Visualisation studies and glomerular filtration in early diabetic rats.

The purpose of this mini-review is to show that more modern multi-photon microscopy approaches allow quantitative glomerular filtration experiments. Modern science has now entered a transition period from light microscopy to multi-photon confocal microscopy. Since the late 20th century, multi-photon microscopy has been applied in the study of organ function. In keeping with observations made in renal physiology and other representative studies throughout this transition period, and in the context of advancing microscopy techniques, this review has been presented as a comment on the glomerular filtration barrier, with a focus on the early aetiopathogenesis of diabetes.

The short-term effects of C-peptide on the early diabetes-related ultrastructural changes to the podocyte slit diaphragm of glomeruli in rats.

OBJECTIVE: This study aimed to investigate the structural changes in the slit diaphragm, caused by early diabetes, and the nephroprotective effect of C-peptide. METHODS: Streptozotocin-induced type 1 diabetic Wistar rats were divided into control, control plus C-peptide, early diabetes, and early diabetes plus C-peptide groups. C-peptide was infused into rats for one day. The slit diaphragm component proteins podocin, CD2AP, and nephrin, were stained immunofluorescently and their distribution quantitatively analyzed by determining the overlapping area ratio. The interfoot process gap length was measured from electron microscopic images. RESULTS: Diabetic duration correlated best with the area ratio of podocin and CD2AP (r = 0.626), followed by other protein combinations, showing progressive change in the slit diaphragm structure. C-peptide-treatment did not alter area ratios. The interfoot process gap length was wider in diabetic rats (p < 0.05) and did not narrow with C-peptide-treatment in either control or diabetic rats (both p < 0.05). CONCLUSIONS: Diabetes widened the interfoot process gap length and distorted the slit diaphragm structure progressively and heterogeneously in rats with early diabetes; this was not altered by C-peptide-treatment. The nephroprotective effect of C-peptide in decreasing the glomerular filtration rate appears to be functional rather than structural.

In vivo quantitative visualization analysis of the effect of C-peptide on glomerular hyperfiltration in diabetic rats by using multiphoton microscopy.

OBJECTIVE: The purpose of this study was to visualize glomerular hyperfiltration in rats at the early stage of diabetes under in vivo conditions and to quantitatively examine the effect of C-peptide on filtration. METHODS: Type 1 diabetes was induced by streptozotocin (50 mg/kg) in Wistar rats. The rats were divided into four groups: control, control plus C-peptide, early diabetes, and early diabetes plus C-peptide. C-peptide was continuously infused (50 pmol/kg/min). Filtration was visualized by a bolus shot of various sizes of dextrans (3 k to 70 k Da) conjugated with Texas Red and observed with a multiphoton microscope under inhaled anesthesia. Relative sieving coefficients were used to quantify filtration. RESULTS: Almost all smaller particles (3­10 k Da) were filtered both in control and diabetic rats. Filtration of larger particles (40­70 k Da) was seen in normal rats but was more apparent in diabetic rats, where it was progressive according to the duration of diabetes. C-peptide administration restored the leakage of larger particles to the level seen for the control. CONCLUSIONS: We visualized and analyzed hyperfiltration and confirmed that C-peptide has a nephroprotective effect. Furthermore, we found that the leakage of larger particles increased as the duration of diabetes increased.

Visualisation of the effects of dilazep on rat afferent and efferent arterioles in vivo.

Although the effects of dilazep hydrochloride (dilazep), a nucleoside transport inhibitor, have been examined, there have been no visualisation studies on the physiological effects of dilazep on the glomerular arterioles. The purpose of this study was to visualise and evaluate the effects of dilazep and consequently the effects of adenosine, which dilazep augments by measuring glomelurar diameters, renal blood flow and resistance in rats in vivo. We time-sequentially examined afferent and efferent arteriolar diameter changes using an intravital videomicroscope and renal blood flow. We administered dilazep at a dose of 300 microg/kg intravenously. To further investigate the effects of dilazep, rats were pre-treated with 8-p-sulfophenyl theophylline (a nonselective adenosine receptor antagonist), 8-cyclopentyl-1,3-dipropylxanthine (an A1 receptor antagonist), or 3,7-dimethyl-1-propargylxanthine (an A2 receptor antagonist). Dilazep constricted the afferent and efferent arterioles at the early phase and dilated them at the later phase, with the same degree of vasoconstrictive and vasodilatory effect on both arterioles. A1 blockade abolished vasoconstriction and augmented vasodilatation at the later phase and A2 blockade abolished vasodilatation and augmented vasoconstriction at the early phase. Non-selective blockade abolished both early vasoconstriction and later vasodilatation. In conclusion, adenosine augmented by dilazep constricted the afferent and efferent arterioles of the cortical nephrons at the early phase and dilated both arterioles at the later phase via A1 and A2 adenosine receptor activation, respectively. That the ratio of afferent to efferent arteriolar diameter was fairly constant suggests that intraglomerular pressure is maintained in the acute phase by adenosine despite the biphasic flow change.

