Detection of low-frequency oscillations in renal blood flow.

Detection of the low-frequency (LF; approximately 0.01 Hz) component of renal blood flow, which is theorized to reflect the action of a third renal autoregulatory mechanism, has been difficult due to its slow dynamics. In this work, we used three different experimental approaches to detect the presence of the LF component of renal autoregulation using normotensive and spontaneously hypertensive rats (SHR), both anesthetized and unanesthetized. The first experimental approach utilized a blood pressure forcing in the form of a chirp, an oscillating perturbation with linearly increasing frequency, to elicit responses from the LF autoregulatory component in anesthetized normotensive rats. The second experimental approach involved collection and analysis of spontaneous blood flow fluctuation data from anesthetized normotensive rats and SHR to search for evidence of the LF component in the form of either amplitude or frequency modulation of the myogenic and tubuloglomerular feedback mechanisms. The third experiment used telemetric recordings of arterial pressure and renal blood flow from normotensive rats and SHR for the same purpose. Our transfer function analysis of chirp signal data yielded a resonant peak centered at 0.01 Hz that is greater than 0 dB, with the transfer function gain attenuated to lower than 0 dB at lower frequencies, which is a hallmark of autoregulation. Analysis of the data from the second experiments detected the presence of approximately 0.01-Hz oscillations only with isoflurane, albeit at a weaker strength compared with telemetric recordings. With the third experimental approach, the strength of the LF component was significantly weaker in the SHR than in the normotensive rats. In summary, our detection via the amplitude modulation approach of interactions between the LF component and both tubuloglomerular feedback and the myogenic mechanism, with the LF component having an identical frequency to that of the resonant gain peak, provides evidence that 0.01-Hz oscillations may represent the third autoregulatory mechanism.

Measurement of nitric oxide formation.

Nitric oxide (NO) is a simple gaseous monoxide that is involved in a variety of biological mechanisms in the mammalian body (1). NO is produced by a group of enzymes, nitric oxide synthases. One of the more important functions of NO is to regulate the tone of blood vessels (2). As one of the most potent vasodilators known, NO modulates the vasoconstrictive action of humoral factors, including angiotensin II (Ang II). Therefore, a well-controlled balance between Ang II and NO is required for normal hemodynamic function in all blood vessels (3-6).

Lacrimal gland fluid secretion and lymphocytic infiltration in the NZB/W mouse model of Sjögren's syndrome.

PURPOSE: The fluid secretory impairment of lacrimal and salivary glands in Sjögren's syndrome (SS) is thought to be related to the extent of lymphocytic infiltration (LI) and subsequent loss of glandular tissue. In this study, we examine the correlation between the extent of tear flow reduction and the extent of LI of lacrimal glands in the NZB/W mouse, a model of SS. METHODS: We stimulated tear production by topical application of carbachol onto the gland while fluid was collected from the lacrimal duct. The lacrimal glands were removed after fluid collection for histology. RESULTS: Fluid secretion in response to carbachol was less in the majority of young NZB/W females compared to C57 control animals and none of the glands showed LI. Fluid secretion was also impaired in the majority of old NZB/W females, and the extent of LI was highly variable. Some of the old SW females also showed blunted fluid secretory responses and some degree of focal LI. Young SW females showed no LI and most animals exhibited normal flow responses. Analysis of paired flow and LI measurements showed no correlation between LI and flow impairment in any of the groups or in the pooled data. Carbachol-stimulated protein secretion from lacrimal gland slices in vitro were similar in young and old SW and NZB/W mice. CONCLUSIONS: These results suggest that LI alone is not sufficient to explain the secretory dysfunction in the NZB/W mouse model of Sjögren's syndrome.

Advanced glycosylation end-products and NO-dependent vasodilation in renal afferent arterioles from diabetic rats.

