INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE PROCESS OF GLOMERULAR filtration and its control by local hemodynamics and the hormonal milieu involve the complex interaction of a number of different cell types. Regulation of glomerular filtration occurs at the juxtaglomerular apparatus (JGA), a complex structure that consists of a number of different cell types, including vascular smooth muscle cells, secretory granular epitheloid cells, vascular endothelium, mesangial cells, and macula densa (MD) tubular epithelial cells (2). MD cells, in the distal nephron, function as specific sensor cells that detect changes in tubular fluid osmolality and/or salt concentration via specific transport mechanisms (3) and send signals through mesangial cells in the JGA. These signals effect afferent arteriolar smooth muscle cells and renin granular cells to control preglomerular vascular resistance [tubuloglomerular feedback (TGF)] and renin release, respectively (1-3, 13). It has been difficult to study these cellular interactions within the JGA in living preparations, given the constraints of existing technologies.

Recently, multiphoton excitation laser scanning fluorescence microscopy has been developed (8, 14) that is particularly applicable to deep optical sectioning of living tissue samples. This technique offers a tremendous increase in image resolution vs. conventional confocal microscopy. Importantly, it can optically section through an entire glomerulus (glomerular diameter 100 µm), thus providing the ability to directly study structures and cellular components that lie deep within the glomerulus. This new technology was used in the present work to visualize the living perfused JGA.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolated, perfused afferent arteriole-cortical thick ascending limb preparations were used as previously described (10, 11). Preparations were visualized using a two-photon laser scanning fluorescence microscope (MRC1024MP, Bio-Rad) composed of a mode-locked titanium-sapphire laser (Tsunami, Spectra Physics), and a photo-diode pump laser (Millennium, Spectra Physics). Individual preparations were transferred to a thermoregulated Lucite chamber mounted on a Zeiss Axiolab inverted microscope and visualized using a ×40 water-immersion lens. The fluorescent membrane-staining dye 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH; 5 µM, Dojin) or the Ca²⁺-sensitive fluorophore indo 1-acetoyxmethyl ester (indo 1-AM; 5 µM, Molecular Probes, Eugene, OR) was added to the tubular and/or arteriolar perfusion solutions (Table 1) for a loading period of 5 min to visualize cellular structures or monitor intracellular Ca²⁺ concentration ([Ca²⁺]_i). After the loading period, TMA-DPH or indo 1-AM was removed. TMA-DPH fluorescence was captured using a band-width emission filter at 430 ± 15 nm in response to a two-photon excitation wavelength of 755 nm. Indo 1 fluorescence was measured at emission wavelengths of 453 and 405 nm in response to two-photon excitation wavelength of 720 nm. A 405 ± 17-nm band-pass filter, placed before detection by a photomultiplier tube (PMT; channel 2), was used to select indo 1 emissions that increase with increasing [Ca²⁺], whereas a 453 ± 2-nm band-pass filter, placed before a second PMT (channel 1) was used to select isosbestic emissions of indo 1 that are independent of [Ca²⁺]. After ~15-min incubation with control perfusion solutions, fluorescence intensities for both wavelengths stabilized at a constant level. Ratiometric (405 nm/453 nm) images were collected and analyzed using a time series function of the LaserSharp1.2 software (Bio-Rad) with the most frequently used application of cycle time 10-s, for 30 cycles.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Glomerular and JGA morphology. We examined three-dimensional (3D) morphology of the perfused, living glomerulus with attached tubular segments using optical Z-sectioning. Figure 1 represents midlevel sections (a complete Z-series in 2-µm steps is available in a supplementary file), using a cell membrane marker (TMA-DPH) or an intracellular fluorophore (indo 1). Both morphology and cytosolic parameters, e.g., [Ca²⁺]_i of individual cells that comprise the glomerulus can be studied with high resolution. Afferent and efferent arterioles, glomerular capillary loops, the proximal tubule and cortical thick ascending limb, and MD cells can be identified and visualized in striking detail. In addition, it was also possible to visualize the process of filtration across the capillary wall into the Bowman's capsule. In terms of the MD cells, we show for the first time (Fig. 2), 3D imaging of a perfused MD plaque, consisting of 24 individual cells.

