Intact Kidney Research Articles

Characterization of the naturally occurring Arg344His variant of the human 5-HT3A receptor

The present study aimed at examining the function and pharmacological properties of the naturally occurring Arg344His variant of the human 5-HT3A receptor, identified in a schizophrenic patient. In intact human embryonic kidney (HEK) 293 cells expressing the wild-type (WT) or the variant receptor, the function was analyzed by indirect measurement of agonist-induced Ca2+ current through the 5-HT3A receptor channel by an aequorin luminescence-based Ca2+ assay. In cell membrane patches cation currents were determined electrophysiologically including technically demanding single channel analyses. The pharmacological properties were analyzed by [3H]GR65630 binding to cell membrane fragments. The density of [3H]GR65630 binding sites in cells expressing the variant receptor was reduced to 55% of that in cells expressing the WT receptor, which, however, was not accompanied by an analogous decrease in 5-HT-induced Ca2+ influx through the receptor channel. However, the single channel analysis suggests an increase in single receptor channel mean open time (which is known to be subject of many variables) but not in unitary current amplitude. Radioligand competition experiments revealed that the affinity of five 5-HT3 receptor agonists and four antagonists for the variant receptor did not differ from that for the WT receptor. In conclusion, the variant receptor resembles the WT receptor in that it forms functional homopentameric 5-HT3A receptors with identical pharmacological properties. In view of the lack of reduction in Ca2+ flux through the variant receptor channels in spite ofthe decrease in its density on the cell membrane, the increase in single receptor channel mean open time appears to compensate for the reduction in variant receptor density.

A New Simple Cell-Based Homogeneous Time-Resolved Fluorescence QRET Technique for Receptor-Ligand Interaction Screening

In this article, a single-label separation-free fluorescence technique is presented as a potential screening method for cell-based receptor antagonists and agonists.The time-resolved fluorescence technique, quenching resonance energy transfer (QRET), relies on a single-labeled binding partner in combination with a soluble quencher. The quencher efficiently suppresses the luminescence of the unbound labeled ligand, whereas the luminescence of the bound fraction is not affected. This approach allows the development of cell-based screening assays in a simple and cost-effective manner. The authors have applied the technique to the screening of beta(2)-adrenoreceptor (beta(2)AR) antagonists and agonists in intact human embryonic kidney HEK293(i) cells overexpressing human beta(2)-adrenergic receptors. Two antagonists (propranolol, alprenolol) and 2 agonists (metaproterenol, terbutaline) for beta(2)AR were investigated in a displacement assay using europium(III)-labeled pindolol ligand. The assay Z' values ranged from 0.68 to 0.78, the coefficient of variation was less than 10%, and the K(i) values were 19 nM for propranolol and alprenolol and 14 and 5.9 microM for metaproterenol and terbutaline, respectively. The QRET technique with beta(2)AR was also applied to LOPAC compound library screening, yielding nearly error-free recognition of known binders. This simple and cost-effective technique can be readily adapted to laboratory and industrial-scale screening.

Heat Shock Protein gp96 Interacts with Protein Phosphatase 5 and Controls Toll-like Receptor 2 (TLR2)-mediated Activation of Extracellular Signal-regulated Kinase (ERK) 1/2 in Post-hypoxic Kidney Cells

Ischemia/reperfusion injury (IRI) induces an innate immune response, leading to an inflammatory reaction and tissue damage that have been attributed to engagement of the Toll-like receptor (TLR) 2 and 4. However, the respective roles of TLR2 and/or TLR4 in mediating downstream activation of mitogen-activated protein kinase (MAPK) pathways during IRI have not been fully elucidated. Here we show that extracellular signal-regulated kinase (ERK)1/2 is activated in both intact kidneys and cultured renal tubule epithelial cells (RTECs) from wildtype and Tlr4 knockout mice, but not those from Tlr2 knockout mice subjected to transient ischemia. Geldanamycin (GA), an inhibitor of heat shock protein 90 and reticulum endoplasmic-resident gp96, and gp96 mRNA silencing (siRNA), did not affect ERK1/2 activation in either post-hypoxic wild-type or Tlr4-deficient RTECs, but did restore its activation in post-hypoxic Tlr2-deficient RTECs. Immunoprecipitation studies revealed that gp96 co-immunoprecipitates with the serine-threonine protein phosphatase 5 (PP5), identified as a negative modulator of the mitogen extracellular kinase (MEK)-ERK pathway, in unstressed wild-type and post-hypoxic Tlr2-deficient RTECs. In contrast, PP5 co-immunoprecipitation with gp96 was strikingly reduced in post-hypoxic wild-type RTECs, suggesting that the inactivation of PP5 resulting from the dissociation of PP5 from gp96 allows the activation of ERK1/2 to occur. Inhibition of PP5 by okadaic acid, and Pp5 siRNA also restored TLR2-mediated phosphorylation of ERK1/2, and apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK)-mediated apoptosis in post-hypoxic Tlr2-deficient RTECs. These findings indicate that gp96 interacts with PP5 and controls TLR2-mediated induction of ERK1/2 in post-hypoxic renal tubule cells.