Systemic pressor responses to acetylcholine (ACh) are reduced in DM, an effect thought to be related to quenching of nitric oxide (NO) by advanced glycosylation end-products (AGE). We studied the effects of AGE in juxtamedullary (JM) afferent arterioles (AA) from rats with 40-50 days diabetes mellitus (DM) induced via streptozotocin. JM AA were perfused in vitro with solutions containing fresh RBCs suspended in either 6% bovine albumin or 6% AGE-albumin in euglycaemic Krebs-Ringer. Autoregulatory responses were evident in the DM vessels: AA constricted 31 +/- 2% (n=9) when perfusion pressure (PP) was raised from 60 to 140 mmHg. ACh (10 microM) caused a 43 +/- 15% dilation and Ca2+-channel blockade elicited a 95 +/- 14% dilation at 100 mmHg PP, indicating substantial basal vascular tone in DM AA. L-NAME (0.1 mM) constricted DM AA by 21 +/- 2% (n=9) at 100 mmHg PP, indicating significant basal NO production in DM vessels. Segments of renal resistance arteries from DM rats perfused in vitro responded to muscarinic stimulation and elevated glucose levels with significant increments in NO production, as measured with an NO-sensitive electrode. This observation shows that the renal endothelial NO system is intact in DM. While AGE in the perfusate dilated control AA, they had no effect on DM AA at all PP levels, although they blunted ACh-induced dilation. Hence, although AGE do appear to have vasoactive properties in the absence of hyperglycaemia, the results of this study are inconsistent with substantial NO quenching by AGE.

Limit-cycle oscillations and tubuloglomerular feedback regulation of distal sodium delivery.

A mathematical model was used to evaluate the potential effects of limit-cycle oscillations (LCO) on tubuloglomerular feedback (TGF) regulation of fluid and sodium delivery to the distal tubule. In accordance with linear systems theory, simulations of steady-state responses to infinitesimal perturbations in single-nephron glomerular filtration rate (SNGFR) show that TGF regulatory ability (assessed as TGF compensation) increases with TGF gain magnitude gamma when gamma is less than the critical value gamma(c), the value at which LCO emerge in tubular fluid flow and NaCl concentration at the macula densa. When gamma > gamma(c) and LCO are present, TGF compensation is reduced for both infinitesimal and finite perturbations in SNGFR, relative to the compensation that could be achieved in the absence of LCO. Maximal TGF compensation occurs when gamma approximately gamma(c). Even in the absence of perturbations, LCO increase time-averaged sodium delivery to the distal tubule, while fluid delivery is little changed. These effects of LCO are consequences of nonlinear elements in the TGF system. Because increased distal sodium delivery may increase the rate of sodium excretion, these simulations suggest that LCO enhance sodium excretion.

New method for imaging innervation of the renal preglomerular vasculature. Alterations in hypertensive rats.

OBJECTIVE: To develop a new method for viewing adrenergic innervation along renal preglomerular vessels; to assess nerve densities and vascular lesions along arcuate arteries (ArcA), arcuate arterial branches (ArcB), and interlobular arteries (ILA) in spontaneously hypertensive rats (SHR) and in angiotensin II (AngII) and in N(G)-nitro-L-arginine methyl ester (L-NAME) hypertensive rats. METHODS: Preglomerular vasculatyres were isolated after HCl maceration and were immunostained against synaptophysin, a membrane protein of synaptic vesicles. Lesions were stained with Sudan black. Longitudinal nerve densities and relative frequencies of ArcA, ArcB, and ILA endowed with sudanophilic lesions were assessed separately. RESULTS: Synaptophysin immunostaining revealed the vascular neural plexus. Nerves were adrenergic, as the plexus was destroyed by treatment with 6-hydroxy dopamine. Vascular lesions were not seen in SHR, and increased nerve density was observed along ArcA and ILA. In L-NAME- and AngII-hypertensive rats, vascular lesions affected predominantly ArcB and ILA, and nerve density was reduced by 12% and 28% (ArcA), 37% and 31% (ArcB), and by 55% and 34% (ILA), respectively, versus normotensive controls. Endothelin-1 receptor blockade did not affect AngII-induced hypertension but prevented both lesion development and reduction of density of the vascular neural plexus. CONCLUSIONS: The method we have devised provides a direct en face view of the vascular adrenergic innervation of isolated preglomerular vasculature. Measurements in hypertensive rat models suggest a link between vascular lesions and reduction in nerve density in hypertension. Endothelin-1 likely plays a key role in mediation both vascular injury and altered vascular nerve density in hypertension.

Carbon monoxide induces vasodilation and nitric oxide release but suppresses endothelial NOS.