View larger version (67K): [in this window] [in a new window]   Fig. 1.   Visualization of various glomerular and juxtaglomerular apparatus (JGA) structures in situ. a: Glomerulus perfused through the afferent arteriole (AA) with attached cortical thick ascending limb (cTAL) and macula densa (MD). Tissue was stained with TMA-DPH. Note the dilation of Bowman's capsule (CAP) and exit of glomerular filtrate via the proximal tubule (PT). b: Ca2+ image of the double-perfused AA and cTAL containing the MD. Tissue was loaded with indo 1. G, glomerulus; EA, efferent ateriole. Bar = 10 µm.

View larger version (66K): [in this window] [in a new window]   Fig. 2.   Sectioning of the MD (arrowheads) plaque stained with 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) in the longitudinal (a), sagittal (b), and horizontal (c) planes. Bar = 10 µm.

Real-time imaging of the JGA during TGF. We visualized MD cells as well as the entire JGA area during activation of the TGF mechanism. Images are shown in Figs. 3 and 4 [videos (time-series images taken every 10 s) of the same preparation are available in supplementary files]. Increasing tubular perfusate osmolality and NaCl concentration ([NaCl]), which mimics normal physiological TGF activation, produced a remarkably fast and reversible MD cell swelling. MD cell swelling is most likely due to a high level of NaCl transport into these cells. It should also be noted that a reversible blebbing or expansion of the MD apical membrane occurred during TGF activation (Fig. 3b). Thus two-photon imaging has revealed what may be a novel rapid process of membrane insertion + retrieval that occurs in MD cells during TGF. Figures 4 and 5 demonstrate morphological changes in the JGA during TGF activation. Parallel with MD cell swelling, swelling/contraction of cells in the final part of the afferent arteriole was observed that caused an almost complete collapse of the arteriolar lumen and a shrinkage of capillary loops and the entire glomerulus. This is the first evidence for a "sphincter-like" response of the terminal intraglomerular afferent arteriole. These intraglomerular morphological changes are absent when the efferent arteriole is perfused, suggesting the lack of efferent arteriolar vasoconstriction during TGF.

View larger version (91K): [in this window] [in a new window]   Fig. 3.   MD cell (arrowheads) swelling in response to increasing tubular NaCl concentration from 25 [osmolality 210 mosmol/kgH2O, NaCl substituted by N-methyl-D-glucamine (NMDG); a] to 135 mM (osmolality 300 mosmol/kgH2O; b). Tissue was stained with TMA-DPH. Note that balloon-like blebbing occurred in several MD cells. Bar = 10 µm.

View larger version (98K): [in this window] [in a new window]   Fig. 4.   Changes in JGA morphology during increasing tubular NaCl concentration from 25 (osmolality 210 mosmol/kgH2O, NaCl substituted by NMDG; a) to 135 mM (osmolality 300 mosmol/kgH2O; b). MD cell (arrowhead) swelling and parallel swelling/contraction of cells in the final part of the AA that causes an almost complete closure of the arteriolar lumen (arrows), collapse of capillary loops (CAP), and shrinkage of the entire glomerulus (G) are shown. Tissue was stained with TMA-DPH. *, Mesangial cells. Bar = 10 µm.

View larger version (98K): [in this window] [in a new window]   Fig. 5.   Comparison of changes in afferent arteriolar diameter in intra- and extraglomerular segments. Tubular NaCl concentration was increased from 25 (osmolality 210 mosmol/kgH2O, NaCl substituted by NMDG; a) to 135 mM (osmolality 300 mosmol/kgH2O; b). Tissue was stained with TMA-DPH. Bar = 10 µm.

