (10(-6)M) in plasma ultrafiltrate and kidney were measured by HPLC. Microperfusion of agmatine into renal interstitium and into the urinary space of surface glomeruli of Wistar-Frömter rats produced reversible increases in nephron filtration rate (SNGFR) and absolute proximal reabsorption (APR). Renal denervation did not alter SNGFR effects but prevented APR changes. Yohimbine (an alpha 2 antagonist) microperfusion into the urinary space produced opposite effects to that of agmatine. Microperfusion of urinary space with BU-224 (microM), a synthetic imidazoline2 (I2) agonist, duplicated agmatine effects on SNGFR but not APR whereas an I1 agonist had no effect. Agmatine effects on SNGFR and APR are not only dissociable but appear to be mediated by different mechanisms. The production and degradation of this biologically active substance derived from arginine constitutes a novel endogenous regulatory system in the kidney.

Temporal adjustment of the juxtaglomerular apparatus during sustained inhibition of proximal reabsorption.

Tubuloglomerular feedback (TGF) stabilizes nephron function by causing changes in single-nephron GFR (SNGFR) to compensate for changes in late proximal flow (VLP). TGF responds within seconds and reacts over a narrow range of VLP that surrounds normal VLP. To accommodate sustained increases in VLP, TGF must reset around the new flow. We studied TGF resetting by inhibiting proximal reabsorption with benzolamide (BNZ; administered repeatedly over a 24-hour period) in Wistar-Froemter rats. BNZ acutely activates TGF, thereby reducing SNGFR. Micropuncture was performed 6-10 hours after the fourth BNZ dose, when diuresis had subsided. BNZ caused glomerular hyperfiltration, which was prevented with inhibitors of macula densa nitric oxide synthase (NOS). Because of hyperfiltration, BNZ increased VLP and distal flow, but did not affect the basal TGF stimulus (early distal salt concentration). BNZ slightly blunted normalized maximum TGF response and the basal state of TGF activation. BNZ sensitized SNGFR to reduction by S-methyl-thiocitrulline (SMTC) and caused the maximum TGF response to be strengthened by SMTC. Sensitization to type I NOS (NOS-I) blockers correlated with increased macula densa NOS-I immunoreactivity. Tubular transport measurements confirmed that BNZ affected TGF within the juxtaglomerular apparatus. During reduced proximal reabsorption, TGF resets to accommodate increased flow and SNGFR through a mechanism involving macula densa NOS.

Identification of human TGF-beta1 signal (leader) sequence polymorphisms by PCR-RFLP.

Links between disease susceptibility and genetically determined variation in human cytokine expression have recently been described. This has led to a demand for simple methods of identifying cytokine gene polymorphisms of potential clinical relevance. Here, we describe a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for identifying two human transforming growth factor beta1 (TGF-beta1) signal (leader) sequence polymorphisms, T869C (Leu10Pro) and G915C (Arg25Pro). This permits simple and robust identification of TGF-beta1 leader sequence genotypes and demonstrates the physical linkage in cis between T869C (Leu10Pro) and G915C (Arg25Pro). The method does not require previously genotyped standards. The efficacy of enzyme digestion is internally controlled by the presence of conserved restriction sites.

Nitric oxide and tubuloglomerular feedback.

Glomerular filtration is closely coupled to tubular reabsorption by a system of tubuloglomerular feedback (TGF). TGF operates within the juxtaglomerular apparatus (JGA) of each nephron, where changes are sensed in the salt content of fluid at the luminal macula densa and that information is transmitted to the glomerular microvasculature to elicit compensatory changes in single nephron glomerular filtration rate (GFR). Type I nitric oxide synthase (NOS) is expressed in the macula densa. Other NOS isoforms may be produced in the mesangium, and glomerular microvessels. These NOSs are strategically positioned to influence each step of the TGF process. However, micropuncture experiments using NOS antagonists have shown that nitric oxide (NO) does not mediate TGF. Instead, local NOS blockade causes the curve that represents TGF to shift leftward and become more steep. Changes in macula densa NO production may underlie the resetting of TGF, which is required in order to keep the TGF curve aligned with ambient tubular flow as tubular flow changes to accommodate physiologic circumstances. Also, macula densa NO production may be substrate limited and dissociated from NOS protein content. The importance of NO to TGF resetting and the substrate dependence of NO production have both been found during changes in dietary salt.

Resetting protects efficiency of tubuloglomerular feedback.

Tubuloglomerular feedback (TGF) may effect long-term protection of total body salt and water or may govern minute-to-minute autoregulation of renal function. The task for which TGF is best suited depends on the orientation of ambient tubular flow relative to the inflection point of the TGF curve and on the tendency of TGF to reset in response to prolonged stimulation. Current data suggest that the TGF curve is coupled closely to ambient flow in individual nephrons such that the system is capable of compensating both negative and positive perturbations in tubular flow. This coupling is mediated by events within the juxtaglomerular apparatus that cause the TGF curve to reset laterally in response to sustained shifts in tubular flow. This resetting of TGF occurs within 30 to 60 minutes of an applied stimulus, suggesting that TGF is better suited to mediate dynamic autoregulation than to account for sustained vasoconstriction during proximal tubular injury.