The vascular effects of carbon monoxide (CO) resemble those of nitric oxide (NO), but it is unknown whether the two messengers converge or exhibit reciprocal feedback regulation. These questions were examined in microdissected perfused renal resistance arteries (RRA) studied using NO-sensitive microelectrodes. Perfusion of RRA with buffers containing increasing concentrations of CO resulted in a biphasic release of NO. The NO response peaked at 100 nM CO and then declined to virtually zero at 10 microM. When a series of 50-s pulses of 100 nM CO were applied repeatedly (150-s interval), the amplitude of consecutive NO responses was diminished. NO release from RRA showed dependence on L-arginine but not D-arginine, and the responses to CO were inhibited by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthases (NOS). CO (100 nM) also suppressed NO release induced by 100 microM carbachol, a potent agonist for endothelial NOS (eNOS). RRA from rats in which endogenous CO production from inducible HO was elevated (cobalt chloride 12 h prior to study) also showed suppressed responses to carbachol. Furthermore, responses consistent with these findings were obtained in juxtamedullary afferent arterioles perfused in vitro, where the vasodilatory response to CO was biphasic and the response to acetylcholine was blunted. Collectively, these data suggest that the CO-induced NO release could be attributed to either stimulation of eNOS or to NO displacement from a cellular storage pool. To address this, direct in vitro measurements with an NO-selective electrode of NO production by recombinant eNOS revealed that CO dose-dependently inhibits NO synthesis. Together, the above data demonstrate that, whereas high levels of CO inhibit NOS activity and NO generation, lower concentrations of CO induce release of NO from a large intracellular pool and, therefore, may mimic the vascular effects of NO.

AT1 receptor inhibition blunts angiotensin II-stimulated nitric oxide release in renal arteries.

Nitric oxide (NO) is known to modulate the vascular effects of angiotensin II (AngII) in the kidney. To investigate the effect of AngII on NO release, a new technique was used that employs an NO-sensitive microelectrode to measure NO release from the vascular endothelium of perfused renal resistance arteries (tertiary branches of the renal artery or primary arcuate arteries) in vitro. The vessels were microdissected from isolated perfused rat kidneys, cannulated, and perfused at constant flow and pressure with Krebs-Ringer bicarbonate solution. The electrode was placed inside the glass collection cannula to measure vessel effluent NO concentration. Addition of AngII to the perfusate stimulated NO release in a dose-dependent manner; 0.1, 10, and 1000 nM AngII increased NO oxidation current by 85+/-18 pA (n=11), 148+/-22 pA (n=11), and 193+/-29 pA (n=11), respectively. These currents correspond to changes in effluent NO concentration of 3.4+/-0.5, 6.1+/-1.1, and 8.2+/-1.3 nM, respectively. The presence of 0.1 mM N(G)-nitro-L-arginine methyl ester in the perfusate significantly reduced the response to 10 nM AngII by 90.5+/-3.4% (n=5). Neither losartan (1 microM) nor candesartan (1 nM) significantly affected basal NO production, but both of these AT1-receptor blockers markedly blunted NO release in response to AngII (10 nM): 77+/-6% inhibition with losartan (n=8) and 63+/-9% with candesartan (n=8). These results demonstrate that AngII stimulates N(G)-nitro-L-arginine methyl ester-inhibitable NO release in isolated renal resistance arteries. Because the response was significantly blunted by AT1 receptor blockade, the findings suggest that endothelium-dependent modulation of AngII-induced vasoconstriction in renal resistance arteries is mediated, at least in part, by AT1 receptor-dependent NO release.

Insulin-like growth factor-I restores microvascular autoregulation in experimental chronic renal failure.

Impairment of autoregulation (AR) is associated with accelerated progression of chronic renal failure (CRF). As the bioavailability of insulin-like growth factor-I (IGF-I) is low in CRF, we investigated the effects of acute luminal application of 10 nM recombinant human IGF-I on AR in juxtamedullary (JM) afferent arterioles (AA) perfused in vitro with a blood solution [(approximately 30% hematocrit (HCT)]. Studies were conducted in AA from adult male rats three to four weeks after five-sixths nephrectomy (Nx) by either surgical excision (N = 7) or infarction (N = 5) of two thirds of the remnant kidney; controls (N = 6) had sham surgery. AA from both Nx groups exhibited marked hypertrophy and impaired AR responses (60 to 140 mm Hg perfusion pressure), features more pronounced in the infarction group. Responses to abluminal acetylcholine (10 microM) were similar in sham and excision groups but were significantly blunted in the infarction group. All groups vasodilated significantly after Ca-channel blockade (10 mM MnCl2). IGF-I restored AR in AA from both Nx groups (P < 0.05, analysis of variance) while it vasodilated AA from controls. These results suggest that IGF-I may protect the glomerulus from injury by maintaining autoregulatory control of renal blood flow, thereby slowing the progression of CRF.

