EDITORIAL www.jasn.org

Illuminating the GlomerularFiltration Barrier, Two Photonsat a Time

William H. FissellBiomedical Engineering, Cleveland Clinic, Cleveland, Ohio

J Am Soc Nephrol 23: ccc–ccc, 2012.doi: 10.1681/ASN.2012010067

The mechanisms by which the kidney retains albumin andlarger proteins in the circulation while excreting smaller sol-utes remain enigmatic despite intense investigation. Althoughthe controversy largely plays out in academic journals, theproblem is anything but academic, because the health of mil-lions of adults hinges on effective treatment of proteinuricCKD. Proteinuria is associated with cardiovascular and all-cause mortality.1,2 Indeed, albuminuria has emerged as thesingle best predictor of progression of CKD, and control ofproteinuria is clearly associated with preservation of GFR.3–6

To illustrate the public health burden of CKD, the work byCoresh et al.7 used National Health and Nutrition Examina-tion Surveys data to estimate that nearly 8% of the US pop-ulation had moderate or severe reductions in GFR and slightlymore than 8%hadmicroalbuminuria. Almost all of the increasedprevalence of albuminuria over the prior decade could be ex-plained by increased prevalence of hypertension, diabetes, andobesity, suggesting that increased prosperity in the so-called de-veloping world will bring an increased burden of proteinuricCKD.8,9 In JASN, the work by Sandoval et al.10 presents a detaileddescription of factors influencing the accuracy of a recently de-veloped technique (two-photon intravital microscopy) that hasunexpectedly lent support to a controversialmodel of glomerularfiltration and tubular reclamation.11 The importance of this pa-per is highlighted by background of the debate.

Although albumin circulates in blood at a concentration of40 g/L,micropuncture studies have repeatedly reported albuminconcentrations in Bowman’s capsule of 10 mg/L or less.6,12–14

Thus, physiologic models of renal protein handling have beenbased on the generally shared tenet that the glomerular capillarywall forms a stringent barrier to albumin passage. Indeed, clin-ical syndromes with proteinuria are characterized by ultra-structural changes to the glomerular capillary wall on electron

microscopic examination. Conversely, tubulointerstitial disordersare typically associated with low-level proteinuria. Finally, thelink between congenital nephropathy of the Finnish type,nephrin, and nephrin’s expression at the slit diaphragm codi-fied the role of the glomerular filtration barrier in the languageof molecular biology.15–17

Albumin seems to enjoy a special status in measurements ofglomerular integrity. Similar-sized polysaccharide molecules,such as ficoll, are hindered by the glomerular capillary wall to alesser extent than albumin.18,19However,measurements ofmac-romolecular transport through well-defined artificial mem-branes or agarose gels have not shown similar discrepancies,and the differential in sieving coefficient between albumin andficoll is much less pronounced in other capillary beds.20–23

Insight into albumin’s special status in the kidney can bederived from studies of albumin metabolism by proximal tu-bule cells and reports of albumin fragments found in urine andthe renal vein.24–27 Antibody-based assays seem to underesti-mate urinary albumin excretion compared with studies thatuse radiolabeled albumin.28,29 Thus, the work by Osickaet al.30 suggested that the low fractional excretion of albuminby the kidney might be attributable to tubular transport andmetabolism rather than the unique properties of the glomerularcapillary wall. However, because clinicopathologic and geneticdataweremost consistent with a glomerular barrier, a significantrole for tubular albumin transport was difficult to integrate withprior models of renal physiology.19,31,32

In an attempt to gain insight into the ongoing controversy byusing a new technique, the work by Russo et al.11 employed therecently developed toolkit of two-photon intravital microscopyto determine the concentration of albumin in Bowman’s capsuleand measured a glomerular sieving coefficient for albumin(GSCA) of about 0.034 (or 50- to 100-fold higher than the co-efficient determined from micropuncture studies). Furthermore,rats treated with puromycin aminonucleoside display reducedproximal tubule brush border albumin uptake and little changein GSCA.

11 The appearance of this study in 2007 stimulated an-imated discussion about the two-photon technique and the sig-nificance of the results, and the readermay follow the controversyin the original literature.11,33–38 There have been technical criti-cisms of the 2007 paper. Others using the two-photon techniquehave obtained more conventional values for the filtration barrier.The high value for GSCA may be an artifact of low signal to noiseratio, inflating the measured values in Bowman’s capsule. Signalsaturation or interference of passing red blood cells in the capillaryartifactually may lower the values in the capillary loop, and atransmission electron micrograph interpreted as showing fusionof a vesicle ladenwith albuminwith the basolateral cellmembranemay have been either a fixation artifact or a chance occurrence.

The work by Sandoval et al.10 marks a significant reinforce-ment of the application of the two-photon technique to

Published online ahead of print. Publication date available at www.jasn.org.

Correspondence: Dr. William H. Fissell, Cleveland Clinic, Biomedical EngineeringND20, 9500 Euclid Avenue, Cleveland, OH 44195. Email: fisselw@ccf.org

Copyright © 2012 by the American Society of Nephrology

J Am Soc Nephrol 23: ccc–ccc, 2012 ISSN : 1046-6673/2303-ccc 1

glomerular physiology, because it highlights control of sourcesof artifact and addresses apparent discrepancies between theirdata and data from others. Amid the details of photodetectorgeometry and polymer polydispersion, two features emergethat deserve attention beyond discussions of technique. First,the two-photon technique reported in the work by Sandovalet al.10measures a GSCA in fasted Frömter strainMunich–Wistarrats that is much closer (GSCA;0.007) to the GSCA obtainedfrommicropuncture studies than the 0.034 measurements (in adifferent strain) published in the 2007 paper.11 These values forGSCAmay overlap values obtained by thework ofOhlson et al.19

using a chilled isolated perfused kidney from Wistar rats. Thisfinding opens the possibility that the differences in measuredvalues for GSCA between techniques might originate in differ-ences between strains and physiologic conditions rather than theexperimental error of which Russo et al.11 have been accused.Second, thework by Sandoval et al.10 presentsmultiple images offluorescent intracellular bodies appearing to approach or fusewith the basolateral membrane, suggesting that the prior obser-vation was not just a freak occurrence but a phenomenon thatmerits more quantitative examination.

The data still require caution in interpretation, but hopefully,they will redirect discourse to the mechanisms by which theapparent GSCA might have much greater spatial and temporalvariation than previously imagined. Perhaps not all glomeruliare created equal, and by its nature, the two-photon techniqueonly probes the behavior of glomeruli exactly at the surface of thekidney just like micropuncture studies.39 The roles of trauma,fasted/fed status, and other short-term physiology in alteringglomerular permeability deserve increased mechanistic atten-tion.40 Other studies support the hypothesis that albumin istaken up by proximal tubule cells and peptides from albuminproteolysis are observed on apical and basolateral sides of theepithelium; however, fluorescence microscopy cannot distin-guish whether the fluorophore was bound to an intact albuminmolecule or an albumin-derived peptide fragment.41

The data and the debate regarding the filtration barriercontinue to accumulate while a record 113,636 patients starteddialysis in the United States in 2009. Patients and practitionersalike are eager for mechanism-based treatments that will delaydisease progression. The relative contributions of endothelium,basement membrane, podocyte, and tubule to renal proteinhandling in health and disease could be better understood just aswe as physicians and researchers might better understand eachother’s results.

DISCLOSURESNone.

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