Bradykinin stimulates renal collecting duct prorenin

Elevated plasma prorenin (PR) is a predictor of diabetic (DM) microvascular complications. We recently identified the renal collecting duct (CD) as the major source of PR in DM and showed that angiotensin II (ANGII) receptor blockade, effectively reduces renal and plasma PR levels. We aimed to compare the effects of the ANGII receptor 1 blocker telmisartan (ARB, T), and the angiotensin converting enzyme inhibitor (ACEI), ramipril (R), on CD PR. PR content in the intact kidney was visualized in control (C) and STZ‐DM MWF rats using in vivo multi‐photon fluorescence microscopy. DM‐rats were treated for 28 days with T (ARB, 3mg/kg/day) or R (ACEI, 1mg/kg/day). Direct effects of ANGII and bradykinin (B,10nM each) on CD PR synthesis were measured using renin immunocytochemistry. STZ‐DM caused a 3‐fold increase in CD PR content which was completely abolished by T, but not by R. Also, immunoblot and renin activity measurements found that renal tissue and plasma PR levels increased 2‐fold in STZ‐DM which were prevented by T (0.8‐fold of C), but not by R (2.2‐fold). Similarly to ANGII, B increased immunoreactive PR content in cultured CD cells 35‐fold which was prevented by the B B2 receptor blocker HOE‐140. In conclusion, elevated CD PR content in DM is most effectively inhibited by ANGII receptor blockade using T and not by the blockade of the ACE using R, which effect is at least in part due to the direct stimulatory effect of B on CD PR.

Multiphoton Imaging of Renal Regulatory Mechanisms

Most physiological functions of the kidneys, including the clearance of metabolic waste products, maintenance of body fluid, electrolyte homeostasis, and blood pressure, are achieved by complex interactions between multiple renal cell types and previously inaccessible structures in many organ parts that have been difficult to study. Multiphoton fluorescence microscopy offers a state-of-the-art imaging technique for deep optical sectioning of living tissues and organs with minimal deleterious effects. Dynamic regulatory processes and multiple functions in the intact kidney can be quantitatively visualized in real time, noninvasively, and with submicron resolution. This article reviews innovative multiphoton imaging technologies and their applications that provided the most complex, immediate, and dynamic portrayal of renal function-clearly depicting as well as analyzing the components and mechanisms involved in renal (patho)physiology.

Independent two-photon measurements of albumin GSC give low values

Independent two-photon measurements of albumin GSC give low values

SGK1-sensitive renal tubular glucose reabsorption in diabetes

The hyperglycemia of diabetes mellitus increases the filtered glucose load beyond the maximal tubular transport rate and thus leads to glucosuria. Sustained hyperglycemia, however, may gradually increase the maximal renal tubular transport rate and thereby blunt the increase of urinary glucose excretion. The mechanisms accounting for the increase of renal tubular glucose transport have remained ill-defined. A candidate is the serum- and glucocorticoid-inducible kinase SGK1. The kinase has been shown to stimulate Na(+)-coupled glucose transport in vitro and mediate the stimulation of electrogenic intestinal glucose transport by glucocorticoids in vivo. SGK1 expression is confined to glomerula and distal nephron in intact kidneys but may extend to the proximal tubule in diabetic nephropathy. To explore whether SGK1 modifies glucose transport in diabetic kidneys, Akita mice (akita(+/-)), which develop spontaneous diabetes, have been crossbred with gene-targeted mice lacking SGK1 on one allele (sgk1(+/-)) to eventually generate either akita(+/-)/sgk1(-/-) or akita(+/-)/sgk1(+/+) mice. Both akita(+/-)/sgk1(-/-) and akita(+/-)/sgk1(+/+) mice developed profound hyperglycemia (>20 mM) within approximately 6 wk. Body weight and plasma glucose concentrations were not significantly different between these two genotypes. However, urinary excretion of glucose and urinary excretion of fluid, Na(+), and K(+), as well as plasma aldosterone concentrations, were significantly higher in akita(+/-)/sgk1(-/-) than in akita(+/-)/sgk1(+/+) mice. Studies in isolated perfused proximal tubules revealed that the electrogenic glucose transport was significantly lower in akita(+/-)/sgk1(-/-) than in akita(+/-)/sgk1(+/+) mice. The data provide the first evidence that SGK1 participates in the stimulation of renal tubular glucose transport in diabetic kidneys.