Effects of insulin-like growth factor I on the renal juxtamedullary microvasculature.

To characterize the effects on the rat renal preglomerular microvasculature of insulin-like growth factor I (IGF-I), experiments were performed using the in vitro blood-perfused juxtamedullary nephron preparation. IGF-I induced a reversible vasodilation of pre- but not postglomerular microvessels in a dose-dependent manner (10(-9)-10(-7) M). The IGF-I-induced vasodilation was similar in all preglomerular vascular segments: interlobular artery, 11.5 +/- 1.2% of control (n = 16); mid-afferent arterioles, 11.6 +/- 1.7% (n = 24); and juxtaglomerular afferent segments, 16.1 +/- 2.8% (n = 19). Renal autoregulatory capacity was not reduced by IGF-I. Pretreatment with the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (10(-4) M) completely inhibited the vasodilatory response to IGF-I. IGF-I induced a rapid increase of NO concentration in intact renal microvessels, monitored by a NO-selective voltametric microelectrode. Pretreatment with the cyclooxygenase inhibitor indomethacin (10(-5) M) not only abrogated the IGF-I-induced dilation, but, moreover, IGF-I elicited a small but significant (approximately 10%) vasoconstriction in all preglomerular vessels. These results indicate that the renal vascular effects of IGF-I involve activation of two endogenous vasodilators (NO and vasodilatory prostaglandins). In addition, IGF-I may also release an undefined vasoconstrictor.

Angiotensin-II stimulates nitric oxide release in isolated perfused renal resistance arteries.

Nitric oxide (NO) has been implicated as a modulator of the vascular effects of angiotensin II (ANG II) in the kidney. We used a NO-sensitive microelectrode to study the effect of ANG II on NO release, and to determine the effect of selective inhibition of the ANG II subtype I receptor (AT1) with losartan (LOS) and candesartan (CAN). NO release from isolated and perfused renal resistance arteries was measured with a porphyrin-electroplated, carbon fiber. The vessels were microdissected from isolated perfused rat kidneys and perfused at constant flow and pressure in vitro. The NO-electrode was placed inside the glass collection cannula to measure vessel effluent NO concentration. ANG II stimulated NO release in a dose-dependent fashion: 0.1 nM, 10 nM and 1000 nM ANG II increased NO-oxidation current by 85+/-18 pA (n = 11), 148+/-22 pA (n = 11), and 193+/-29 pA (n = 11), respectively. These currents correspond to changes in effluent NO concentration of 3.4+/-0.5 nM, 6.1+/-1.1 nM, and 8.2+/-1.3 nM, respectively. Neither LOS (1 muM) nor CAN (1 nM) significantly affected basal NO production, but both AT1-receptor blockers markedly blunted NO release in response to ANG II (10 nM): 77+/-6% inhibition with LOS (n = 8) and 63+/-9% with CAN (n = 8). These results are the first to demonstrate that ANG II stimulates NO release in isolated renal resistance arteries, and that ANG II-induced NO release is blunted by simultaneous AT1-receptor blockade. Our findings suggest that endothelium-dependent modulation of ANG II-induced vasoconstriction in renal resistance arteries is mediated, at least in part, by AT1-receptor-dependent NO release.

Nonlinear filter properties of the thick ascending limb.

A mathematical model was used to investigate the filter properties of the thick ascending limb (TAL), that is, the response of TAL luminal NaCl concentration to oscillations in tubular fluid flow. For the special case of no transtubular NaCl backleak and for spatially homogeneous transport parameters, the model predicts that NaCl concentration in intratubular fluid at each location along the TAL depends only on the fluid transit time up the TAL to that location. This exact mathematical result has four important consequences: 1) when a sinusoidal component is added to steady-state TAL flow, the NaCl concentration at the macula densa (MD) undergoes oscillations that are bounded by a range interval envelope with magnitude that decreases as a function of oscillatory frequency; 2) the frequency response within the range envelope exhibits nodes at those frequencies where the oscillatory flow has a transit time to the MD that equals the steady-state fluid transit time (this nodal structure arises from the establishment of standing waves in luminal concentration, relative to the steady-state concentration profile, along the length of the TAL); 3) for any dynamically changing but positive TAL flow rate, the luminal TAL NaCl concentration profile along the TAL decreases monotonically as a function of TAL length; and 4) sinusoidal oscillations in TAL flow, except at nodal frequencies, result in nonsinusoidal oscillations in NaCl concentration at the MD. Numerical calculations that include NaCl backleak exhibit solutions with these same four properties. For parameters in the physiological range, the first few nodes in the frequency response curve are separated by antinodes of significant amplitude, and the nodes arise at frequencies well below the frequency of respiration in rat. Therefore, the nodal structure and nonsinusoidal oscillations should be detectable in experiments, and they may influence the dynamic behavior of the tubuloglomerular feedback system.

