Combined Structural and Functional Imaging of the Kidney Reveals Major Axial Differences in Proximal Tubule Endocytosis

BASIC RESEARCH www.jasn.org

Claus D. Schuh,1 Marcello Polesel,1 Evgenia Platonova,2 Dominik Haenni,1,2 Alkaly Gassama,3 Natsuko Tokonami,3 Susan Ghazi,1 Milica Bugarski,1 Olivier Devuyst,3 Urs Ziegler,2 and Andrew M. Hall1,4

1Institute of Anatomy, 2Center for Microscopy and Image Analysis, and 3Institute of Physiology, University of Zurich, Zurich, Switzerland; and 4Department of Nephrology, University Hospital Zurich, Zurich, Switzerland

ABSTRACT Background The kidney proximal convoluted tubule (PCT) reabsorbs filtered macromolecules via receptor-mediated endocytosis (RME) or nonspecific fluid phase endocytosis (FPE); endocytosis is also an entry route for disease-causing toxins. PCT cells express the protein ligand receptor megalin and have a highly developed endolysosomal system (ELS). Two PCT segments (S1 and S2) display subtle differences in cellular ultrastructure; whether these translate into differences in endocytotic function has been unknown. Methods To investigate potential differences in endocytic function in S1 and S2, we quantified ELS protein expression in mouse kidney PCTs using real-time quantitative polymerase chain reaction and immunos- taining. We also used multiphoton microscopy to visualize uptake of fluorescently labeled ligands in both living animals and tissue cleared using a modified CLARITY approach. Results Uptake of proteins by RME occurs almost exclusively in S1. In contrast, dextran uptake by FPE takes place in both S1 and S2, suggesting that RME and FPE are discrete processes. Expression of key ELS proteins, but not megalin, showed a bimodal distribution; levels were far higher in S1, where intracellular distribution was also more polarized. Tissue clearing permitted imaging of ligand uptake at single- organelle resolution in large sections of kidney cortex. Analysis of segmented tubules confirmed that, compared with protein uptake, dextran uptake occurred over a much greater length of the PCT, although individual PCTs show marked heterogeneity in solute uptake length and three-dimensional morphology. Conclusions Striking axial differences in ligand uptake and ELS function exist along the PCT, independent of megalin expression. These differences have important implications for understanding topographic patterns of kidney diseases and the origins of proteinuria.

J Am Soc Nephrol 29: 2696–2712, 2018. doi: https://doi.org/10.1681/ASN.2018050522

Adult humans filter approximately 170 L of fluid per isolation or as part of a generalized solute transport day in the kidneys. Contained within the filtrate are defect (renal Fanconi syndrome).1–5 Conversely, hundreds of low mol wt proteins (LMWPs) and some albumin. Many of these LMWPs have impor- Received May 18, 2018. Accepted September 18, 2018. tant physiologic functions and need to be reclaimed to prevent urinary wasting. Reabsorption of pro- C.D.S. and M.P. contributed equally to this work. teins takes place by endocytosis in the proximal Published online ahead of print. Publication date available at convoluted tubule (PCT), which is highly adapted www.jasn.org. to this purpose. Numerous insults—both genetic Correspondence: Dr. Andrew M. Hall, Institute of Anatomy, and acquired—can affect protein uptake in the University of Zurich, Winterthurerstrasse 190, 8057 Zurich, PCT leading to elevated urinary levels, a condition Switzerland. Email: [email protected] known as tubular proteinuria, which can occur in Copyright © 2018 by the American Society of Nephrology