Rho-kinase inhibition reduces pressure-mediated autoregulatory adjustments in afferent arteriolar diameter.

Preglomerular resistance is regulated by calcium influx- and mobilization-dependent mechanisms; however, the role of Rho-kinase in calcium sensitization in the intact kidney has not been carefully examined. Experiments were performed to test the hypothesis that Rho-kinase inhibition blunts pressure-mediated afferent arteriolar autoregulatory behavior and vasoconstrictor responses evoked by angiotensin II and P2X1 receptor activation. Rat kidneys were studied in vitro using the blood-perfused juxtamedullary nephron technique. Autoregulatory behavior was assessed before and during Rho-kinase inhibition with Y-27632 (1.0 microM; n = 5). Control diameter averaged 14.3 +/- 0.8 microm and increased to 18.1 +/- 0.9 microm (P < 0.05) during Y-27632 treatment. In the continued presence of Y-27632, reducing perfusion pressure to 65 mmHg slightly increased diameter to 18.7 +/- 1.0 microm. Subsequent pressure increases to 130 and 160 mmHg yielded afferent arteriolar diameters of 17.5 +/- 0.8 and 16.6 +/- 0.6 microm (P < 0.05). This 11% decline in diameter is significantly smaller than the 40% decrease obtained in untreated kidneys. The inhibitory effects of Y-27632 on autoregulatory behavior were concentration dependent. Angiotensin II responses were blunted by Y-27632. Angiotensin II (1.0 nM) reduced afferent diameter by 17 +/- 1% in untreated arterioles and by 6 +/- 2% during exposure to Y-27632. The P2X1 receptor agonist, alpha, beta-methylene ATP, reduced afferent arteriolar diameter by 8 +/- 1% but this response was eliminated during exposure to Y-27632. Western blot analysis confirms expression of the Rho-kinase signaling pathway. Thus, Rho-kinase may be important in pressure-mediated autoregulatory adjustments in preglomerular resistance and responsiveness to angiotensin II and autoregulatory P2X1 receptor agonists.

Hypoxia Differently Modulates Gene Expression of Inositol 1,4,5‐Trisphosphate Receptors in Mouse Kidney and HEK 293 Cell Line

Hypoxia is a state of insufficient oxygen supply of the tissue or cell. Kidney tissue is highly sensitive to oxygen deprivation and easily develops renal ischemic injury. Calcium transporters very sensitively react to oxygen deficiency. We investigated whether hypoxia affects the gene expression of intracellular calcium transporters in the intact kidney, and we compared the response to that of HEK 293 cells. Our results showed that, while in mouse kidney tissue hypoxia elevates mRNA for inositol 1,4,5-trisphosphate receptors (IP3R) type 1 (IP3R1) and ryanodine receptors (RyR) type 2 (RyR2), in culture of HEK 293 cells the gene expression of all IP3Rs decreased without affecting viability of the cells. RyR2 mRNA in HEK 293 cells was not significantly changed, but RyR1 gene expression was significantly increased by hypoxia. The different response of kidney tissue and HEK 293 cells to hypoxia could be due to unequal differentiation state of the cells in intact tissue and cultured embryonic cell line. The physiological relevance of this observation remains to be determined.