Spectral properties of the tubuloglomerular feedback system.

A simple mathematical model was used to investigate the spectral properties of the tubuloglomerular feedback (TGF) system. A perturbation, consisting of small-amplitude broad-band forcing, was applied to simulated thick ascending limb (TAL) flow, and the resulting spectral response of the TGF pathway was assessed by computing a power spectrum from resulting TGF-regulated TAL flow. Power spectra were computed for both open- and closed-feedback-loop cases. Open-feedback-loop power spectra are consistent with a mathematical analysis that predicts a nodal pattern in TAL frequency response, with nodes corresponding to frequencies where oscillatory flow has a TAL transit time that equals the steady-state fluid transit time. Closed-feedback-loop spectra are dominated by the open-loop spectral response, provided that gamma, the magnitude of feedback gain, is less than the critical value gamma c required for emergence of a sustained TGF-mediated oscillation. For gamma exceeding gamma c, closed-loop spectra have peaks corresponding to the fundamental frequency of the TGF-mediated oscillation and its harmonics. The harmonics, expressed in a nonsinusoidal waveform for tubular flow, are introduced by nonlinear elements of the TGF pathway, notably TAL transit time and the TGF response curve. The effect of transit time on the flow waveform leads to crests that are broader than troughs and to an asymmetry in the magnitudes of increasing and decreasing slopes. For feedback gain magnitude that is sufficiently large, the TGF response curve tends to give a square waveshape to the waveform. Published waveforms and power spectra of in vivo TGF oscillations have features consistent with the predictions of this analysis.

Production and physiological actions of anandamide in the vasculature of the rat kidney.

The endogenous cannabinoid receptor agonist anandamide is present in central and peripheral tissues. As the kidney contains both the amidase that degrades anandamide and transcripts for anandamide receptors, we characterized the molecular components of the anandamide signaling system and the vascular effects of exogenous anandamide in the kidney. We show that anandamide is present in kidney homogenates, cultured renal endothelial cells (EC), and mesangial cells; these cells also contain anandamide amidase. Reverse-transcriptase PCR shows that EC contain transcripts for cannabinoid type 1 (CB1) receptors, while mesangial cells have mRNA for both CB1 and CB2 receptors. EC exhibit specific, high-affinity binding of anandamide (Kd = 27.4 nM). Anandamide (1 microM) vasodilates juxtamedullary afferent arterioles perfused in vitro; the vasodilation can be blocked by nitric oxide (NO) synthase inhibition with L-NAME (0.1 mM) or CB1 receptor antagonism with SR 141716A (1 microM), but not by indomethacin (10 microM). Anandamide (10 nM) stimulates CB1-receptor-mediated NO release from perfused renal arterial segments; a similar effect was seen in EC. Finally, anandamide (1 microM) produces a NO-mediated inhibition of KCl-stimulated [3H]norepinephrine release from sympathetic nerves on isolated renal arterial segments. Hence, an anandamide signaling system is present in the kidney, where it exerts significant vasorelaxant and neuromodulatory effects.

Role of mesangial cells in macula densa to afferent arteriole information transfer.

1. Extraglomerular mesangial cells are strategically positioned between the macula densa and the afferent arteriole. These cells form a syncytium and are connected with glomerular mesangial cells via gap junctions. The model of immunoablation of mesangial cells in anti-Thy-1 glomerulonephritis carries the promise for understanding the function of mesangial cells as potential transmitters of information between the macula densa and the afferent arteriole. 2. The above anatomical relations between structures in the juxtaglomerular apparatus predict several possible routes of information flow. This review charts some hypothetical routes. 3. Research into the messenger systems involved in the transfer of signals from the macula densa to mesangial cells and from mesangial cells to the afferent arteriole suggests several candidate molecules to function in this capacity. Mechanisms of action for each candidate are discussed. 4. The oscillating nature of the afferent signal and efferent function in the tubuloglomerular feedback system, as well as other discoveries, offer a fertile field for future studies.