2696 ISSN : 1046-6673/2911-2696 JAmSocNephrol29: 2696–2712, 2018 www.jasn.org BASIC RESEARCH endocytosis provides an entry route into PCT cells for toxins Significance Statement that frequently cause kidney damage.6 Meanwhile, the detection of albuminuria is widely used as a screening test for glo- Defective reabsorption of filtered molecules by endocytosis in the merular pathology, but the role of the PCT in determining kidney proximal convoluted tubule (PCT) occurs in numerous albumin excretion remains controversial.7 Therefore, to im- diseases, and endocytosis is also an important entry route for prove understanding of diseases in the PCT and the origins of nephrotoxic substances. PCT cells have a highly developed endo- fi lysosomal system (ELS), but relatively little is known about its proteinuria, it is rst necessary to increase knowledge regard- function in vivo. The authors measured ELS protein expression and ing normal handling of macromolecules by this nephron used advanced imaging techniques to demonstrate that early and segment. late PCT segments show striking differences in ligand uptake Uptake of macromolecules across the apical PCT cell and ELS function. They found that the early segment is highly spe- membrane occurs via either specific receptor-mediated en- cialized to perform protein reabsorption via receptor-mediated fi fl docytosis (RME) or nonspecific fluid phase endocytosis endocytosis, whereas nonspeci c uid phase endocytosis occurs along the entire PCT. These findings have important implications 8 (FPE). The main receptors for RME are megalin and cubi- for understanding PCT function, topographic patterns of renal lin, two large proteins that can bind a wide range of LMWPs9 disease, and the origins of tubular proteinuria. and require adaptor proteins such as Disabled-2 (Dab2) for their normal function.10 Once internalized into early endosomes, proteins are trafficked to late endosomes and even- show distinct reabsorption patterns throughout the cortex, tually lysosomes, to undergo degradation and/or transport but that PCTs show marked heterogeneity in three-dimen- to the baso-lateral membrane.7 Thecriticalroleoftheen- sional morphology and solute uptake length. In summary, we dolysosomal system (ELS) in LMWP uptake and receptor present evidence of substantial axial differences in PCT ligand recycling is illustrated by hereditary forms of renal Fanconi uptake and ELS function, independent of megalin expression, ’ and that the S1 segment is highly specialized to reabsorb pro- syndrome, such as Dent s disease and Lowe syndrome, where via the causative mutations have been localized to genes encod- teins RME. ing proteins important for endosomal acidification and traf- fi 11 cking. METHODS The proximal tubule is classically divided into three – segments (S1 3) on the basis of differences in cell ultra- Dyes and Reagents structure, including the ELS, observable with electron mi- Both dextran and albumin were purchased prelabeled from the 12,13 fi croscopy (EM). S1 cells are found in the rst part of the manufacturer (#D1976, #D22914, #A13100, #A34785; PCTaftertheglomerulus,S2inthelaterPCT,andS3 Thermo Fisher). Recombinant human lysozyme and exclusively in the straight portion. Whereas structural dif- b-lactoglobulin (#L1667, #L3908; Sigma-Aldrich) were la- ferences between S1 and S3 cells are quite striking, the dif- beled with either Atto-565 or Atto-647N, according the 13 ferences between S1 and S2 cells are much subtler, and manufacturer’s protocol (#AD 565–31, #AD 647N-31; 14 megalin is thought to be expressed in both segments, so Atto-Tec). Labeled protein was purified using benchtop fi whether there are signi cant functional differences between PD midiTrap G-25 Sephadex chromatography columns them in ligand uptake was unclear. Intravital multiphoton (#28–9180–08; GE Healthcare) and concentrated with fl imaging of uorescently labeled ligands offers the possibil- 3-kD Amicon Ultra-4 centrifugal filters (#Z740186; ity to visualize renal solute handling in real time. This Sigma-Aldrich) in PBS. Purity and labeling ratio were mea- approach has been used in numerous previous studies to sured by liquid chromatography mass spectrometry and fi 15–17 investigate albumin ltration and uptake, but less so absorption spectroscopy (Libra S70; Biochrom). Labeled to study LMWPs, partly due to a paucity of suitable labeled solutes were dissolved in saline and injected intravenously ligands. Moreover, depth of imaging is limited to the outer in a volume of 0.12 ml. For control experiments with free fi fi cortex and ndings from super cial PCTs may not be dye, mice were injected with a mixture of unlabeled albu- representative. min (A7030; Sigma-Aldrich) and Alexa Fluor 488 carbox- — Here, using intravital imaging and a variety of ligands ylic acid (A33077; Thermo Fisher Scientific), the inert including LMWPs conjugated to very bright and photo- nonreactive form of the dye, at the same concentra- stable fluorophores—we show that uptake of protein by tions used for labeled albumin. Monochlorobimane RME occurs almost exclusively in S1 PCT segments, which (MCB) (#635; AAT Bioquest) was dissolved in DMSO and have a very high capacity for reabsorption and a far higher expression of ELS machinery. In contrast, uptake of dex- injected as a 1-mg/ml solution. trans via FPE occurs to a similar extent in S1 and S2. By utilizing a tissue-clearing approach, we show that it is pos- Animals sible to image solute uptake in PCTs at high resolution in All experiments were performed on 6–12-week-old male C57Bl/ large kidney sections, at depths way beyond what is achiev- 6J-Rj mice (supplied by Janvier, Le Genest, France), and were able with intravital microscopy. Analysis of individually performed in accordance with the regulations of The Zurich segmented nephrons revealed that LMWPs and dextrans Cantonal Veterinary Office. Unless stated otherwise, animals