Micropuncturing the nephron

To achieve the role of the kidney in maintaining body homeostasis, the renal vasculature, the glomeruli, and the various segments of the nephron and the collecting duct system have to fulfill very diverse and specific functions. These functions are dependent on a complex renal architecture and are regulated by systemic hemodynamics, hormones, and nerves. As a consequence, to better understand the physiology of the kidney, methods are necessary that allow insights on the function of these diverse structures in the physiological context of the intact kidney. The renal micropuncture technique allows direct access to study superficial nephrons in vivo. In this review, the application of micropuncture techniques on the single nephron level is outlined as an approach to better understand aspects of glomerular filtration, tubular transport, and tubulo-glomerular communication. Studies from the author's lab, including experiments in gene-targeted mice, are briefly presented to illustrate some of the approaches and show how they can further advance our understanding of the molecular mechanisms involved in the regulation of kidney function.

Hand-Assisted Laparoscopic Donor Nephrectomy: Comparison to Pure Laparoscopic Donor Nephrectomy

Hand-assisted laparoscopic surgery is assumed to be easier to learn than the standard approach and simplifies intact kidney removal. Herein we have presented our experience performing hand-assisted laparoscopic donor nephrectomy (HALDN) compared with contemporary pure laparoscopic donor nephrectomy (LDN). We retrospectively analyzed 55 patients who underwent LDN. Among the procedures, 21 were HALDN and 34 were pure LDN. We compared the two groups with regard to operative time, warm ischemic time (WIT), estimated blood loss, conversion rate, postoperative stay, and complications. For the HALDN group, the mean operative time was 191 minutes, WIT varied from 2 to 11 minutes, and bleeding estimates varied from 100 to 4000 mL. The overall complication rate of 28.6% included: vessel injury, urinary leakage, and paralytic ileus. In the LDN group, the mean operative time was 184 minutes, WIT varied from 2 to 10 minutes, and bleeding estimated varied 100 to 3000 mL. Three patients (8.8%) had complications including ureteral obstruction ( n = 1) and vessel injury ( n = 2). There was no significant difference between the two groups about the procedure and the complications. Our series suggested that HALDN and LDN were similar, with a tendency toward better results in LDN group, which also shows lower costs.

Hemodynamic and Molecular Changes in the Solitary Kidney in Response to Partial Ureteral Obstruction

PURPOSE In conditions with unilateral ureteric obstruction the function of the obstructed kidney is impaired. In addition, the non-obstructed intact kidney suffers during severe unilateral obstruction. The main aim of the present study is to eliminate any compensation in the contralateral kidney so that all observed changes in the kidney tissue occurred in kidneys with a partially obstructed ureter. MATERIAL AND METHODS Newborn rats were subjected to UNX (unilateral left nephrectomy) within the first 48 hr of life and a subset of unilateral nephrectomized rats was subjected to right severe PUO (Partial Ureteral Obstruction) at day 14. Renal blood flow (RBF), Total Kidney Volume (TKV) was measured using MRI at week 10; moreover renal functional and metabolic data were analysed. The renal expression of Na, K-ATPase, Na-Pi2, NHE3 and AQP1, AQP3, AQP2 were examined by immunoblotting and immunocytochemistry. RESULTS PUO resulted in upregulation of type3 Na +/H + exchanger (NHE3) to 157±8%, the NaPi-2 cotransporter to 134±16%, Na, K-ATPase to 139±7%. Furthermore, in the obstructed kidney immunoblotting revealed a significant increase in the expression of AQP2 to 112±3%, AQP3 to 141±6%. In contrast, AQP1 expression markedly decreased to 75±10% of sham level (p<0.05). Additionally, PUO caused a reduction in kidney glomerular filtration rate, urine osmolality, urine sodium concentration and body weight. However, fractional sodium excretion, plasma creatinine, kidney weight, and water intake were elevated in these partially obstructed rats. CONCLUSIONS The contralateral kidney plays a very important compensatory role in regulation of key renal transporters and channels in response to urinary tract obstruction kidney functions.

Uric acid and incident kidney disease in the community.

