Spatiotemporal organisation of protein processing in the kidney

Abstract

The kidney regulates plasma protein levels by eliminating them from the circulation. Proteins filtered by glomeruli are endocytosed and degraded in the proximal tubule and defects in this process result in tubular proteinuria, an important clinical biomarker. However, the spatiotemporal organization of renal protein metabolism in vivo was previously unclear. Here, using functional probes and intravital microscopy, we track the fate of filtered proteins in real time in living mice, and map specialized processing to tubular structures with singular value decomposition analysis and three-dimensional electron microscopy. We reveal that degradation of proteins requires sequential, coordinated activity of distinct tubular sub-segments, each adapted to specific tasks. Moreover, we leverage this approach to pinpoint the nature of endo-lysosomal disorders in disease models, and show that compensatory uptake in later regions of the proximal tubule limits urinary protein loss. This means that measurement of proteinuria likely underestimates severity of endocytotic defects in patients.

Fig. 1. Filtered proteins are endocytosed, degraded and released from S1.

a Early (S1) and late (S2) segments of the proximal tubule (PT) are distinguished in vivo by autofluorescence signals excited at 850 nm (representative image of three independent experiments). b Fluorescence unquenching was used to detect protein degradation (#) in S1: signal increase was proportional to labeling density (*=uptake phase). An intermediate labeling density (arrowed) was used for subsequent experiments. Data were derived from regions of interest (ROIs) drawn around whole tubular segments (n = 3 mice). c–l Uptake and degradation of Lactoglobulin^(4XAt532) and β2-microglobulin^At532 along the PT. c, h 10 min after injection, filtration and S1 uptake are complete. d, i Degradation then commences in S1 and a delayed fluorescence signal subsequently appeared in S2 segments devoid of initial uptake; these phenomena were severely dampened by a lysosomal cathepsin inhibitor (e64) (e, f and j, k). g, l ROIs were drawn around tubular segments and the plots depict fluorescence intensity over time (mean value ± SEM; n = 3 mice per group). Example single plane images are depicted from the indicated time points (representative of three independent experiments). m A delayed signal increase also occurred in the lumen of the distal tubule (DT), with kinetics reflecting S1 degradation (mean value ± SEM; n = 3 mice per group). n Summary diagram: filtered proteins are degraded in S1 cells, with release of fragments, some of which undergo a second wave of reabsorption in S2. Scale bars = 20 μm.

Fig. 2. Small peptides bypass S1 and are reabsorbed in S2.

a Enzymatically-digested lactoglobulin peptide fragments of different sizes were labeled with Atto 532 and purified, and their renal handling post intravenous injection was investigated with intravital imaging (mAU milli-absorbance unit). b The small peptide ALK^At532 (331 Da) was reabsorbed in S2 (representative of three independent experiments). Scale bar = 20 μm. c Conversely, a larger peptide VYVEELK^At532PTPEGDLEILLQK (2313 Da) triggered S1 uptake (representative of three independent experiments). Scale bars = 20 μm. d, e ROIs were drawn around tubular segments and the plots depict fluorescence intensity over time post injection (mean value ± SEM; n = 3 mice per group). f, g Subsequent injection of intact lysozyme^Atto647N showed a clearly distinct uptake pattern from the small peptide, but not the larger species (representative of three independent experiments; arrowheads = S1). h, i Single experiments showing uptake intensities along the proximal tubule 20 min post injection, with individual ROIs ordered according to lysozyme^Atto647N signal intensity (from highest to lowest). j–l Overview of the kidney on cross-section post fixation showing uptake of lysozyme^At647N and ALK^At532 in early and late segments of the proximal tubule, respectively. Scale bars = 500 μm. Single example image planes are depicted (representative of three independent experiments). m Higher resolution image of cortical region showing lysozyme^At647N in S1 segments leaving the glomerulus (G), and ALK^At532 in segments staining positive for the S2 marker OAT1. Nuclei were labeled with Hoechst (blue). Scale bar = 20 μm. Single example image planes are depicted (representative of three independent experiments).

Fig. 3. Expression of genes related to protein and peptide handling along the mouse proximal tubule.

a The multi-ligand receptors megalin and cubilin, the endocytotic adaptor protein Dab2, and protein degrading cathepsins are all highly expressed in S1. b In contrast, peptidases are more abundant in S2, and the expression of the major renal peptide transporter (PEPT2) increases progressively from S1 to S3. Data depict mRNA levels (TPM values for RNA-Seq) in micro-dissected nephron segments and were derived from ref. . c Proposed schematic, based on protein/peptide uptake experiments and gene expression data, depicting the spatial arrangement of protein metabolism along the PT.

