SPECIAL ARTICLE www.jasn.org A High-Powered View of the Filtration Barrier Ja´nos Peti-Peterdi and Arnold Sipos Departments of Physiology and Biophysics and Medicine, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, California ABSTRACT Multiphoton excitation fluorescence microscopy is a powerful noninvasive imaging (PAN) model of focal segmental glo- technique for the deep optical sectioning of living tissues. Its application in several merulosclerosis (FSGS). intact tissues is a significant advance in our understanding of organ function, including renal pathophysiological mechanisms. The glomerulus, the filtering unit in the kidney, is one good example of a relatively inaccessible and complex structure, MULTIPHOTON IMAGING SHOWS with cell types that are otherwise difficult to study at high resolution in their native NEW DETAILS OF environment. In this article, we address the application, advantages, and limita- JUXTAGLOMERULAR FUNCTION tions of this imaging technology for the study of the glomerular filtration barrier and the controversy it recently generated regarding the glomerular filtration of One of the main research interests of our macromolecules. More advanced and accurate multiphoton determinations of the laboratory has been the function of the glomerular sieving coefficient that are presented here dismiss previous claims on juxtaglomerular apparatus (JGA), the the filtration of nephrotic levels of albumin. The sieving coefficient of 70-kD mechanisms of basic physiologic pro- dextran was found to be around 0.001. Using a model of focal segmental glomer- cesses by which cells of the macula densa ulosclerosis, increased filtration barrier permeability is restricted only to areas of in the distal nephron control GFR, renal podocyte damage, consistent with the generally accepted role of podocytes and blood flow, and the renin–angiotensin the glomerular origin of albuminuria. Time-lapse imaging provides new details and system.9 Confocal and multiphoton im- important in vivo confirmation of the dynamics of podocyte movement, shedding, aging helped to identify new details of the replacement, and the role of the parietal epithelial cells and Bowman’s capsule in macula densa–mediated, tubular salt/ the pathology of glomerulosclerosis. flow–induced afferent arteriole vaso- constriction (tubuloglomerular feed- J Am Soc Nephrol 21: 1835–1841, 2010. doi: 10.1681/ASN.2010040378 back [TGF])2,7,8,10,11 and allowed the direct visualization of renin release from the JGA.8,12,13 The advanced confocal imaging tech- cient (GSC) for macromolecules. New One interesting feature of TGF, which nique of two- or three-photon, collec- data will be presented using more ad- may have great implications for the func- tively called multiphoton, excitation flu- vanced and accurate multiphoton tech- tion of the GFB, is the cell-to-cell calcium orescence microscopy was established1 niques, suggesting that conventional flu- signaling that propagates as a calcium and first applied to the kidney several orescence imaging applications have wave in the JGA and beyond. Increased years ago.2,3 The basic principles and ad- serious limitations for the measurement salt content or tubular fluid flow at the vantages of the technology, and examples of GSC and providing evidence that the macula densa triggers elevations in of multiphoton studies in kidney research, amount of filtered macromolecules in [Ca2ϩ] in the adjacent mesangial and have been reviewed previously.2–8 In this the Bowman’s space is extremely low. special article, we focus on the application Also, time-lapse images and supple- Published online ahead of print. Publication date of multiphoton imaging for the study of mentary video material provide new available at www.jasn.org. the glomerular filtration barrier (GFB). details and important in vivo confirma- Correspondence: Dr. Ja´nos Peti-Peterdi, Depart- Here we briefly discuss select, recent find- tion of podocyte functions in their in- ment of Physiology and Biophysics and Department ings in juxtaglomerular physiology that tact environment during health and of Medicine, Zilkha Neurogenetic Institute, Univer- are relevant to the function of the GFB disease. These include the dynamics of sity of Southern California, 1501 San Pablo Street, ZNI 335, Los Angeles, CA 90033. Phone: 323-442- that were obtained using the multiphoton podocyte migration, shedding, re- 4337; Fax: 323-442-4466; E-mail: petipete@usc. imaging approach. Then we address the con- placement, and the role of the parietal edu troversy regarding the multiphoton de- epithelial cells and Bowman’s capsule Copyright © 2010 by the American Society of termination of glomerular sieving coeffi- in the puromycin aminonucleoside Nephrology J Am Soc Nephrol 21: 