Uric acid may mediate aspects of the relationship between hypertension and kidney disease via renal vasoconstriction and systemic hypertension. To investigate the relationship between uric acid and subsequent reduced kidney function, limited-access data of 13,338 participants with intact kidney function in two community-based cohorts, the Atherosclerosis Risks in Communities and the Cardiovascular Health Study, were pooled. Mean baseline serum uric acid was 5.9 +/- 1.5 mg/dl, mean baseline serum creatinine was 0.9 +/- 0.2 mg/dl, and mean baseline estimated GFR was 90.4 +/- 19.4 ml/min/1.73 m(2). During 8.5 +/- 0.9 yr of follow-up, 712 (5.6%) had incident kidney disease defined by GFR decrease (>or=15 ml/min/1.73 m(2) with final GFR <60 ml/min/1.73 m(2)), while 302 (2.3%) individuals had incident kidney disease defined by creatinine increase (>or=0.4 mg/dl with final serum creatinine >1.4 mg/dl in men and 1.2 mg/dl in women). In GFR- and creatinine-based logistic regression models, baseline uric acid level was associated with increased risk for incident kidney disease (odds ratio 1.07 [95% confidence interval 1.01 to 1.14] and 1.11 [95% confidence interval 1.02 to 1.21] per 1-mg/dl increase in uric acid, respectively), after adjustment for age, gender, race, diabetes, systolic BP, hypertension, cardiovascular disease, left ventricular hypertrophy, smoking, alcohol use, education, lipids, albumin, hematocrit, baseline kidney function and cohort; therefore, elevated serum uric acid level is a modest, independent risk factor for incident kidney disease in the general population.

Regulation of Human Cytidine Triphosphate Synthetase 1 by Glycogen Synthase Kinase 3

Cytidine triphosphate synthetase (CTPS) catalyzes the rate-limiting step in the de novo synthesis of CTP, and both the yeast and human enzymes have been reported to be regulated by protein kinase A or protein kinase C phosphorylation. Here, we provide evidence that stimulation or inhibition of protein kinase A and protein kinase C does not alter the phosphorylation of endogenous human CTPS1 in human embryonic kidney 293 cells under the conditions tested. Unexpectedly, we found that low serum conditions increased phosphorylation of endogenous CTPS1 and this phosphorylation was inhibited by the glycogen synthase kinase 3 (GSK3) inhibitor indirubin-3'-monoxime and GSK3beta short interfering RNAs, demonstrating the involvement of GSK3 in phosphorylation of endogenous human CTPS1. Separating tryptic peptides from [(32)P]orthophosphate-labeled cells and analyzing the phosphopeptides by mass spectrometry identified Ser-574 and Ser-575 as phosphorylated residues. Mutation of Ser-571 demonstrated that Ser-571 was the major site phosphorylated by GSK3 in intact human embryonic kidney 293 cells by GSK3 in vitro. Furthermore, mutation of Ser-575 prevented the phosphorylation of Ser-571, suggesting that phosphorylation of Ser-575 was necessary for priming the GSK3 phosphorylation of Ser-571. Low serum was found to decrease CTPS1 activity, and incubation with the GSK3 inhibitor indirubin-3'-monoxime protected against this decrease in activity. Incubation with an alkaline phosphatase increased CTPS1 activity in a time-dependent manner, demonstrating that phosphorylation inhibits CTPS1 activity. This is the first study to investigate the phosphorylation and regulation of human CTPS1 in human cells and suggests that GSK3 is a novel regulator of CTPS activity.

Evidence for restriction of fluid and solute movement across the glomerular capillary wall by the subpodocyte space

The glomerular filtration barrier (GFB) is generally considered to consist of three layers: fenestrated glomerular endothelium, glomerular basement membrane, and filtration slits between adjacent podocyte foot processes. Detailed anatomic examination of the GFB has revealed a novel abluminal structure, the subpodocyte space (SPS), identified as the labyrinthine space between the underside of podocyte cell body/primary processes and the foot processes. The SPS covers 50-65% of the filtration surface of the GFB, indicating that SPS may influence glomerular permeability. We have examined the contribution of the SPS to the permeability characteristics of the GFB using multiphoton microscopy techniques in isolated, perfused glomeruli and in the intact kidney in vivo. SPS were identified using this technique, with comparable dimensions to SPS examined with electron microscopy. The passage of the intermediate-weight molecule rhodamine-conjugated 10-kDa dextran, but not the low-weight molecule lucifer yellow ( approximately 450 Da), accumulated in SPS-covered regions of the GFB, compared with GFB regions not covered by SPS ("naked regions"). Net lucifer yellow flux (taken to indicate fluid flux) through identifiable SPS regions was calculated to be 66-75% of that occurring through naked regions. These observations indicate both ultrafiltration and hydraulic resistance imparted by the SPS, demonstrating the potential physiological contribution of the SPS to glomerular permeability.