Direct measurement of nipradilol-derived nitric oxide in the vascular wall of canine femoral arteries.

Nipradilol (NP: 3,4-dihydro-8-[2-hydroxy-3-isopropylamino]propoxy-3-nitroxy-2H-1-benzopyran) shows not only beta-adrenoreceptor-blocking effects but also nitroglycerin-like vasodilatory action. We aimed to directly measure NP-derived nitric oxide (NO) in the vascular wall. An NO-sensitive microelectrode was inserted into the vascular media (the vasodilatory action site of NO) of isolated perfused canine femoral arteries. Each vessel was perfused with 15 microM NP in the presence or absence of 1 mM N-ethylmaleimide (NEM; a thiol alkylator). Intravascular-wall NO concentration increased 181+/-34 nM during NP perfusion (P<0.001 vs basal, n=10) with an average base-to-peak reaction time of 1.5+/-0.1 min (P<0.0001, n=8). Concomitant perfusion of NEM with NP attenuated the intravascular-wall NO production significantly (P<0.0001 vs NP only). It is concluded that NP is metabolized to NO in the vascular wall of an isolated canine femoral artery in large part through a metabolic process involving thiols with a base-to-peak reaction time of about 1.5 min.

Direct observation of epicardial coronary capillary hemodynamics during reactive hyperemia and during adenosine administration by intravital video microscopy.

Using high-resolution intravital charge-coupled device video microscopy, we visualized the epicardial capillary network of the beating canine heart in vivo to elucidate its functional role under control conditions, during reactive hyperemia (RH), and during intracoronary adenosine administration. The pencil-lens video-microscope probe was placed over capillaries fed by the left anterior descending artery in atrioventricular-blocked hearts of open-chest, anesthetized dogs paced at 60-90 beats/min (n = 17). In individual capillaries under control conditions, red blood cell flow was predominant during systole or diastole, indicating that the watershed between diastolic arterial and systolic venous flows is located within the capillaries. Capillary flow increased during RH and reached a peak flow velocity (2.1 +/- 0.6 mm/s), twice as high as control (1.2 +/- 0.5 mm/s), with enhancement of intercapillary cross-connection flow and enlargement of diameter (by 17%). With adenosine, capillary flow velocity significantly increased (1.8 +/- 0.7 mm/s). However, the increase in volumetric capillary flow with adenosine estimated from red blood cell velocity and diameter was less than the increase in arterial flow, whereas that during RH was nearly equivalent to the increase in arterial flow. There was a time lag of approximately 1.5 s for refilling of capillaries during RH, indicating their function as capacitance vessels. In conclusion, the coronary capillary network functions as 1) the major watershed between diastolic-dominant arterial and systolic-dominant venous flows, 2) a capacitor, and 3) a significant local flow amplifier and homogenizer of blood supply during RH, but with adenosine the increase in capillary flow velocity was less than the increase in arterial flow.

Exogenous NO suppresses flow-induced endothelium-derived NO production because of depletion of tetrahydrobiopterin.

Exogenous nitric oxide (NO) suppresses endothelium-derived NO production. We were interested in determining whether this is also the case in flow-induced endothelium-derived NO production. If so, then is the mechanism because of intracellular depletion of tetrahydrobiopterin [BH4; a cofactor of NO synthase (NOS)], which results in superoxide production by uncoupled NOS? Isolated canine femoral arteries were perfused with 100 microM S-nitroso-N-acetylpenicillamine (SNAP; an NO donor) and/or 64 microM BH4. Perfusion of SNAP suppressed flow-induced NO production, which was evaluated as a change in the slope of the linear relationship between perfusion rate and NO production rate (P < 0.02 vs. control; n = 7). Subsequent BH4 perfusion returned the slope to the control level. Concomitant perfusion of SNAP and BH4 retained the control-level NO production (n = 7). Concomitant perfusion of SNAP and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron; 1 mM; a membrane-permeable superoxide scavenger) also retained the control-level NO production (n = 7), whereas perfusion of Tiron after SNAP could not return the NO production to the control level (P < 0.02 vs. control; n = 7). We also found a significant decrease in BH4 concentration in the endothelial cells after SNAP perfusion. In conclusion, these results indicate that exogenous NO suppresses the flow-induced, endothelium-derived NO production by superoxide released from uncoupled NOS because of intracellular BH4 depletion.