Branching patterns and autoregulatory responses of juxtamedullary afferent arterioles.

The spatial organization of autoregulatory responses (AR) was assessed in couples of afferent arterioles (AA), either grouped as anatomic pairs or branched sequentially along the same arcuate arterial branch (ArcB). With blood-perfused juxtamedullary nephron (JMN) preparations, AR were elicited by raising blood perfusion pressure from 60 to 120 mmHg and quantified by videomicroscopy as pressure-induced constrictions. Paired AA had unequal lengths (long-to-short ratio, 1.9 +/- 0.1; n = 36); however, no statistical difference in AR was found between long and short AA at juxtaglomerular or early AA (EAA) sites. Sequentially branched AA had the same length heterogeneity as paired AA (proximal-to-distal AA length ratio, 2.0 +/- 0.2; n = 30). However, AR exhibited a significant axial gradient, being higher in distal than in proximal AA or ArcB sites. In both AA branching patterns, EAA and nearby sites of the feed arteries had similar AR. Hence, our results are consistent with hemodynamic coupling in paired JMN. Around branching sites, AR are spatially organized in a way consistent with electrotonic vascular coupling.

Decreased hepatic insulin-like growth factor (IGF)-I and increased IGF binding protein-1 and -2 gene expression in experimental uremia.

The imbalance between normal insulin-like growth factor-I (IGF-I) and markedly increased IGF binding protein (IGFBP) plasma levels plays a pathogenic role for growth retardation and catabolism in children with chronic renal failure. To investigate the mechanism of these alterations, experiments were performed in an experimental model of uremia in rats (5/6 nephrectomy) and in pair-fed and ad libitum-fed sham-operated controls Using a specific solution hybridization/RNase protection assay, we observed a marked reduction of hepatic IGF-I messenger RNA (mRNA) abundance at steady state in uremic animals (37 +/- 5% of control) compared both with pair-fed (65 +/- 10%) and ad libitum-fed controls (100 +/- 11%) (P < 0.001). Reduced IGF-I gene expression was clearly organ-specific; it was most pronounced in liver (significant vs., pair-fed controls) and lung and muscle tissue (significant vs., ad libitum-fed controls); no change was observed in kidney and heart tissue. To determine a potential mechanism of reduced hepatic IGF-I gene expression in uremia, the hepatic GH receptor gene expression in the same experimental animals was analyzed by specific solution hybridization/RNase protection assay. Uremic animals had a 20-30% reduction of hepatic GH receptor mRNA abundance compared with controls. Hepatic GHBP expression in uremia was decreased in parallel. Despite the reduction of hepatic IGF-I mRNA abundance, plasma IGF-I levels in uremia were not different from ad libitum-fed controls. This discrepancy is explained by an increased concentration of IGFBPs in uremic plasma. By RIA, plasma IGFBP-1 levels in uremia were increased 4-fold; by Western immunoblot, plasma IGFBP-2 levels were increased 7-fold and plasma IGFBP-4 levels were increased 2-fold compared with both control groups. Intact IGFBP-3 (M(r), approximately 48 kDa) and low molecular IGFBP-3 fragments were not significantly different among the three groups. By Northern blot analysis, hepatic IGFBP-1 mRNA levels in uremia were 2-fold higher than in controls. IGFBP-2 mRNA abundance in liver tissue was increased 4-fold, whereas in kidney there was a significant reduction of IGFBP-2 mRNA (30% of control). IGFBP-4 mRNA was increased by 50% in kidney but not in liver. Plasma insulin and corticosterone levels were not different among the groups. Our study shows that hepatic IGF-I gene expression was specifically reduced in uremia, partially as the consequence of a reduced hepatic GH receptor gene expression. One of the mechanisms contributing to increased IGFBP levels in uremia is increased hepatic gene expression of IGFBP-1 and IGFBP-2. The imbalance between reduced hepatic IGF-I production and increased hepatic IGFBP-1 and 2 production is likely to play a pathogenic role for catabolism and growth failure in CRF.