Tubuloglomerular feedback activity after acute reductions in renal mass.

Removal of one kidney results in prompt increases in urinary excretion of NaCl and water from the remaining kidney, followed rather soon thereafter by increases in glomerular filtration rate (GFR). At 12-15 h and 24 h after contralateral nephrectomy, the single nephron filtration rate (SNGFR) is increased, accompanied by parallel increases in absolute proximal tubular reabsorption, late proximal tubular and early distal tubular flow rates, suggesting that these events might be critical to the increased urinary excretion. However, micropuncture studies 2-4 h after contralateral nephrectomy demonstrate that increased SNGFR and even increased distal tubular flow rates are not requirements for augmented urinary excretion, suggesting that decreased tubular reabsorption in the most distal nephron segments causes the increase in urinary excretion. Analysis of TGF profiles by stop-flow pressure response at 2-4 h after contralateral nephrectomy have suggested suppression of TGF as assessed by a rightward shift in the turning point with increasing late proximal tubular perfusion. However, our studies have examined SNGFR responses and shown no suppression of TGF profiles but a downward shift in the operating point, suggesting activation of TGF and a modest reduction in SNGFR, determined from distal tubular collections. Although SNGFR was increased at all late proximal perfusion rates 12 h after nephrectomy, the turning point (V 1/2) was not altered. TGF profiles are not suppressed within 12 h after nephrectomy while SNGFR is increased. Suppression of TGF does not cause the increase in SNGFR after nephrectomy and TGF activity is maintained and adapts to increases in SNGFR caused by TGF-independent mechanisms.

Adrenergic influences and interactions with angiotensin II.

Increasing evidence indicates the existence of a complex interplay between the angiotensin and adrenergic nervous systems within the kidney. Since both of these vasoconstrictor systems are integrally involved in the maintenance of systemic blood pressure and fluid and electrolyte homeostasis, it is not surprising that each might influence the other vis-a-vis their mutual capacity to alter the physiologic determinants of glomerular filtration.

Chest tube removal after cardiac surgery.

Prompt remove of chest tubes by RNs has allowed earlier and more aggressive ambulation of our patients and, along with other interventions, has decreased length of stay by 1.5 days while improving quality of care. Proper education, both didactic and clinical, is the key component in preparing RNs to safely and effectively perform this procedure.

Tubuloglomerular feedback.

Increasing evidence is accumulating that the tubuloglomerular feedback system exercises significant control over the glomerular filtration rate. We present our current understanding of the mechanisms underlying the operation of the TGF system and discuss several situations applicable to clinical medicine where altered TGF responses are likely to be manifested as perceivable changes in overall renal function.

Homeostatic efficiency of tubuloglomerular feedback in hydropenia, euvolemia, and acute volume expansion.

We assessed the homeostatic efficiency of the tubuloglomerular feedback (TGF) system in Inactin-anesthetized Munich-Wistar rats by use of perturbation analysis in closed-loop micropuncture studies. Nephrons were studied in vivo under conditions of hydropenia (HYD, n = 17), euvolemia (EUV, n = 23), and acute isoncotic extracellular volume expansion (EXP, n = 15). Proximal tubular flow was perturbed in free-flowing nephrons with a microperfusion apparatus. Flow rate (VM) was measured upstream from the perturbation (VH) by a noninvasive optical technique. The dependence of VM on VH was estimated by polynomial regression. By using fractional compensation (C = -dVM/dVH), as an index of homeostatic efficiency, we constructed efficiency profiles (C vs. VH). At VH = 0, C tended toward higher values with decreasing volume status, although the effect did not achieve significance. The maximum value of C did not differ between groups. The efficiency profiles shifted leftward with each increment in volume (P < 0.03, HYD vs. EXP), suggesting that the TGF system adapts to acute increments in volume by shifting the efficiency profile in favor of a vasodilatory role.

Alpha 2-adrenoceptors determine the response to nitric oxide inhibition in the rat glomerulus and proximal tubule.

Arginine-derived nitric oxide exerts control over the processes of glomerular filtration and tubular reabsorption. The tonic influence of nitric oxide over both of these is eliminated by renal denervation. The hypothesis that the renal nerves function, in this regard, via the activation of alpha 2-adrenoceptors was tested by renal micropuncture. The physical determinants of glomerular filtration and proximal tubular reabsorption were assessed in Munich-Wistar rats before and during the administration of the nitric oxide synthase inhibitor NG-monomethyl L-arginine (L-NMMA). In one set of studies, the systemic infusion of the alpha 2-agonist B-HT 933 rendered nephron GFR, nephron plasma flow, and proximal reabsorption sensitive to reduction by L-NMMA after renal denervation. In a second set of studies, the infusion of the alpha 2 receptor antagonist, yohimbine, to rats with renal nerves intact was found to suppress the effects of L-NMMA on nephron plasma flow and proximal reabsorption. The effects of L-NMMA on nephron GFR and nephron plasma flow, afferent and efferent arteriolar resistances, and proximal reabsorption correlated with the level of underlying alpha 2-adrenergic activity. The activation of renal alpha 2-adrenoceptors increases the influence of arginine-derived nitric oxide in the glomerulus and proximal tubule.