J Am Soc Nephrol 29: 2696–2712, 2018 Proximal Tubule Endocytosis In Vivo 2697 BASIC RESEARCH www.jasn.org were anesthetized with 2.5% isoflurane (Attane; Provet AG, detectionofsinglePCRproductaccumulation.RT-qPCR Switzerland) in 100 ml/min oxygen. analyses were performed in duplicate with 100 nM of both sense and antisense primers in a final volume of 20 ml using Antibody Staining in Fixed Kidney Tissue iQTM SYBR Green Supermix. Specific primers were designed Kidney tissue was perfusion fixed via the aorta with 3% para- using Primer3 (Supplemental Table 1).18 The PCR prod- formaldehyde in a phosphate-buffered solution. For lysozyme ucts were sequenced with the BigDye Terminator kit uptake experiments, Atto-565–labeled protein was injected (Perkin Elmer Applied Biosystems). The multiScreen via tail vein injection 1 hour before fixation. Immune staining SEQ384 Filter Plate (Millipore) and Sephadex G-50 DNA was performed on 5-mm-thick cryosections incubated over- Grade Fine (Amersham Biosciences) dye terminator removal night with the following antibodies: rabbit anti-Rab5 (C8B1) were used to purify sequence reactions before analysis on an (#3547; Cell Signaling Technology), rabbit anti-Rab11 (D4F5) ABI3100 capillary sequencer (Perkin Elmer Applied Biosys- (#5589; Cell Signaling Technology), rabbit anti-Rab7 tems). The relative changes in target gene/GAPDH mRNA ratio (EPR7589) (ab137029; Abcam), rat anti-Lamp1 (1D4B) were determined using the 2DDct formula.19 (ab25245; Abcam), mouse anti-Lrp2 (CD7D5) (NB110– 96417; Novus Biologicals), polyclonal sheep anti-Cubilin EM (AF3700; R&D Systems), and rabbit anti-RBP4 (EP3657) Kidneys were perfusion fixed with 3% paraformaldehyde in a (ab109193; Abcam). The following secondary conjugated an- phosphate-buffered solution via the abdominal aorta. Tissue tibodies were used (all at 1:500): Dylight488 goat anti-rat was stored at 4°C overnight in 2.5% glutaraldehyde, before (A110–242D2; Bethyl Laboratories/Lubioscience), Alexa647 fixation with 1% osmium tetroxide in cacodylate buffer. The donkey anti-rabbit (711–606–152; Jackson Immuno- samples were enhanced for contrast in 1% uranyl acetate, de- Research), and AbberiorStar635-P goat anti-mouse (a kind hydrated with graded ethanol and 100% propylene oxide, and gift from The Zurich Centre for Microscopy and Image Anal- embedded in 100% Epon/Araldite. Ultrathin sections (70 nm) ysis). Brush border actin filaments were stained with ActinRed were cut on an ultra-microtome (Reichert Ultracut), mounted 555 ReadyProbes reagent (R37112; Invitrogen) according to on formvar-coated grids, and stained with lead citrate. Images the manufacturer’s instructions. Images were acquired using a were acquired with a CM 100 (FEI/Philips) equipped with a Leica SP8 upright confocal microscope. To quantify signals, Gatan Orius 1000 CCD camera. regions of interest were drawn around individual PCT segments in cortical labyrinths. Intravital Imaging The left kidney was externalized for imaging using previously Gene Expression Analysis in Microdissected Tubular established protocols.20 The internal jugular vein was cannu- Segments lated to allow intravenous injections of dyes and reagents. Mice were anesthetized with intraperitoneal injection of ket- Body temperature was monitored throughout experiments. amine (100 mg/kg body wt; Streuli Pharma) and xylazine Imaging was performed on a custom-built multiphoton mi- (20 mg/kg body wt; Streuli Pharma). The left kidney was per- croscope operating in an inverted mode.20 A broadband tun- fused, removed, and cut into small pieces before incubation for able laser (InSight DS Dual; Spectraphysics, Santa Clara) was 40 minutes at 37°C in a solution with 40 mg/ml liberase used as excitation source. The following excitation wave- (Roche), containing: HBSS, HEPES 15 mM, D-glucose lengths were used: MCB 780 nm, Cascade-blue 800 nm, 10 mM, glycine 5 mM, and alanine 1 mM. Tubular segments Atto-565 850 nm, Alexa-488 950 nm, Alexa-647 and Atto- were dissected on ice according to anatomic and morphologic 647N 1180 nm, and Alexa-680 1280 nm. Image processing characteristics (S1: PCTs that attach to glomeruli; S2: distal was performed using FIJI and Imaris version 9.0 (Bitplane and thicker PCTs following from S1; S3: proximal straight AG, Zurich, Switzerland). Starting shortly before intravenous tubules from the outer medullary region). Isolated tubules injection of ligands, single plane intravital images were ac- were lysed in the RNA extraction buffer from the RNAqueous quired every 15 seconds to visualize PCT handling in real Total RNA Isolation Kit (Invitrogen). Total RNAwas extracted time. Analysis of uptake was performed using images acquired immediately using the RNAqueous-Micro kit following the 30–40 minutes postinjection, when the fluorescence signal manufacturer’s protocol (Ambion). Quality and concentra- from LMWPs or dextran was no longer detectable in the vas- tion of the isolated RNA preparations were analyzed using culature, thus signifying complete filtration of the molecules. the 2100 BioAnalyzer (RNA Pico chip from Agilent Technol- ogies). Total RNA samples were stored at 280°C, and the same Urine Analysis quantity of RNAwas used to perform the reverse transcription Urine samples from albumin-injected and control animals reaction with iScript TM cDNA Synthesis Kit (Bio Rad). were separated by a 10% SDS-PAGE. Protein bands were visualized Changes in target gene mRNA levels were determined by either by fluorescence using a Gel scanner ChemiDoc Touch relative real-time quantitative polymerase chain reaction (Biorad) with the settings for 488-nm dyes, or by Coomassie with a CFX96TM Real-Time PCR Detection System (Bio- staining with ProtoBlue Safe (EC-722; National Diagnostics) Rad), using iQ TM SYBR Green Supermix (Bio-Rad) and scanning with an Odyssey CLx gel scanner (Li-Cor).

2698 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2696–2712, 2018 www.jasn.org BASIC RESEARCH