Alternative splicing of CREB and CREM mRNAs in an immortalized germ cell line.

Alternative splicing of CREB (cAMP response element binding protein) and CREM (cAMP response element modulator) mRNAs in separated pachytene spermatocyte, round spermatid, and elongated spermatid fractions and the germ cell-derived immortalized cell line GC-2spd(ts) was studied by reverse transcription polymerase chain reaction (RT-PCR). Both primary germ cells and the GC-2spd(ts) cell line expressed the testis-specific CREB splice variant containing exon W. In the CREB C-E exon region, both primary germ cells and GC-2spd(ts) cells produced RT-PCR products that included exon Y. RT-PCR using CREM primers produced multiple bands in primary germ cells. The truncated CREAM deltaC-G form was found in all the germ cell fractions. The smaller splice forms of CREM were more prominent in the GC-2spd(ts) cells. GC-2spd(ts) cells resembled F9 teratocarcinoma cells more closely than primary germ cells with respect to the relative expression of both CREB and CREM alternative splice products. In Sertoli cells, RT-PCR products of CREB exon lacking W and the product corresponding to CREM delta C-G were most prominent. These data show that the GC-2spd(ts) cell line retains some qualitative characteristics of primary germ cells with respect to alternative splicing of CREB and CREM mRNA.

Capillary electrophoresis for the determination of glomerular filtration rate using nonradioactive iohexol.

High-performance liquid chromatography (HPLC) has been used as an alternative to the isotopic method to calculate glomerular filtration rate (GFR). With the HPLC method, serum iohexol or iothalamate levels are measured, and the plasma clearance rate of the compound is used as a surrogate for GFR. However, HPLC is a labor-intensive procedure, which limits its usefulness in the clinical setting. Capillary electrophoresis, a newer technique in which electrophoretic separations are performed in capillary tubes, is easier and faster than HPLC. We used capillary electrophoresis for the determination of serum iohexol levels and the calculation of GFR. Patients underwent a simultaneous 125I-iothalamate clearance test and a plasma iohexol clearance test to determine GFR. Mean GFR (+/-SD) was 70.9 +/- 29.9 mL/min (range, 14.5 to 131 mL/min) in 52 patients as determined by standard iothalamate clearance methods. For iohexol clearance, the correlation coefficient and standard error were 0.93 and 10.9 mL/min, respectively, using capillary electrophoresis compared with the iothalamate method. Capillary electrophoresis is a simple, rapid method that can be used to calculate GFR and provides results at least as accurate as those obtained by HPLC and x-ray fluorescence.

Neuropathic pain in rats is associated with altered nitric oxide synthase activity in neural tissue.

Peripheral nerve injury may lead to a chronic neuropathic pain state that results from an increase in excitability of central neurons. This central sensitization is mediated via an N-methyl-D-aspartic acid (NMDA) receptor and may involve the production of nitric oxide (NO). As NO is suggested to play a role in nociceptive transmission following nerve injury, we examined for altered NO synthase activity at multiple levels of peripheral and spinal neural tissue in a rat model of neuropathic pain. Peripheral neuropathy was induced in rats (N = 12) by ligation of the left L5 and L6 nerve roots. Six other rats had sham surgery. An ipsilateral decrease in paw withdrawal threshold to mechanical stimuli confirmed the presence of a neuropathic pain state. Samples of the lumbar and thoracic spinal cords, L4, L5, and L6 dorsal root ganglia (DRGs), and the sciatic nerves were obtained from the lesioned and contralateral sides at 2 and 4 weeks after neuropathic surgery (N = 6 per group). In the lumbar spinal cord, a bilateral decrease in nitric oxide synthase (NOS) activity was observed 2 and 4 weeks after neuropathic surgery. NOS activity was increased in the ipsilateral L5 and 6 DRGs 2 weeks following neuropathic surgery. An increase in NOS activity in the DRG may be an early mechanism for inducing more central changes. The bilaterally decreased NOS activity in the lumbar spinal cord may be secondary to a negative feedback mechanism resulting from increased NO production in the spinal dorsal root ganglia. Multiple alterations in expression of NOS activity that occur in both peripheral and central processing may play a role in the pain behavior resulting from peripheral nerve injury. (Preliminary results of these studies have been presented in abstract form at the annual meetings of the Society for Neuroscience, 1994, and the American Society of Anesthesiologists, 1994).