advances in renal (patho)physiology using multiphoton microscopy

Advances in renal (patho)physiology using multi-photonmicroscopy

Arnold Sipos1,2, Ildikó Toma1, Jung Julie Kang1, László Rosivall2, and János Peti-Peterdi1,2

1 Departments of Physiology and Biophysics and Medicine, Zilkha Neurogenetic Institute, University ofSouthern California, Los Angeles, CA 90089

2 Hungarian Academy of Sciences and Semmmelweis University Nephrology Research Group, Institute ofPathophysiology, Semmelweis University Faculty of Medicine, Budapest, Hungary, 1089

AbstractMulti-photon excitation fluorescence microscopy is a state-of-the-art confocal imaging techniqueideal for deep optical sectioning of living tissues. It is capable of performing ultra-sensitive,quantitative imaging of organ functions in health and disease with high spatial and temporalresolution that other imaging modalities can not achieve. For more than a decade, multi-photonmicroscopy has been successfully used with various in vitro and in vivo experimental approaches tostudy many functions of organs, including the kidney. This mini-review focuses on recent advancesin our knowledge of renal (patho)physiological processes made possible by the use of this imagingtechnology. Visualization of cellular variables like cytosolic calcium, pH, cell-to-cell communicationand signal propagation, interstitial fluid flow in the juxtaglomerular apparatus (JGA), real-timeimaging of tubuloglomerular feedback (TGF) and renin release mechanisms are reviewed. Briefsummary is provided how one can perform quantitative imaging of kidney functions in vivo includingglomerular filtration and permeability, concentration, dilution, and activity of the intra-renal renin-angiotensin system using this minimally invasive approach. New visual data challenge a number ofexisting paradigms in renal (patho)physiology. Also, quantitative imaging of kidney function withmulti-photon microscopy has excellent potential to eventually provide novel non-invasive diagnosticand therapeutic tools for future applications in clinical nephrology.

Imaging of JGA functions in vitroMulti-photon fluorescence microscopy offers the advantages of deep confocal sectioning ofliving tissue with minimal phototoxicity and high, subcellular resolution.1 In combination within vitro experimental models, both cortical and medullary structures freshly dissected from therabbit kidney have been successfully visualized.2 Since multi-photon microscopy can sectionthrough an entire glomerulus (about 100 μm in diameter), it has been used with the isolatedmicroperfused afferent arteriole-glomerulus technique to study dynamic processes of (juxta)glomerular structures.2–8 Fig. 1A shows a representative preparation and illustrates how thecombination of conventional transmitted light (DIC) detection with two-photon fluorescenceimaging of cellular or subcellular structures (for example renin granular content) providesadditional, enhanced detail. Since the low cytotoxicity of multi-photon excitation allowscontinuous imaging of living tissues, real-time imaging of the tubuloglomerular feedback(TGF) and renin release mechanisms became possible.5–6 This visual approach providedsignificant new information on these fundamental mechanisms regulating renal blood flow,

Corresponding Author: Janos Peti-Peterdi, University of Southern California, Zilkha Neurogenetic Institute, 1501 San Pablo Street, ZNI335, Los Angeles, CA 90033, [email protected], Phone: (323) 442-4337, Fax: (323) 442-4466.

NIH Public AccessAuthor ManuscriptKidney Int. Author manuscript; available in PMC 2008 November 1.

Published in final edited form as:Kidney Int. 2007 November ; 72(10): 1188–1191.