Preparation and Imaging of Cleared Kidney Tissue was similar in all segments (Figure 1, A–C). In contrast, cubi- Tissue clearing was performed using a modified CLARITY lin expression decreased from S1 to S3, but was highly variable approach.21 Kidneys were harvested immediately after intra- in S2; however, the ratio of megalin-to-cubilin expression was vital imaging experiments and were dissected transversally. significantly higher in S2 than in S1 (Supplemental Figure 3). Resulting sections were fixed overnight in 4% paraformalde- Expression levels of the adaptor protein Dab2 and established hyde at 4°C and incubated overnight in 4% acrylamide markers for early endosomes (Rab5), late endosomes (Rab7), (161–0140; Bio-Rad) and 0.25% VA-044 (017–19362; Nova- recycling endosomes (Rab11), and lysosomes (Lamp1)22 were chem) in PBS at 4°C. They were then incubated for 3 hours at all higher in S1, and Lamp1 expression correlated closely with 37°C for acrylamide polymerization, washed overnight in that of NaPi2a (Figure 1, A–C, Supplemental Figure 3). His- clearing solution (200 mM SDS (L3371; Sigma-Aldrich) and tograms of expression intensities in PCTs revealed a unimodal 200 mM boric acid (L185094; Sigma-Aldrich), pH 8.5), and distribution for megalin, but bimodal for Rab7 and Lamp1, electrophoresed in clearing solution using an X-CLARITY consistent with the existence of two discrete populations of Tissue Clearing System (Logos Biosystems) for 8 hours at tubules, most likely corresponding to S1 and S2 segments 1.5 A constant current, temperature,37°C, and 100 rpm (Figure 1B). pump speed. The sections were sectioned to 1-mm thickness, Expression of ELS proteins within cells in early PCT seg- incubated in approximately 88% Histodenz (D2158; Sigma- ments was observed predominantly in the subapical region Aldrich) solution in PBS (refractive index adjusted to 1.46) (Figure 2A). In contrast, in late segments the distribution of overnight, and mounted in the same solution. Imaging was ELS proteins was more diffuse. To confirm this impression, we performed on a TCS SP8 upright multiphoton microscope calculated the SD of signal intensity distributions within indi- equipped with an NA 1.0 253 water objective, HyD-RLD de- vidual PCT cells. Using this approach, we found that the SDs tectors (Leica Microsystems, Germany), and an InSight DS of signal intensities for Rab7 and Lamp1 showed bimodal dis- Dual NIR laser tuned to 1040 (Atto 565 excitation) and 1250 tributions and a close correlation (Figure 2B). In contrast, the nm (Alexa 647 excitation). The high-resolution data were pre- SD for megalin showed a unimodal distribution, and did not processed using Leica HyVolution2; all data were segmented correlate with either Rab7 or Lamp1. and single nephrons were semiautomatically traced using Ima- In summary, we found evidence of striking axial differences ris v9.0 for the length calculations. in expression levels and subcellular distribution of key ELS proteins along the PCT, independent of megalin expression, Statistical Analyses which supports the existence of two discrete functional seg- All data were analyzed in Graphpad Prism 7. Differences in ments (i.e., S1 and S2), and could point to major differences in RNA data were investigated using one-way ANOVA with ligand uptake capacity. Tukey’s multiple comparison test. Differences in fluorescence intensity were investigated with multiple t tests using the Uptake of Filtered Proteins by RME Occurs Almost Holm–Sidak method. Exclusively in S1 To investigate functional uptake of filtered molecules via endocytosis in the PCT, we used intravital multiphoton mi- RESULTS croscopy and intravenous injections of three different fluorescently labeled ligands: (1) lysozyme, a 14-kD LMWP that is Expression of Endolysosomal Proteins, but Not rapidly filtered, and is a classic ligand for megalin-mediated Megalin, Is Higher in S1 than in S2 RME23;(2) albumin, a large protein that is only partially First, we used EM to confirm the existence of morphologic filtered, and then taken up by RME via megalin and the differences in the ELS between early (S1) and late (S2) PCT FcRn receptor7;and(3) a small dextran (10 kD), which is segments in our mice. S1 cells were characterized by the pres- rapidly filtered and taken up by FPE. Commercially available ence of large subapical vacuoles, whereas S2 cells contained prelabeled forms of albumin and dextran were used. To label electron-dense lysosomes (Supplemental Figure 1). Immuno- lysozyme we used atto dyes, which are extremely bright and staining for the apical solute transporter NaPi2a revealed highly photo-stable,24 thus enabling high-resolution imaging higher expression in early than in late PCT segments (Supple- in vivo. Experiments with each ligand were repeated with two mental Figure 1), further supporting the notion that these are different labels to exclude a confounding effect of the fluoro- discrete transporting portions. To investigate axial patterns of phores on uptake. ELS protein expression along the PCT, we performed both At an injected amount of 10 mg, which should result in a immuno-staining of fixed tissue and RT-qPCR gene expres- plasma concentration .1003 physiologic levels,25 we ob- sion analysis of microdissected segments. Expression of stan- served that lysozyme disappeared rapidly from the vasculature dard markers confirmed the purity of isolated tissue, and that (within 30 minutes), consistent with glomerular filtration. S2 segments were discrete from S3 (Supplemental Figure 2). Regardless of the fluorescent label used, subsequent uptake Both early and late PCT segments displayed a highly de- of lysozyme occurred exclusively in S1 PCT segments, with veloped actin brush border (Figure 1A). Megalin expression no observable distal tubular wasting (Figure 3, A, D, and G).

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A Actin Megalin Rab7 Lamp1

G # # # Overview # Early PCT Late PCT

B Megalin Rab7 Lamp1 30 20 40

15 30 20

10 20

10 Number of ROIs Number of ROIs 5 Number of ROIs 10

0 0 0 60 70 80 90 100 80 90 100 110 120 60 70 80 90 100 Mean ROI Intensity Mean ROI Intensity Mean ROI Intensity

C Megalin Rab7 Lamp1 200 200 200 **

150 150 ** 150 *

100 100 100 % mRNA levels % mRNA levels % mRNA levels 50 50 50 (/GAPDH mRNA) (/GAPDH mRNA) (/GAPDH mRNA)

0 0 0 S1 S2 S3 S1 S2 S3 S1 S2 S3

2700 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2696–2712, 2018 www.jasn.org BASIC RESEARCH