Leukocytes Induce Epithelial to Mesenchymal Transition after Unilateral Ureteral Obstruction in Neonatal Mice

Urinary tract obstruction during renal development leads to tubular apoptosis, tubular atrophy, and interstitial fibrosis. Epithelial to mesenchymal transition (EMT) has been proposed as a key mechanism of myofibroblast accumulation in renal fibrosis. We studied the interplay of leukocyte infiltration, tubular apoptosis, and EMT in renal fibrosis induced by unilateral ureteral obstruction (UUO) in neonatal mice. We show that leukocytes mediate tubular apoptosis and EMT in the developing kidney with obstructive nephropathy. Blocking leukocyte recruitment by using the chemokine receptor-1 antagonist BX471 protected against tubular apoptosis and interstitial fibrosis, as evidenced by reduced monocyte influx, a decrease in EMT, and attenuated collagen deposition. EMT was rapidly induced within 24 hours after UUO along with up-regulation of the transcription factors Snail1 and Snail2/Slug, preceding the induction of alpha-smooth muscle actin and vimentin. In the presence of BX471, the expression of chemokines, as well as of Snail1 and Snail2/Slug, in the obstructed kidney was completely attenuated. This was associated with reduced macrophage and T-cell infiltration, tubular apoptosis, and interstitial fibrosis in the developing kidney. Our findings provide evidence that leukocytes induce EMT and renal fibrosis after UUO and suggest that chemokine receptor-1 antagonism may prove beneficial in obstructive nephropathy.

Tissue-specific Regulation of Sodium/Proton Exchanger Isoform 3 Activity in Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) Null Mice: cAMP INHIBITION IS DIFFERENTIALLY DEPENDENT ON NHERF1 AND EXCHANGE PROTEIN DIRECTLY ACTIVATED BY cAMP IN ILEUM VERSUS PROXIMAL TUBULE

The multi-PDZ domain containing protein Na(+)/H(+) Exchanger Regulatory Factor 1 (NHERF1) binds to Na(+)/H(+) exchanger 3 (NHE3) and is associated with the brush border (BB) membrane of murine kidney and small intestine. Although studies in BB isolated from kidney cortex of wild type and NHERF1(-/-) mice have shown that NHERF1 is necessary for cAMP inhibition of NHE3 activity, a role of NHERF1 in NHE3 regulation in small intestine and in intact kidney has not been established. Here a method using multi-photon microscopy with the pH-sensitive dye SNARF-4F (carboxyseminaphthorhodafluors-4F) to measure BB NHE3 activity in intact murine tissue and use it to examine the role of NHERF1 in regulation of NHE3 activity. NHE3 activity in wild type and NHERF1(-/-) ileum and wild type kidney cortex were inhibited by cAMP, whereas the cAMP effect was abolished in kidney cortex of NHERF1(-/-) mice. cAMP inhibition of NHE3 activity in these two tissues is mediated by different mechanisms. In ileum, a protein kinase A (PKA)-dependent mechanism accounts for all cAMP inhibition of NHE3 activity since the PKA antagonist H-89 abolished the inhibitory effect of cAMP. In kidney, both PKA-dependent and non-PKA-dependent mechanisms were involved, with the latter reproduced by the effect on an EPAC (exchange protein directly activated by cAMP) agonist (8-(4-chlorophenylthio)-2'O-Me-cAMP). In contrast, the EPAC agonist had no effect in proximal tubules in NHERF1(-/-) mice. These data suggest that in proximal tubule, NHERF1 is required for all cAMP inhibition of NHE3, which occurs through both EPAC-dependent and PKA-dependent mechanisms; in contrast, cAMP inhibits ileal NHE3 only by a PKA-dependent pathway, which is independent of NHERF1 and EPAC.

Frequency modulation of renal myogenic autoregulation by perfusion pressure

Recent studies of renal autoregulation have shown modulation of the faster myogenic mechanism by the slower tubuloglomerular feedback and that the modulation can be detected in the dynamics of the myogenic mechanism. Conceptual and empirical considerations suggest that perfusion pressure may modulate the myogenic mechanism, although this has not been tested to date. Here we present data showing that the myogenic operating frequency, assessed by transfer-function analysis, varied directly as a function of perfusion pressure in the hydronephrotic kidney perfused in vitro over the range from 80 to 140 mmHg. A similar result was obtained in intact kidneys in vivo when renal perfusion pressure was altered by systemic injection of N(G)-nitro-L-arginine methyl ester (L-NAME). When perfusion pressure was not allowed to increase, L-NAME did not affect the myogenic operating frequency despite equivalent reduction of renal vascular conductance. Blood-flow dynamics were assessed in the superior mesenteric artery before and after L-NAME. In this vascular bed, the operating frequency of the myogenic mechanism was not affected by perfusion pressure. Thus the operating frequency of the renal myogenic mechanism is modulated by perfusion pressure independently of tubuloglomerular feedback, and the data suggest some degree of renal specificity of this response.