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glomerular filtration rate and activating the renin-angiotensin system. Novel, TGF-associatedmorphological findings include significant cell volume changes of the macula densa underisotonic3 or hypertonic conditions4, the existence of bulk fluid flow in the JGA8, a sphincter-like contraction of the terminal, intraglomerular afferent arteriole2,3, and the TGF-associatedcontraction of not only the afferent arteriole, but the entire intraglomerular mesangium.3,5Spreading of the TGF vasoconstrictor signal in the JGA and beyond involves an extracellularATP-mediated purinergic calcium wave5. This wave was directly visualized with confocalmicroscopy (Fig. 1B), propagating from the macula densa and extraglomerular mesangial areato the afferent arteriole, along the vasculature to adjacent glomeruli and also to all cells of theglomerulus including the most distant podocytes.5 Propagation of the TGF calcium wave fromafferent arteriole smooth muscle cells to the underlying endothelium was also observed in thesestudies.5 This phenomenon may provide a negative feedback and helps to balance the TGFvasoconstriction by triggering endothelium-derived vasodilator mechanisms. These imagingstudies further emphasized the roles of both gap junctional communication and extracellularATP as integral components of TGF. In addition, these studies provided functional evidencethat complementing the afferent arteriolar vasoconstriction, all cells of the glomerulus activelyparticipate in TGF by contracting the glomerular tuft, thereby helping to reduce the rate ofglomerular filtration. The unexpected finding that the calcium wave of TGF was mediated byextracellular ATP provided further support that ATP itself is directly involved in TGF and notonly through its breakdown to adenosine.5

Renin release is the first, and at least initially, the rate-limiting step in the activation of therenin-angiotensin system which helps to maintain body salt and water balance. Additionaldetails of the renin release mechanism were also observed using the multi-photon imagingapproach. Acidotropic fluorophores including quinacrine and LysoTracker dyes (Invitrogen)are highly membrane permeant weak base compounds that rapidly accumulate in acidic cellularorganelles. They have been successfully used to label renin granular content both in vitro6–7and in vivo7–10, and even as a counter stain on histological sections.6 Imaging the entiregranular content as opposed to labeling specific molecules of interest (renin itself) is a greatadvantage when studying the mechanism and regulation of renin granule exocytosis. Forexample, there is a renewed interest in prorenin which is part of the granular content andtherefore its release is also visualized, but it can not be detected by existing assays measuringrenin activity since it is enzymatically inactive. Renin exocytosis has been visualized in real-time and on the individual renin granule level in response to a number of physiological stimuliincluding beta-adrenergic activation, low perfusion pressure and the macula densa mechanism.2,6 Dimming and disappearance of the entire granular content (quantal release) was observedwithin 2–300 ms.6 A significant number of renin granules released into the interstitial side ofthe JGA, in addition to the vascular lumen.6 Not only the degranulation process, but enzymeactivity of the released renin (angiotensin I generation) was visualized in real-time using aFRET-based renin substrate.6,9 Together with imaging the actual renin content, this approachis very useful to monitor the status of the intra-renal renin-angiotensin system, an importanttarget of anti-hypertensive therapy.9

Quantitative imaging of kidney functions in vivoThe first years after multi-photon microscopy became commercially available (around 1995)was the “awe” period of in vivo organ imaging. The emphasis however, soon shifted from justgenerating pretty images to the development of quantitative imaging techniques for theevaluation of organ function. Studies aimed to establish new procedures or to extend existingmethods in fluorescence imaging to directly observe and quantify basic physiologicalparameters of the kidney10–18 including single nephron glomerular filtration rate (SNGFR),glomerular permeability, blood flow, tubular flow, tubular reabsorption, urinary concentration/dilution, renin content and release, as well as more integrated and complex functions like the

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tubuloglomerular feedback (TGF)-mediated oscillations in glomerular filtration and tubularflow.10 Fig. 2 provides examples for some of these approaches. Using multi-photon techniques,dynamic processes such as glomerular filtration13,18, proximal tubule endocytosis15,apoptosis13, microvascular function13,14, protein expression16, renal cysts17 have beenvisualized and studied down to the subcellular level. A ratiometric intravital two-photonmicroscopy technique based on the generalized polarity concept has been recently applied toquantify glomerular filtration and tubular reabsorption.18 Yu et al also reported a newratiometric measurement technique based on intravital fluorescence microscopy that allowsrapid evaluations of renal function in rodent models.19 By using this technique, plasmaclearance rates of a fluorescent GFR marker can be measured in less than five minutes followinga bolus infusion of a fluorescent dye mixture into the blood stream. Intravital multi-photonimaging provided evidence for intense proximal tubular reabsorption of negatively chargedmacromolecules (albumin, dextran) which provided further explanation for their low amountsin the urine.14,20 Also, the glomerular filter appears to normally leak albumin at nephroticlevels. 20 These important findings question the paradigms of charge selectivity of theglomerular filtration barrier and the mechanism of albuminuria. Instead, they strongly supportthe new concept that albuminuria does not occur because of the filtered albumin load is avidlybound and retrieved by the proximal tubule. Dysfunction of this retrieval pathway leads toalbuminuria. 20