To exclude the possibility that lysozyme is not representative injection of lysozyme. Using this approach, we confirmed of other LMWPs, we also injected fluorescently labeled that lysozyme uptake occurred in S1 segments, matching the b-lactoglobulin, which showed a similar uptake pattern high expression of Rab7 and Lamp1 in this region (Figure 4A). (Supplemental Figure 4). Organic anion transport is a spe- In contrast, in spite of clear evidence of megalin expression, we cialized function of S2 and can be measured using MCB, did not observe lysozyme signal in late segments. To exclude which forms a fluorescent adduct with intracellular the possibility that exogenous injected LMWPs are handled thiols.26 We observed an increased transport of MCB in ly- differently from endogenous ligands, we performed immuno- sozyme-negative PCT segments (Supplemental Figure 4), staining for endogenous retinol-binding protein (RBP4), consistent with an S2 identity. The identity of S1 and S2 which localized exclusively to early S1 segments (Figure 4B, PCT segments was also confirmed by their characteristic au- Supplemental Figure 4). Thus, the uptake pattern of LMWPs tofluorescence patterns (Supplemental Figure 4).27 matches the expression of ELS machinery, rather than that of Albumin was injected at an amount of 500 mg, on the basis megalin. of previous studies suggesting this is sufficient to observe PCT uptake in vivo in rodents.28 As expected, albumin remained S1 Proximal Tubule Segments Have a Very High visible in the vasculature far longer than lysozyme, consistent Capacity for Lysozyme Uptake with a slow rate of filtration. Uptake of albumin also occurred Because the uptake of lysozyme is apparently highly efficient in predominantly in S1 segments, with very little S2 uptake ob- S1 PCT segments, we considered that significant S2 uptake served (Figure 3, B, E, and H). To exclude the possibility that might only occur under conditions when S1 uptake is satu- the uptake in S1 reflected free dye cleaved in the circulation, rated. To investigate this, we injected a 103 higher dose of we performed injections of the dye alone, which only labeled lysozyme (100 mg), which rapidly disappeared from revealed a very low level of uptake (Supplemental Figure 5). the vasculature, suggesting no rate-limiting effect of glomer- Despite the relatively slow rate of albumin filtration, fluores- ular filtration. At this high amount we again observed reab- cence signal was clearly observed in distal tubule lumens, sorption predominantly in S1 segments (Figure 5, A and B), consistent with wasting of either labeled albumin or cleaved and although some lysozyme wasting was observed in more free dye. We were unable to detect evidence of intact fluores- distal nephron segments, uptake in S2 remained minimal. Be- cently labeled albumin on gel electrophoresis of urine samples cause of practical issues with saturation of fluorescence sig- collected from injected mice. However, by Coomassie stain nals, we were not able to use injections of labeled lysozyme both albumin-injected and control mice displayed bands con- .100 mg. Because we established that 300 mg was the maxi- sistent with urinary albumin excretion (Supplemental Figure mum dose that the animals could tolerate without showing an 5), suggesting that the uptake capacity in vivo is close to the adverse reaction, to further increase lysozyme delivery we filtered load. injected a combination of 290 mg unlabeled and 10 mg labeled. Uptake of 10-kD dextran was found to be far less efficient Even at this very high amount, uptake levels in S1 and S2 were than that of proteins, with prominent distal tubular wasting, similar to injecting 10 mg alone (Figure 5, C and D), and high- and despite rapid filtration a relatively high amount of 300 mg resolution confocal imaging in fixed tissue confirmed minimal was required to clearly visualize uptake in PCT cells. In stark lysozyme signal in S2 (Figure 5E). contrast to the proteins, reabsorption of dextran occurred in In summary, our findings suggest that the S1 PCT segment S1 and S2 segments at a similar level (Figure 3, C, F, and I), has a very high capacity for LMWP reabsorption that is way suggesting that FPE occurs in both. beyond the normal filtered load, but that very little uptake occurs in S2 segments even under saturating conditions. Uptake of Lysozyme in the Proximal Tubule Matches the Expression of Endolysosomal Proteins Three-Dimensional Imaging of Solute Uptake in the To further investigate the anatomic relationship between Kidney Cortex Using Tissue Clearing LMWP uptake and the expression of ELS proteins in the A major constraint of intravital kidney microscopy is that the PCT, we perfusion fixed kidneys from mice 1 hour post depth of imaging is limited to the very outer cortex. We there-

Figure 1. Expression of endolysosomal proteins is higher in early segments of the proximal tubule. (A) Immuno-staining of mouse kidney cortex revealed that all PCT segments displayed a highly developed actin brush border, and a similar expression level of megalin. In contrast, expression of Rab7 and Lamp1 was markedly higher in early (#) PCT segments than late (*). The identity of early segments was confirmed by their emergence from glomeruli (G). Scale bars, 50 mm in the top panel, and 10 mm in the lower panels. (B) To quantitatively analyze signal intensities, regions of interest (ROIs) were drawn around early or late sections of PCTs (in a total of three large field images), and mean intensity histograms for each segment are depicted. The histograms for Rab7 and Lamp1 expression showed bimodal distributions, corresponding to early and late segments, whereas megalin expression showed a unimodal distribution. (C) Gene expression analysis by RT-qPCR confirmed that megalin expression was similar in all proximal tubule segments. In contrast, Rab7 and Lamp1 expression decreased from S1 to S3 (*P,0.05, **P,0.01).

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A Rab7 Lamp1 Megalin

Early PCT Late PCT Early PCT Late PCT Early PCT Late PCT Tubular cross-section Inlay

8 8 8 8 4 4 6 6 6 6 3 3 4 4 4 4 2 2 2 2 2 2 1 1 Cumulative Intensity (AU) BL Ap 010101010101 Normalized Normalized Normalized Normalized Normalized Normalized Distance (AU) Distance (AU) Distance (AU) Distance (AU) Distance (AU) Distance (AU)

40 40

35 35

30 30

25 25 Rab7 Intensity SD Megalin Intensity SD 20 20

20 25 30 35 40 20 25 30 35 40 Lamp1 Intensity SD Lamp1 Intensity SD

Figure 2. Expression patterns of endolysosomal proteins within cells differ between early and late segments of the proximal tubule. (A) Analysis of signal intensity across PCTs cut in cross-section revealed that expression of the endolysosomal proteins Rab7 and Lamp1 was concentrated predominantly in the subapical region of cells in early segments, but was relatively uniform across cells in later segments. In contrast, megalin was expressed in the apical brush border region of all PCT segments. Scale bars, 10 mm. The plots (bottom panels) depict the cumulative intensity profiles for the segments drawn in the top panels. The segments have different lengths and were therefore normalized to arbitrary units (AU), with 0 being the baso-lateral side of the cell (BL), and 1 the apex (Ap). (B) The SD of the Lamp1 signal within regions of interest drawn around sections of PCTs correlated closely with that of Rab7 (R=0.93; P,0.001), and two distinct clusters were observed, corresponding to early (solid line) and late (dashed line) segments, respectively. In contrast, no positive correlation was observed between the SD of the Lamp1 and megalin signals (R=20.47; P,0.001). fore developed a new protocol, whereby upon completion of fluorescence signal from ligands taken up by PCTs. We were intravital experiments the very same kidneys were fixed and then able to image solute uptake at single-organelle resolution chemically cleared to increase transparency, while retaining the throughout the full thickness of the cortex in three dimensions,

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Lysozyme 15 kD Albumin 66 kD Dextran 10 kD