Effects of ANG II. In another effort to apply this new technology to the JGA, we measured morphological and dimensional changes in arterioles and glomerular capillary loops in response to the vasoactive peptide ANG II in the presence or absence of the type 1 ANG II receptor (AT₁) blockade with candesartan. We also tested the effects of another vasoconstrictor hormone, norepinephrine (Fig. 6). ANG II (10⁸ M), added to the arteriolar perfusate, significantly constricted afferent (AA) and efferent arterioles (EA). These vascular effects were prevented by coadministration of 10⁶ M candesartan. ANG II also caused a significant reduction in the glomerular capillary loop diameter (Fig. 6). Vascular reactivity was well preserved in these studies, because addition of 10⁶ M norepinephrine at the end of each experiment significantly constricted both AA and EA.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Effects of ANG II alone or with AT1 receptor blockade candesarten (Cand) and norepinephrine (NE) on AA, EA, and glomerular capillary loop (GCL) internal diameter (ID). *P < 0.05, nonsignificant compared with control (ctrl) in each group (n = 5 each).

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6

View larger version (63K): [in this window] [in a new window]   Fig. 7.   Ca2+ image of the perfused AA-glomerulus (G)-EA complex before (a) and 2 min after (b) the addition of ANG II to the lumen. Tissue was loaded with indo 1. Bar = 10 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Despite the key role that the JGA plays in the regulation of renin release, TGF, and control of glomerular filtration and blood flow (1, 2, 13), it has been very difficult to examine single cells of the JGA even with conventional videomicroscopy (4-7). However, we now report that with two-photon microscopy, one can visualize single cells of the living JGA and glomerulus in real time and in striking detail.

With simultaneous increases in tubular osmolality and [NaCl], MD cells produced a significant cell swelling. This was primarily due to an increase in cell height (Fig. 3), consistent with an earlier work (4). However, in contrast to the studies of Gonzalez et al. (4) we found much larger increases in MD cell volume. Remarkably, these cells are capable of almost doubling their initial cell volume within 30 s despite a concomitant increase in luminal osmolality. This cell swelling is primarily due to NaCl entry into the cell, because the same change in the tubular perfusate osmolality at constant [NaCl] did not produce any significant change in cell volume.

We also examined TGF activation on intraglomerular elements. For the first time, we observed a sphincter-like constriction of the afferent arteriole in the terminal intraglomerular segment of this vessel. During TGF, the vascular diameter of this sphincter decreased to ~0. Compared with this strong vasoconstrictor response, we observed only minor changes in vascular diameter in more proximal extraglomerular segments of the afferent arteriole (Fig. 5). These studies indicate that the principal effector site for TGF occurs in the terminal-intraglomerular afferent arteriole. These studies also suggest that measurements of proximal afferent arteriolar diameter, as used by others (5, 12), may not accurately reflect TGF responses. One can further speculate that the minor vasoconstriction seen in the proximal afferent arteriole may be, at least in part, a myogenic response that is a consequence of the terminal-TGF-mediated sphincter activity. Also, the TGF-mediated reduction in intraglomerular afferent arteriolar diameter is more consistent with the magnitude of in vivo TGF responses obtained with micropuncture (9). Further studies are needed to identify the cell type in the afferent arteriole that constituents this sphincter-like structure.

In other experiments, we tested the effects of ANG II on glomerular and JGA morphology, because ANG II is a well-known modulator of renal hemodynamics and kidney function (9). In addition to the expected arteriolar vasoactive effects, to our knowledge this is the first study that demonstrates an increase in mesangial cell Ca²⁺ in the intact JGA. Thus these studies suggest that ANG II, perhaps through activation of mesangial cells and podocytes, may modify the glomerular capillary filtration surface area and that these effects are inhibited by AT₁ receptor blockade with candesartan. This approach has allowed us to identify a novel intraglomerular sphincter-like effector site for TGF. It has also permitted us to begin to define the intraglomerular effects of ANG II.

In summary, multiphoton excitation fluorescence microscopy in combination with isolated perfused JGA offers a powerful new tool for investigating the structural and cellular components that regulate the process of glomerular filtrate formation and renal hemodynamics.