Multi-photon quantitative imaging techniques were applied to disease models including renalischemia20, diabetes9,10, cystic kidney17 and also to directly visualize and quantify drugdelivery21 and the effects of therapeutic interventions9,21. For example, increased SNGFRand glomerular permeability were observed in untreated STZ-diabetic rats10 (Fig. 2A).Angiotensin II AT1 receptor inhibition improved many functional parameters12, but alsocaused a significant increase in JGA renin content9 (olmesartan, Fig. 2B).

Intravital multi-photon microscopy was used to directly visualize fenestration of the afferentarteriole endothelium in the renin expressing segment, bulk fluid flow in the JGA originatingfrom the afferent arteriolar ultrafiltration of plasma into the JGA interstitium as well as theflow of glomerular filtrate in the Bowman’s space back into the extraglomerular mesangium.8 Labeling the afferent arteriole endothelium by the endocytosis of the fluid marker luciferyellow revealed heterogeneity of the preglomerular vasculature (Fig. 2C). LY stained theproximal and JG AA endothelium segments differently. In the proximal portion, theendothelium appeared as a solid and intensely fluorescent line, but it was weakly fluorescentwith a discontinuous line pattern in the JG segment (Fig. 2C). These studies concluded thatsignificant and dynamic fluid flow exists in the JGA which may help filter the released renininto the renal interstitium (endocrine function). It may also modulate TGF and renin signals inthe JGA (hemodynamic function). These findings challenge the existing paradigm of the stableand isolated JGA environment.

Imaging cytosolic ion concentrations in vivoIn vitro model systems, cell cultures and isolated, microperfused renal tissue techniques havebeen widely used in combination with fluorescence imaging to measure cytosolic ionconcentrations and variables including pH, Ca2+, Na+, Cl−, cell volume, etc.22 However, itmay be desirable to confirm if these measurements, cellular processes are relevant to in vivoconditions and also exist in the intact kidney. One or two-photon excitation confocal imaging,depending on the tissue depth, is ideally suited to achieve this task with close to real-time,subcellular resolution. Fig. 3 demonstrates that it is possible to measure cytosolic ionconcentrations (for example pH and calcium) in superficial tubular segments (both in proximaland distal tubules) with conventional one-photon fluorescence excitation. For opticalsectioning of deeper structures, two-photon may be required. Loading of tubular epithelial cells

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with a fluorophore is technically the easiest in the distal nephron (Fig. 3A) taking advantageof the high luminal dye concentrations there attained by the renal concentrating mechanism.Using mice, a single iv bolus injection of the calcium sensitive dye Fluo-4 results in sufficientloading of the distal nephron (Invitrogen, 50 μg AM form dissolved in 1 μl DMSO and dilutedin 50 μl Ringer solution, excitation at 488 nm, emission at 520 nm). In preliminary experimentsa number of cells in the cortical collecting duct showed intense calcium oscillations (Fig. 3A).In contrast, in cortical structures where blood flow or tubular flow and consequently washoutof the dye are high (glomerulus, proximal tubule), injecting fluorophores locally, under therenal capsule may be a better loading strategy. Fig. 3B exemplifies loading of proximal tubulecells with the pH sensitive dye BCECF injected under the renal capsule (prepared the sameway as above for Fluo-4, excitation at 488 nm by the Argon laser or at 800 nm by the MP laser,emission at 530 nm). In preliminary assays in mice, loading required ~5–10 min, during whichtime BCECF fluorescence intensity stabilized at values of at least one order of magnitudegreater than background fluorescence. Fig. 3B shows that BCECF fluorescence intensity wasthe highest (indicating high, alkalotic pHi) in the brush-border membrane area, consistent withsignificant bicarbonate reabsorption into the relatively small cytosolic volume of apicalmicrovilli. Developing a reproducible method to measure Na+/H+ exchanger activity in vivowill be an important tool to directly assess the role of the proximal tubule in salt and waterreabsorption under various conditions.