A Atto B Alexa C Cascade 565 488 Blue

# #

D E F Atto # Alexa Cascade 565 488 Blue #

# #

G H I Atto Alexa Alexa 647N 680 647

# #

Figure 3. Intravital microscopy reveals different uptake patterns for proteins and dextrans in the proximal tubule. Real-time intravital multiphoton imaging of solute handling in the kidney revealed that the proteins lysozyme (A, D, and G) and albumin (B, E, and H), which are both substrates for RME, were predominantly reabsorbed in S1 (#) PCTs. In contrast, dextran (10 kD) (C, F, and I), a marker for FPE, was taken up in both S1 and S2 (*) segments. Albumin was visible in the vasculature far longer than the smaller molecules (arrowheads), consistent with a slower rate of filtration. Luminal wasting of albumin and dextran in distal tubules was observed (arrows), but not lysozyme. Two different fluorescent markers were used for each solute. Images were acquired 30–40 minutes postinjection. Scale bars, 50 mm. at depths approximately 103 the normal limit of intravital longer visible, suggesting a step change in function at the S1/S2 microscopy (Figure 6). interface. Quantitative analysis revealed that the length of Using this approach, we could confirm that the basic pattern lysozyme uptake ranged from 600 to 1100 mmwithinasingle of solute uptake observed in superficial nephrons held true kidney, whereas dextran uptake length ranged from 900 to throughout the cortex; namely, lysozyme uptake was localized 2600 mm (Figure 7, D and E). After a 103 larger lysozyme exclusively to early PCT segments, whereas dextran was also injection (10 mg labeled plus 290 mg unlabeled), the histogram reabsorbed in later segments (Figure 7, A–C, Supplemental uptake length was only slightly right shifted, providing further Material). Moreover, detailed imaging of the uptake pattern evidence against a substantial increase in S2 uptake (Figure 8). of lysozyme along individually segmented nephrons typically In addition to variable uptake length of solutes, individ- revealed a distinct cut-off region beyond which signal was no ually segmented PCTs displayed striking heterogeneity in

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A Exogenous Lysozyme

Rab7 Lysozyme (10  g injected) Overlay

Lamp1 Lysozyme (10  g injected) Overlay

G #

Megalin Lysozyme (10  g injected) Overlay

B Endogenous Retinol-binding protein

Lamp1 eRBP4 Overlay

Figure 4. Proteinuptakeoccursinregionsofproximaltubules with high expression of endolysosomal proteins. (A) One hour post intravenous injection of fluorescently labeled lysozyme, kidneys were perfusion fixed and stained for endolysosomal markers and megalin. Lysozyme (magenta in overlay) was visible in the subapical region of early (#) PCT cells expressing high levels of Rab7 and Lamp1. No uptake was detected in late (*) PCT segments, where expression levels of these markers were low, in spite of a high expression of megalin. Early segments could be observed directly leaving the glomerulus (G), thus confirming their identity. (B) Immuno-staining for the endogenous protein retinol-binding protein (RBP4, magenta in overlay) also revealed an early S1 localization. Scale bars, 10 mm.

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A Lysozyme (10  g) B Lysozyme (100 g)

# #

C Lysozyme (10+290  g) D Mean Lysozyme fluorescence 1000

800 10 g 600 10+290 g

# 400

200 # 75 Fluorescence (AU) 50 #

0 S1 S2

E Lysozyme (10  g) Lysozyme (10+290 g) S1 S2 S1 S2

Figure 5. The S1 segment of the proximal tubule has a very high capacity for protein uptake. (A–D) Intravital imaging of lysozyme uptake in the PCT in vivo. (A) After intravenous injection of 10 mgofﬂuorescently labeled lysozyme, uptake was only observed in S1

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A B In vivo

100 m

G G

1000 m

C Cleared fixed tissue G G

G G

G G 1500

850 600

Figure 6. Tissue clearing allows high resolution imaging of lysozyme uptake in deep proximal tubules. (A) Example image of lysozyme uptake in superficial PCTs, 35 mm below the capsule, acquired using intravital microscopy. (B) Overview of a 150038503600-mm section of the same kidney after fixation and tissue clearing, showing lysozyme uptake in PCTs throughout the entire thickness of the cortex. The dotted line denotes the typical depth of imaging limit for intravital microscopy (100 mm). (C) High-resolution imaging of a single glomerulus and early PCT segment from (B), approximately 1000 mm below the organ surface, showing single endosomes containing lysozyme. Scale bars, 20 mm in (A and C), and 100 mm in (B). G, glomeruli. three-dimensional morphology, with some being highly con- additional information compared with standard intravital im- voluted,whereasotherswererelativelystraight,leading tomajor aging approaches. differences in space-occupying volume (Figure 8, Supplemental Material). PCTs characterized morphologically by an elonga- tion in the cortico-medullary axis of the space-occupying DISCUSSION volume were typically found in deeper regions of the cortex, whereas PCTs occupying a more symmetrical space were Reabsorption of filtered macromolecules by endocytosis is typically located superficially. a major function of the PCT, and impairment of this process In summary, using tissue clearing it is possible to image is the hallmark of the many forms of renal disorders. More- the uptake of different solutes at high resolution throughout over, endocytosis provides an entry route for toxins that the entire thickness of the cortex, which provides substantial cause kidney damage. Using fluorescently labeled ligands

segments (#). (B) Uptake remained predominantly in S1 (#) after a 103 higher injection. At this dose some luminal wasting was observed, but uptake in S2 PCT cells (*) remained low. (C and D) At an even higher dose of lysozyme (10 mg labeled+290 mg unlabeled), uptake patterns in S1 (#) and S2 (*) were similar to the 10-mg injection. Data depicted are mean fluorescence intensities within regions of interest in the apical vesicles of PCT cells (insert in (C)). The intensity values in S2 segments predominantly reflect nonspecific background fluorescence signals. AU, arbitrary units. (E) High-resolution confocal microscopy of fixed kidney specimens 1 hour post injection of lysozyme confirmed that uptake was predominantly confined to S1, with only a small level of uptake detectable in S2 at 300 mg. Scale bars, 50 mmin(A–C), and 5 mmin(E).