1835–1841, 2010 ISSN : 1046-6673/2111-1835 1835 SPECIAL ARTICLE www.jasn.org vascular smooth muscle cells, which prop- confirm the findings of these initial mor- CONTROVERSY REGARDING agate not only to the afferent arteriole caus- phometric measurements and show that GLOMERULAR SIEVING OF ing vasoconstriction but simultaneously the SPS constitutes a highly significant MACROMOLECULES: toward all cells of the glomerulus including restriction to flow.23 The small connec- LIMITATIONS OF THE the most distant podocytes.11 Although the tors between the SPS and the Bowman’s FLUORESCENCE APPROACH issue of active podocyte contraction is space, so-called exit pores,22 may have controversial,14 elevations in podocyte important functional implications in A few years ago, pioneers in the renal intra- [Ca2ϩ] and simultaneous reductions in physiologic (filter backwash, podocyte- vital applications of multiphoton imaging glomerular capillary diameter during to-endothelium crosstalk), as well as made an interesting observation that at the TGF2,11 that exist even in nonperfused pathologic processes (glomerulosclero- time was a potentially paradigm-shifting glomeruli11 are consistent with their sis) and needs further study. discovery regarding the function of the contractile function in vivo. Also well es- Quantitative multiphoton imaging GFB. They observed mass glomerular fil- tablished is the importance of podocyte techniques can directly visualize major tration (high levels of fluorescence in the cell membrane calcium channels (TRPC6 physiologic functions on the single Bowman’s space) of fluorophore-conju- for example) in maintaining normal nephron level, for example, the classic, gated albumin and 70-kD dextran in nor- [Ca2ϩ] dynamics, which produce new oscillatory nature of single nephron mal kidneys (with their glomerular siev- pathology, particularly glomerulosclero- GFR (SNGFR) and tubular fluid flow.6 ing coefficient [GSC] in the range of 0.02 sis, when dysfunctional.15 Real-time imaging and measurement to 0.04) and its rapid endocytosis in the Propagation of the TGF calcium wave of these and other glomerular and tu- proximal tubule.27 Based on these mul- to glomerular podocytes and their in- bular functions have been published tiphoton studies, the authors formulated volvement in the control of GFR suggests earlier and are included in supplemen- a hypothesis that the GFB normally leaks that podocytes may be functionally part tary video material.6,8 For example, regu- albumin at nephrotic levels, and this fil- of the JGA. Because of the bidirectional lar periods of glomerular contraction– tered albumin load is avidly bound and propagation of the TGF calcium wave, relaxation are observed, resulting in retrieved by cells along the proximal tu- podocyte [Ca2ϩ] elevations and contrac- oscillations of filtration (SNGFR) and tu- bule. tions coincide with afferent arteriole bular flow rate. This is measured by This new concept on the tubular ori- contraction. The timing of this synchro- changes in fluorescence intensity of the gin of proteinuria challenged the old par- nized podocyte contraction is especially filtered plasma marker 70-kD dextran- adigm that proteinuria is of glomerular interesting because it occurs when intra- rhodamine B in Bowman’s space and origin.15–21,29 The same authors followed glomerular capillary pressure is at its tubular fluid, as well as by changes in up on their initial observation by apply- lowest. Together with the function of the tubular diameter.6,8 The oscillations ing the multiphoton imaging approach newly discovered subpodocyte space (SPS) clearly show two components: faster in the diabetic kidney and found basi- and the oscillatory feature of glomerular (frequency of 0.12 Hz and cycle time cally the same high level of glomerular function, as detailed below, this raises the about 10 seconds) and slower (fre- filtration but failed reabsorption of albu- possibility that a temporary filter back- quency of 0.023 Hz and cycle time min in the proximal tubule, giving rise to wash mechanism exists in the GFB that about 45 seconds) elements that are diabetic albuminuria.28 Because the value of contributes to the maintenance of a clean consequences of the afferent arteriole albumin GSC measured by multiphoton glomerular filter. Clearly, the location of myogenic and TGF mechanisms, re- microscopy was 50 times greater than the major filtration barrier, glomerular spectively.24,25 previously measured by micropunc- basement membrane versus the slit dia- In addition, real-time multiphoton ture29 or calculated30 (GSC in the 0.0006 phragm, and the reasons why the filtra- imaging of the intact kidney in vivo con- range), these multiphoton studies gener- tion barrier does not clog are still highly firms