Synergistic effects of C-peptide and insulin on coronary flow in early diabetic rats.

The aims of the present study are (1) to examine whether coronary flow is increased and (2) to examine the role of C-peptide in relation to nitric oxide (NO) production and coronary flow in a rat heart (Wistar) during the early stages of type 1 diabetes. Coronary flow increased by 36.4% +/-10.6% (P <.05) during the early stages of streptozotocin-induced diabetes of isolated perfused rat hearts, but NO production increased without significance. C-peptide alone did not change coronary flow, but increased NO production in diabetes. In the presence of insulin, C-peptide reversed both flow and NO production to the control level of normal rats (P <.05). In conclusion, during the early stages of type 1 diabetes, coronary flow was increased, and C-peptide in the presence of insulin synergistically normalized the excessive flow and NO production induced by C-peptide to the control level of normal rats.

In vivo visualization of subendocardial arteriolar response in renovascular hypertensive hearts.

Time-sequential responses to endothelium-dependent and -independent vasodilators and angiotensin-converting enzyme (ACE) inhibitors were studied in the subendocardial arterioles (Endo) of canine renovascular hypertension (HT) compared with subepicardial arterioles (Epi; both <120 microm) by charge-coupled device intravital microscope. Vascular responses to acetylcholine, papaverine, and cilazaprilat were compared between normotensive (NT) and HT dogs [4 wk and 12 wk of HT (4wHT and 12wHT)]. The acetylcholine-induced vasodilation of Endo in both 4wHT and 12wHT was smaller than that of NT (both P < 0.01 vs. 4wHT and 12wHT), and that of Epi was smaller than that of NT only in 12wHT (P < 0.05). The papaverine-induced vasodilation of Endo, but not Epi, was impaired only in 12wHT (both P < 0.01 vs. NT and 4wHT). Vasodilation by cilazaprilat remained unchanged at 4wHT and 12wHT in both Epi and Endo. In conclusion, at the early stage, the endothelium-dependent response of Endo was impaired, whereas at the later stage, the endothelium-dependent and -independent responses of Endo and the endothelium-dependent response of Epi were impaired. However, the vasodilatory responses to the ACE inhibitor were maintained in both Endo and Epi of HT.

A health monitoring system for elderly people living alone.

We have developed a health monitoring system for elderly people living alone. We monitored the in-house movements of eight subjects (average age 81 years) by placing infrared sensors in each room of their homes. Because their movements were unrestricted, monitoring could last longer than other forms of monitoring. Continuous monitoring was performed for 80 months in total. We found that each subject had a specific pattern of movements. We estimated their health condition by comparing the duration of stays in specific rooms, such as the lavatory, with previously recorded data. If after analysis an unusual state was detected, we informed the family of the incident. Final decisions should be made by the family members, not automatically by computer software. For example, after contacting the subject or a neighbour by telephone, family members could call for an ambulance or arrange a visit by a doctor or home help. Thus, this system reduced anxiety for both the elderly subjects living alone and their family members.

Evaluation of basic performance and applicability of a newly developed in vivo nitric oxide sensor.

Direct measurement of nitric oxide (NO) is of great importance and value for both in vitro and in vivo studies on dynamic NO bioactivity. Here, we evaluated the basic performance of a newly developed NO sensor (Innovative Instruments, Inc.). Unlike other NO sensors, the new NO sensor has a highly durable, gas-permeable coating and is affected much less by electrical interference due to its integrated structure where working and reference electrodes are combined in a single element. Calibration with NO gas showed high sensitivity of about 580 pA per nmol-NO l(-1) (the detection limit 0.08 nmol-NO l(-1), S/N = 3). This sensor also showed high selectivity (25,000 times and more) to NO, compared with NO-related reagents such as L-arginine, N(G)-monomethyl-L-arginine, acetylcholine, nitroglycerin (NTG) and tetrahydrobiopterin as well as dissolved oxygen. As an in vivo application, the sensor was located in the anaesthetized rat abdominal aorta to measure NTG-derived plasma NO. lntra-aortic infusion of 0.5 mg NTG caused a measurable increase in plasma NO level (2.0 +/- 2.2 nmol l(-1), mean +/- SD, n = 3). In conclusion, the new NO sensor demonstrated a satisfying performance for both in vitro and in vivo applications.