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B D Lysozyme-Atto565 a b c 6 4

(counts) 2 Nephron number

500 1000 1500 2000 2500 3000 Lysozyme uptake length (m)

C E Lysozyme-Atto565 and Dextran-AlexaFluor647

a b c 3 2

(counts) 1 Nephron number

500 1000 1500 2000 2500 3000 Dextran uptake length ( m)

Figure 7. Quantiﬁcation of PCT uptake lengths in cleared kidney tissue reveals differences between lysozyme and dextran. (A) Combined imaging of lysozyme (red) and dextran (white) revealed that uptake patterns in PCTs were markedly different throughout the entire thickness of the kidney cortex. (B and C) High-resolution imaging of three individually segmented PCTs from (A), showing that uptake of lysozyme typically ends abruptly, but that uptake of dextran continues beyond this point (* denotes the position of the glomerulus). (D and E) Histograms of PCT uptake lengths for both lysozyme and dextran revealed considerable heterogeneity within the same kidney (n=18 tubules). Scale bars, 200 mm in (A), and 100 mmin(BandC).

and novel imaging approaches we have discovered evidence this segment (Figure 9). In contrast, uptake of FPE substrates of major axial differences in PCT ligand uptake and ELS occurred along the entire PCT. Moreover, using tissue clearing, function. We found that, although megalin is expressed we showed that these basic patterns are preserved throughout along the PCT, uptake of both albumin and LMWPs by the renal cortex, but that individual PCTs are highly heteroge- RME occurred almost exclusively in S1, which closely neous in terms of ligand uptake length and three-dimensional matched a much higher expression of ELS machinery in morphology. These ﬁndings have important implications for

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AB a bcd

e f g a

b e c d

h C 20 g f h 10 (counts) Nephron number

400 800 1200 1600 2000 Lysozyme uptake length (m) D

1 hd2 3 a

Bounding box 1 2 3

Size (m) 238h 700w 125d 133h 258w 184d 202h 255w 168d

Volume ( m3) 20.8 x 106 6.3 x 106 8.7 x 106

Segmented Lysozyme-Atto565 123

Length (m) 1160 758 781

Mean diameter ( m) 44 36 31

Volume ( m3) 1.8 x 106 0.8 x 106 0.8 x 106

Figure 8. PCTs show striking heterogeneity in three-dimensional structure. (A) Overview image of a kidney section showing uptake of lysozyme in PCTs after a high-dose injection (300 mg). (B) Segmentation of individual PCTs from superficial (a–c), intermediate (d–f), and deep (g and h) cortical regions from (A) revealed evidence of considerable heterogeneity in three-dimensional structure. Highly convoluted tubules were typically found in superficial regions, whereas more elongated tubules were located in deeper regions. (C) Histogram of lysozyme uptake length in PCTs within the same kidney (n=61 tubules). (D) Quantitative morphometrics of three segmented PCTs from (A) demonstrating the effect of three-dimensional structure on the space-occupying volume (bounding box). Scale bars, 200 mm. h, height, w, width, d, depth. understanding integrated structure and function of the PCT differences in function between the subsegments. Consistent in vivo. with this, older micropuncture studies revealed that the bulk The PCT represents a highly specialized transporting epi- of fluid reabsorption postfiltration occurs in S1.31 Recent thelium, which is responsible for reabsorbing and studies have also reported variation in transcriptomes32 and transporting a range of different solutes. Axial differences in metabolic autofluorescence signals along the PCT.33 However, expression levels of sodium-coupled transporters and organic although differences in the ultrastructure of the ELS between anion/cation transporters are well recognized,29,30 suggesting S1 and S2 cells have been described,13 and could be observed in

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Pars convoluta S2

Pars recta

S1 S3

LMWPs Early endosome Lysosome in S1 Actin Late endosome Lysosome in S2 Megalin Recycling endosome Nucleus

Figure 9. Proximal tubules display axial differences in ELS structure and function. The majority of uptake of filteredLMWPsoccursin the cells of the S1 segment of the proximal tubule, which are specialized to perform this task and express high levels of ELS proteins. Relatively little LMWP uptake occurs in S2 cells, which accordingly display a much lower expression of endolysosomal proteins. our mice by EM, these are fairly subtle. Both segments have a unclear, but possible explanations from our data include a de- highly developed apical brush border and express megalin, creased cubilin-to-megalin ratio and a decreased expression of and the vast majority of studies of endocytosis have considered the adaptor protein Dab2. Of note, a recent study has suggested the PCT as a single entity.32 Therefore, it was previously un- that knockdown of Dab2 in PCT cells leads to a decrease in clear whether there are significant axial differences in ELS endocytosis and alterations in Rab5 and Rab7 expression.37 At function, and to what extent RME and FPE play discrete roles present, we can only speculate as to the role of megalin in S2 in ligand uptake. cells, but it might be important in facilitating the trafficking of In this study, we used lysozyme as an archetypal LMWP, transporters to and from the apical membrane.38 because it is an endogenous plasma protein that has a high Our experiments suggest that the S1 segment has a very glomerular-sieving coefficient,34 and is an established ligand high capacity to reabsorb LMWPs, far exceeding the normal for megalin.23 To visualize uptake at high resolution in vivo we filtered load, perhaps to prevent urinary wasting when plasma labeled lysozyme with very bright and photo-stable fluoro- levels and/or filtration rapidly increase. In contrast, in spite of a phores.24 We found that uptake of lysozyme and albumin much lower glomerular-sieving coefficient, we observed evi- occurred almost exclusively in S1 segments. Although it is dence of albumin wasting, which is in agreement with previous somewhat intuitive that uptake of filtered molecules would studies suggesting that the maximal capacity for albumin re- be higher in the first segment immediately after the glomeru- absorption is close to the normal filtered load.34 Because fil- lus, the magnitude of the S1/S2 differences was surprising. tered proteins have to pass through the dense apical brush Moreover, S2 uptake did not increase substantially even when border to come into contact with membrane invaginations S1 uptake was saturated and the delivery of filtered proteins was in PCT cells and undergo internalization, size and charge increased. These observations point to a step change in func- could play rate-limiting roles in determining the overall effi- tion between two discrete and specialized segments, a concept ciency of the uptake process.39 The uptake of dextran in PCTs that was further reinforced by a bimodal distribution of key was considerably lower than that of proteins, consistent with ELS proteins. Previous studies have described higher expres- previous experiments using radio-labeled ligands in rats,40 sion of lysosomal proteases in S1 segments in rats35 and a low suggesting that RME is a far more efficient uptake process uptake of lysozyme in perfused S2 segments isolated from rab- than FPE in vivo. In contrast to proteins, we found clear evi- bits,36 suggesting these differences are not unique to mice. dence of dextran uptake in both S1 and S2 segments, suggest- Given that both S1 and S2 segments express megalin, the ing that capacity for FPE is similar along the PCT. However, reason(s) for the low uptake capacity in S2 are currently because most fluid reabsorption occurs in S1, the luminal