Validation of the labeled bicarbonate technique for measurement of short-term energy expenditure in the mouse.

The energy expenditure of free-living animals has been studied extensively by the doubly-labeled water (DLW) technique. This method provides a reasonably accurate estimate of daily energy needs. However, there is considerable interest in the energy demands of animals over much shorter timescales, for which the DLW technique is less useful. We examined the possibility of measuring the expenditure of small animals over these shorter timescales from the washout kinetics of a bolus dose of 13C labeled bicarbonate. The study involved 19 laboratory mice which were injected either i.p. or s.c. with 0.2 ml of 13C labeled bicarbonate in water. Mice were placed in a standard respirometry system, maintained at different temperatures to precipitate a 3 fold variation in metabolism. Samples of breath were collected from the chamber into vacutainers at one minute intervals for approximately 40 minutes to an hour. Samples were analyzed by admission to a mass spectrometer (VG Optima) via a GC interface which identified and admitted the CO2 peak. The log converted isotope elimination was linear (r2 > 98% in all cases) indicating a single pool was involved. We evaluated the pool size from a dilution series of the injectate in equilibrium with CO2 gas. Conventional compartmental analysis produced an estimate which on average across the 19 individuals provided a reasonable estimate of the CO2 production. Individual estimates were however imprecise and the overall correlation between isotope and calorimeter estimates had an r2 of only 15%. Reasons for this discrepancy are unclear. Nevertheless an empirical model, using the elimination gradient, pool size and route of isotope administration as predictors explained 86% of the variation in CO2 production. Elimination of a bolus dose of 13C labeled bicarbonate provides a useful tool for estimating the energy metabolism of mice over intervals between 15 and 40 minutes.

Absorption of visible spectrum radiation by the wing membranes of living pteropodid bats.

The wing membranes of bats present a large surface area upon which radiation might be taken up, increasing heat load to the animals. This, combined with the high amount of heat produced during flight, has been advanced as one hypothesis explaining the fact that bats are almost exclusively nocturnal. The proportion of short-wave (visible) radiation absorbed by bat wing membrane has previously been measured at between 0.7 and 0.92. These measurements were made on pieces of membrane taken from the wings of dead, mainly insectivorous bats from temperate regions. Here we examined the amount of light transmitted through and reflected off the wing membranes of four species of live pteropodid bats. There were significant differences in wing reflection between species. At 0.68, the average proportion of light absorbed into the wing membranes was lower than previously reported. This might be because we worked with live animals or because ours were tropical bats which are routinely exposed to tropical sun when roosting. Variation in wing tension strongly affected light absorption. It was predicted that the relaxed state of wing membrane through part of the wing beat cycle would increase the absorption of light into the wings of day-flying bats. The proportion of light absorbed into wings was shown to be an important factor in the heat balance of hypothetical bats flying during the day. Our results raise the predicted temperature at which bats flying during the day might experience hyperthermia by approximately 2 degrees C and suggest that variation in albedo of wings between species may make some species more susceptible to overheating than others.

Tubuloglomerular feedback responses to acute contralateral nephrectomy.

After unilateral nephrectomy adaptive events must occur in the remaining kidney within the first 12-14 h in anticipation of an increase in glomerular filtration rate (GFR) and eventual renal hypertrophy. Utilizing micropuncture and microperfusion techniques in the rat, we have examined tubuloglomerular feedback (TGF) and single-nephron GFR (SNGFR) responses while the late proximal tubule was microperfused [late proximal tubule flow (VLP)] from 0 to 40 nl/min in 10 nl/min intervals at 2-4 and 12 h after contralateral nephrectomy. Urinary excretion increased, but SNGFR derived from distal collections was reduced, and early distal flow rate remained constant 2-4 h after nephrectomy. The operating point was shifted, suggesting activation of TGF. The turning point half-maximal activity (V1/2) and slope were not statistically different when all nephron data were submitted to a curve-fitting procedure, but group mean data suggested a quantitatively lower V1/2 and steeper slope of the TGF profile. Twelve to fourteen hours after contralateral nephrectomy, values for SNGFR at all microperfusion rates were increased, as were late proximal and early distal flow rates. The values for V1/2 and slope of TGF were not statistically different from control values. We conclude that TGF activity and sensitivity are not suppressed at 2 and 12 h after nephrectomy. Increased urinary excretion does not require TGF alterations. Changes in TGF may be adaptive to increases in SNGFR and may not be causal to the increase in filtration rate after nephrectomy.