Fig. 4. Three-dimensional reconstruction of the S1 endo-lysosomal system.

a 2-D electron microscopy (EM) overview images of S1 and S2 segments of the proximal tubule, from stitched tile sets (boxes), showing large apical vacuoles (LAVs, arrowheads) only in S1. Lysosomes (Lys, orange circles) are more homogeneous than LAVs and appear electron dense in S2. Images acquired from a single animals. Scale bar: 10 μm. b, c Reconstruction of the S1 endo-lysosomal system (ELS) in 3-D with FIB-SEM allowed segmentation of different structures (denoted with colors). Deep invaginations of the apical membrane (gold volume) bring the primary urine into close proximity to early endosomes (EEs). Recycling tubules exist in a network between EEs/LAVs and the apical surface. LAVs have a complex, irregular structure, whereas adjacent lysosomes are smaller and more spherical. Vesicles of the trans-Golgi network (TGN) are observed in the basolateral region of the cell. Images depicted were acquired from a single animal. d Fixed kidney tissue sections derived from mice injected with lysozyme^At565 1 h prior to fixation were stained for established structural markers: actin (apical membrane brush-border), megalin (endocytotic receptor), rab11 (recycling tubules), lamp1 (LAVs/lysosomes), and mannose 6-phosphate receptor (M6PR [TGN]). Single example image planes are depicted (representative of three independent experiments). Scale bars: 10 μm. e Line scan analysis revealed distinctive intracellular distributions of the signals, which displayed substantial spatial overlap. The spatial shape is depicted in the position axes (0 = apical side, 1 = basolateral). Amplitudes of markers are from cumulative intensities normalized to 1 (n ≥ 2 mice).

Fig. 5. Derivation of protein processing kinetics in S1 cells in vivo.

a High resolution single plane images of S1 cells post intravenous injection of lactoglobulin^At565 (representative of three independent experiments). b Line scan ROIs were drawn across cells using sum images (representative of three independent experiments). c Representative raw intensity waveform showing normalized evolution of intracellular fluorescence distribution over time (0 = apical side, 1 = basolateral). d Reconstruction from three independent base vectors derived from SVD analysis. e–g The spatial shape and time evolution of the individual base vectors is depicted (0 = apical side, 1 = basolateral), under control conditions (f), and with a cathepsin inhibitor (e64) (g). Cross-referencing to the 3-D reconstruction from FIB-SEM suggests that base vectors localize to major endo-lysosomal structures (LAV large apical vacuoles, EE early endosomes, Lys lysosomes). h Plots depicting kinetic changes in amplitude of SVD1-3 (mean value ± SEM; n = 3 mice per group), and showing loss of lysosomal degradation and fragment release with the cathepsin inhibitor (°°°p < 0.001). i, j SVD1 displays biphasic kinetics. i Post injection of a low-labeled lactoglobulin^(1xAt532), the first component of SVD1 was clearly visible, but not the second. j In contrast, post injection of a high-labeled lactoglobulin^(6xAt532), the second component of SVD1 was accentuated, whereas the amplitude of the first component and SVD2 were markedly attenuated, due the low fluorescence signal emitted from the protein pre-digestion. Following unquenching, a large increase in fluorescence signal was observed in small, punctate vesicles (arrowheads), which subsequently became more diffuse. Single plane images are shown at different time points. Data depicted in plots (mean value ± SEM) are from three experiments. k High resolution single plane images (representative of three independent experiments) depicting unquenching of high-labeled lactoglobulin^(6xAt532) in small punctate vesicles (upper panel, arrow), which resemble the structure of lysosomes in proximal tubules in fixed kidney tissue stained for cathepsin L (lower panel, arrowhead). Nuclei were labeled with Hoechst (blue). Scale bars = 10 μm.

Fig. 6. Alterations in endo-lysosomal function and compensatory remodeling in disease.

a Plots depict fluorescence intensity in S1 segments post injection of lactoglobulin^At565. Uptake was decreased in OCRL knockout (KO) mice compared to wild type (WT) (mean value ± SEM; time × genotype ***p < 0.001; n = 3 mice per group). b Normalized raw intensity waveform derived from line scan ROIs showing intracellular evolution of fluorescence in KO mice (0 = apical, 1 = basolateral). Base vectors derived from SVD analysis in wild type (c) and KO (d). e Kinetics of SVD1-3 in WT and KO mice (mean value ± SEM; n = 3 mice per group). SVD2 shows a small delay in transition through EEs (^$$$p < 0.001). SVD3 (lysosomal protein degradation) also displays a small right shift (°°°p < 0.001). f, g Protein uptake length was increased along the proximal convoluted tubule (PCT) in KO mice. Single plane example images (representative of three independent experiments) are depicted 20 min post injection. Scale bars = 20 μm. Histogram depicts the fluorescence signal (mean value ± SD; n = 3 mice per group) in ROIs drawn around individual PCT segments, ordered from highest to lowest. Data depicted were from single plane images acquired 20 min post injection of lactoglobulin^At565. h, i Antibody staining for the late endosomal/lysosomal markers Rab7 and Lamp1. Example single plane images are depicted (representative of five independent experiments). Scale bars = 10 μm. j Intracellular signal distribution was assessed by the standard deviation (Std. Dev.). In WT mice, signal is condensed in apical vesicles in early PCT segments (high Std. Dev.), and more diffuse in late (low Std. Dev.). This axial pattern was lost in KO mice, resulting in a lack of linear correlation (R value: 0.89 in WT, 0.53 in KO; p < 0.05; n = 5 mice per group). k Summary diagram depicting axial redistribution of protein reabsorption along the PCT in KO mice; increased uptake in later segments can compensate for severe defects in endocytosis.