J Am Soc Nephrol 29: 2696–2712, 2018 Proximal Tubule Endocytosis In Vivo 2709 BASIC RESEARCH www.jasn.org concentration of dextran may be increased by S2, which could structure and function. Elegant previous attempts to study also contribute to the high uptake in this region. three-dimensional nephron morphology have relied on re- Our findings have several important implications for un- construction from serial two-dimensional images,49 which is derstanding kidney diseases. First, because LMWP uptake oc- demonstrably possible, but considerably more labor intensive. curs predominantly in S1, by extrapolation it is likely that Our study hasseveral potential limitations. First, the process patients with tubular proteinuria have damage in this segment. of labeling solutes with fluorescent molecules could alter their We did not observe evidence of compensatory uptake in S2 uptake in the kidney.50 Second, although we have studied when S1 uptake was acutely saturated, but it is possible that several ligands for RME, including both endogenous and ex- some axial plasticity might occur in disease states where S1 ogenous proteins, our findings might not be representative of function is chronically impaired,41 perhaps initiated by all LMWPs, because size and charge can play important roles binding to megalin in S2. A very recent study suggested that in the efficiency of uptake. Moreover, we cannot currently knockdown of megalin in S1 and S2 leads to a downstream explain why megalin and cubilin are expressed in S2. Third, increase in S3 uptake, but this has only a minimal effect on tissue clearing can lead to swelling of the kidney, which could protein excretion.42 Second, the very high capacity for LMWP affect the accuracy of morphologic measurements. Finally, our uptake in S1 reinforces the need for biomarkers that can detect experiments were performed in male mice and might not be PCT damage at an early stage, before the appearance of pro- applicable to females or other species. teinuria.43 Finally, axial differences in uptake could determine In summary, we have found new evidence of major axial various topographic patterns of damage observed in response differences in PCT ligand uptake and ELS function, which have to certain toxins, such as endotoxin,27 light chains,44 contrast important implications for understanding topographic patterns of agents, and volume expanders.45 kidney diseases, the origins of proteinuria, and how individual A major historical problem for intravital kidney microscopy cell function relates to the complex three-dimensional archi- has been the limited depth of imaging possible. We showed tecture of the organ. previously that this can be increased by using far red excitation and probes,20 but even with this improvement the majority of cortex remains beyond view. Tissue-clearing techniques substantially increase transparency and have been used recently in ACKNOWLEDGMENTS the kidney to assess total glomerular number and structural damage in PCTs from cisplatin.46,47 The major practical chal- The authors gratefully acknowledge the assistance of Huguette De- lenge with such approaches is to retain fluorescence signals baix, Andres Kaech, and Monique Carrel. and directly relate structural information to functional read- A.M.H. is supported by The Swiss National Centre for Competence outs. We have developed a protocol whereby the fluorescence in Research Kidney Control of Homeostasis and by a Swiss National signal from labeled solutes taken up by the PCT is still pre- Science Foundation project grant (31003A 166507). O.D. is supported served postclearing. Individual nephrons can then be by a Swiss National Science Foundation project grant (31003A segmented to quantitatively investigate how cellular ligand 169850). The authors also acknowledge support from The Clinical “ ” uptake relates to the three-dimensional architecture of the Research Priority Program Molecular Imaging Network Zurich, organ. Moreover, findings can be directly crossreferenced to and The Zurich Centre for Integrative Human Physiology. dynamic observations made from intravital imaging in the C.D.S., D.H., O.D., U.Z., and A.M.H. designed the study. C.D.S., very same organ. M.P., E.P., A.G., N.T., S.G., and M.B. carried out experiments. C.D.S., Using this approach, we confirmed that uptake patterns of M.P., E.P., D.H., A.G., N.T., and S.G. analyzed the data. A.M.H. drafted fi protein and dextran are markedly different along nephrons the manuscript. All authors approved the nal version of the throughout the entire cortex. However, individual PCTs dis- manuscript. played striking heterogeneity in solute uptake length and three- dimensional morphology, demonstrating that substantial DISCLOSURES additional information can be acquired with this methodology. None. We observed a relatively sharp cut-off point between protein and dextran uptake, probably corresponding to the interface of fi S1/S2, but in the absence of de nitive anatomic markers we REFERENCES cannot be sure of this. Nevertheless, by continuously following individual PCTs from the glomerulus we could show that in 1. Sirac C, Bridoux F, Essig M, Devuyst O, Touchard G, Cogné M: Toward most cases protein uptake is limited to the initial 1 mm or so. understanding renal Fanconi syndrome: Step by step advances through Uptake distance was typically greater in deeper than in super- experimental models. Contrib Nephrol 169: 247–261, 2011 ficial PCTs, consistent with previous micropuncture studies 2. 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