Small-animal CT: Its difference from, and impact on, clinical CT

For whole-body computed tomography (CT) images of small rodents, a voxel resolution of at least 10 −3 mm 3 is needed for scale-equivalence to that currently achieved in clinical CT scanners (∼1 mm 3) in adult humans. These “mini-CT” images generally require minutes rather than seconds to complete a scan. The radiation exposure resulting from these mini-CT scans, while higher than clinical CT scans, is below the level resulting in acute tissue damage. Hence, these scans are useful for performing clinical-type diagnostic and monitoring scans for animal models of disease and their response to treatment. “Micro-CT”, with voxel size <10 −5 mm 3, has been useful for imaging isolated, intact organs at an almost cellular level of resolution. Micro-CT has the great advantage over traditional microscopic methods in that it generates detailed three-dimensional images in relatively large, opaque volumes such as an intact rodent heart or kidney. The radiation exposure needed in these scans results in acute tissue damage if used in living animals. Experience with micro-CT is contributing to exploration of new applications for clinical CT imaging by providing insights into different modes of X-ray image formation as follows: (1) Spatial resolution should be sufficient to detect an individual Basic Functional Unit (BFU, the smallest collection of diverse cells, such as hepatic lobule, that behaves like the organ), which requires voxels ∼10 −3 mm 3 in volume, so that the BFUs can be counted. (2) Contrast resolution sufficient to allow quantitation of: (a) New microvascular growth, which manifests as increased tissue contrast due to X-ray contrast agent in those vessels’ lumens during passage of injected contrast agent in blood. (b) Impaired endothelial integrity which manifests as increased opacification and delayed washout of contrast from tissues. (c) Discrimination of pathological accumulations of metals such as Fe and Ca, which occur in the arterial wall following hemorrhage or tissue damage. (3) Micro-CT can also be used as a test bed for exploring the utility of several modes of X-ray image formation, such as the use of dual-energy X-ray subtraction, X-ray scatter, phase delay and refraction-based imaging for increasing the contrast amongst soft tissue components. With the recent commercial availability of high speed, multi-slice CT scanners which can be operated in dual-energy mode, some of these micro-CT scanner capabilities and insights are becoming implementable in those CT scanners. As a result, the potential diagnostic spectrum that can be addressed with those scanners is broadened considerably.

Corticotropin-releasing factor (CRF) and CRF-binding protein expression in and release from the head kidney of common carp: evolutionary conservation of the adrenal CRF system

Corticotropin-releasing factor (CRF) plays a central role in the regulation of the stress axis. In mammals, CRF as well as its receptors and its CRF-binding protein (CRF-BP) are expressed in a variety of organs and tissues outside the central nervous system. One of these extrahypothalamic sites is the adrenal gland, where the paracrine actions of adrenal CRF influence cortical steroidogenesis and adrenal blood flow. Although the central role of CRF signaling in the initiation and regulation of the stress response has now been established throughout vertebrates, information about the possible peripheral presence of CRF in earlier vertebrate lineages is scant. We established the expression of CRF, CRF-BP, and the CRF receptor 1 in a panel of peripheral organs of common carp (Cyprinus carpio). Out of all the peripheral organs tested, CRF and CRF-BP are most abundantly expressed in the carp head kidney, the fish equivalent of the mammalian adrenal gland. This expression localizes to chromaffin cells. Furthermore, detectable quantities of CRF are released from the intact head kidney following in vitro stimulation with 8-bromo-cAMP in a superfusion setup. The presence of CRF and CRF-BP within the chromaffin compartment of the head kidney suggests that a pathway homologous to the mammalian intra-adrenal CRF system is present in the head kidney of fish. It follows that such a system to locally fine-tune the outcome of the centrally initiated stress response has been an integral part of the vertebrate endocrine system since the common ancestor of teleostean fishes and mammals.