ConclusionMulti-photon excitation fluorescence microscopy is an excellent imaging technique for thestudy of the complex renal (patho)physiological mechanisms in the living kidney both invivo and in vitro. New visual data challenge a number of existing paradigms in renal physiology.Also, quantitative imaging of kidney function with multi-photon microscopy has excellentpotential to eventually provide novel non-invasive diagnostic and therapeutic tools for futureapplications in clinical nephrology.

Acknowledgements

This work was supported by grants from NIH DK064324, and AHA Established Investigator Award 0640056N toJPP. We thank the Sankyo Company in Tokyo, Japan for providing olmesartan. L. Rosivall was a Fulbright Fellowat the University of Southern California during parts of these studies.

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Figure 1.Multi-photon imaging of the juxtaglomerular apparatus using the microperfused afferentarteriole (AA)-glomerulus (G)-attached macula densa (MD) preparation. A: Visualization ofindividual renin granules and exocytosis of granular content in JG granular cells usingquinacrine (green). Differential interference contrast (DIC) overlay. B: Fluo-4 and Fura redratiometric calcium imaging. High ratio values indicate significant elevations in [Ca2+]i in bothAA and intraglomerular cells after TGF activation. Bar is 20 μm. EA: efferent arteriole.

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Figure 2.In vivo multi-photon imaging of the intact rodent kidney (A–B: rat, C: mouse). Rhodamine B-conjugated 70 kD dextran (red) was given iv to label the cortical vasculature (plasma).Quinacrine (green) is a strong marker of renin granular content in the afferent arteriole (AA)and it also weakly stains renal tubules (proximal tubule, PT). A: In vivo imaging of the STZ-diabetic kidney and visualization of glomerular permeability. A sclerotic (arrow) and ahyperfiltering glomerulus (G) are shown. Note the intense ultrafiltration of the high molecularweight (70 kD) dextran-rhodamine B (red) from the plasma into the Bowman’s space in thesclerotic, but not in the hyperfiltering glomerulus. B: In vivo imaging of the juxtaglomerularrenin content in the diabetic kidney treated with an angiotensin II type 1 receptor (AT1) blocker.Note the significantly increased renin content around the juxtaglomerular portion of the afferentarteriole (AA). C: Multi-photon image of the proximal (P) and juxtaglomerular (JG) segmentsof the afferent arteriole (AA) in mouse. Content of individual renin granules is labeled byquinacrine (green). The vascular endothelium is labeled by the endocytosis of lucifer yellow(yellow). Note the intense and continuous yellow labeling in the proximal AA segmentcompared to the weakly fluorescent dash-dot pattern in the JG, renin-positive part. MD: maculadensa. Scale is 20 μm.

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Figure 3.In vivo imaging of cell [Ca2+] in the intact mouse cortical collecting duct (CCD) (A) and cellpH in the proximal tubule (PT) (B). Fluo-4 or BCECF was loaded in the living kidney tomeasure cell [Ca2+] and pH, respectively. Note the diffuse cytosolic distribution of Fluo-4fluorescence in the CCD, but the primarily apical, microvillar BCECF fluorescence in the PT.Intense [Ca2+] oscillations were observed in some CCD cells (arrow). On panel A, the intense,patchy labeling in the PT is mainly tissue autofluorescence, typical for this tubular segment.Rhodamine B-conjugated 70 kD dextran (red) was given iv to label peritubular capillaries(plasma). DT: distal tubule containing highly fluorescent, concentrated tubular fluid. Scale is20 μm.

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advances in renal (patho)physiology using multiphoton microscopy

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