Mtor Regulates Endocytosis and Nutrient Transport in Proximal Tubular Cells

BASIC RESEARCH www.jasn.org mTOR Regulates Endocytosis and Nutrient Transport in Proximal Tubular Cells

† ‡ † Florian Grahammer,* Suresh K. Ramakrishnan, Markus M. Rinschen, Alexey A. Larionov, † | Maryam Syed, Hazim Khatib,§ Malte Roerden,§ Jörn Oliver Sass, ¶ Martin Helmstaedter,* Dorothea Osenberg,* Lucas Kühne,* Oliver Kretz,* Nicola Wanner,* Francois Jouret,** ‡ ††‡‡ † Thomas Benzing, Ferruh Artunc,§ Tobias B. Huber,* and Franziska Theilig

*Department of Medicine IV, Medical Center—University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany; †Institute of Anatomy, Department of Medicine, University of Fribourg, Fribourg, Switzerland; ‡Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; §Department of Medical IV, Sektion Nieren- und Hochdruckkrankheiten, University of Tübingen, Tübingen, Germany; |Bioanalytics and Biochemistry, Department of Natural Sciences, Bonn Rhein Sieg University of Applied Sciences, Rheinbach, Germany; ¶Division of Clinical Chemistry and Biochemistry and Children’s Research Centre, University Children’s Hospital Zürich, Zurich, Switzerland; **Groupe Interdisciplinaire de Génoprotéomique Appliquée, Cardiovascular Sciences, University of Liège, Liege, Belgium; and ††BIOSS, Centre for Biological Signalling Studies and ‡‡FRIAS, Freiburg Institute for Advanced Studies and ZBSA, Center for Biological System Analysis, Albert Ludwigs University of Freiburg, Freiburg, Germany

ABSTRACT Renal proximal tubular cells constantly recycle nutrients to ensure minimal loss of vital substrates into the urine. Although most of the transport mechanisms have been discovered at the molecular level, little is known about the factors regulating these processes. Here, we show that mTORC1 and mTORC2 specifically and synergistically regulate PTC endocytosis and transport processes. Using a conditional mouse genetic approach to disable nonredundant subunits of mTORC1, mTORC2, or both, we showed that mice lacking mTORC1 or mTORC1/mTORC2 but not mTORC2 alone develop a Fanconi-like syndrome of glucosuria, phosphaturia, aminoaciduria, low molecular weight proteinuria, and albuminuria. Interestingly, proteomics and phosphoproteomics of freshly isolated kidney cortex identified either reduced expression or loss of phosphorylation at critical residues of different classes of specific transport proteins. Functionally, this resulted in reduced nutrient transport and a profound perturbation of the endocytic machinery, despite preserved absolute expression of the main scavenger receptors, MEGALIN and CUBILIN. Our findings highlight a novel mTOR–dependent regulatory network for nutrient transport in renal proximal tubular cells.

J Am Soc Nephrol 28: 230–241, 2017. doi: 10.1681/ASN.2015111224

With the transition from water to land, mammalian affect several transport systems, leading to a Fanconi kidneys developed into a high–pressure filtration syndrome.5,6 In addition to aminoaciduria, system combining excess ultrafiltration with highly Received November 12, 2015. Accepted May 14, 2016. efficient tubular reabsorption to minimize nutrient and protein loss.1 The vast amount of the ultrafil- T.B.H. and F.T. contributed equally to this work. trate (approximately 60%–70%), including virtu- Published online ahead of print. Publication date available at ally all nutrients, is already being reabsorbed by the www.jasn.org. 2 first nephron segment, the proximal tubule (PT). Correspondence: Dr. Franziska Theilig, Department of Medi- Specific loss of PT amino acid or phosphate trans- cine, University of Fribourg, Route Albert-Gockel 1, 1700 Fri- fi bourg, Switzerland or Dr. Tobias B. Huber, Medical Center, port function is associated with genetically de ned University of Freiburg, Center of Medicine, Renal Division, disease entities, such as Hartnup disorder or hypo- Breisacherstrasse 66, 79106 Freiburg, Germany. Email: franziska. phosphatemic rickets.3,4 In contrast, disrupted en- [email protected] or [email protected] ergy metabolism or toxic injury can simultaneously Copyright © 2016 by the American Society of Nephrology

230 ISSN : 1046-6673/2801-230 J Am Soc Nephrol 28: 230–241, 2017 www.jasn.org BASIC RESEARCH glucosuria, and phosphaturia, Fanconi syndrome is charac- genes (Raptorfl/fl 3 Rictorfl/fl 3 Pax8rtTA 3 TetOCre;subse- D terized by low molecular weight proteinuria.7 This is caused quently termed RapRic Tubule)(Figure1,A–C). Protein abun- by dysfunction of receptor-mediated endocytosis, which is dance or reduced phosphorylation of known downstream targets mediated by the two scavenger receptors, MEGALIN and was used to assess efficiency of our knockout approach (Figure 1, CUBILIN, followed by clathrin-mediated internalization of D–F). In comparison with the respective control group, all exam- D the ligand-receptor complex.8 Fusion with early endosomes ined parameters were at least reduced by 70% (Rap Tubule: delivers the complex to the endosomal-lysosomal com- RAPTOR, 274.6%64.6%; p-S6P/S6P, 278.9%66.4%; P,0.05; D partment, leading to recycling or degradation of retrieved n=6; Ric Tubule:RICTOR,284.5%68.4%; p-NDRG1/ D proteins.8 The prominent endocytotic activity of the PT is NDRG1, 271.1%63.7%; P,0.05; n=6; RapRic Tubule:RAPTOR, important to retrieve vital plasma proteins and maintain 278.3%66.2%; RICTOR, 269.1%65.3%; p-S6P/S6P, 289.2% body homeostasis.9 Although individual components of this 64.7%; p-NDRG1/NDRG1, 271.7%69.8%; P,0.05; n=6) (Fig- system have been recently described, little is known on the ure 1, D–F). organ-specific regulation of these transport processes. Measurement of urinary glucose and phosphate excretion D The mammalian homolog of TOR1 (mTORC1) and in RapRic Tubule mice revealed 3.3- and fivefold increases in mTORC2 are intracellular multiprotein complexes regulating urinary losses of glucose and phosphate, respectively, in- tissue–specific cellular functions.10 The regulatory associated dicating impaired nutrient reabsorption (Figure 1H). In protein of mammalian target of rapamycin (RAPTOR) is im- addition, SDS-PAGE of urinary samples revealed a slight D portant to assemble mammalian target of rapamycin (mTOR) increase in albuminuria in Rap Tubule,whichwassignificant D complex 1, and its function can be blocked by rapamycin. In in RapRic Tubule (Figure 1, I and K). This observation was contrast, mTORC2, containing the rapamycin-insensitive confirmed by direct measurement of mouse albumin in uri- companion of mammalian target of rapamycin (RICTOR) nary samples (Figure 1L). Furthermore, an increase in low as a scaffolding protein, seems to be insensitive to short– molecular mass (LMW) proteinuria between 25 and 68 kD D term rapamycin treatment.11 mTORC1 integrates a wide va- could be detected in RapRic Tubule (+126%637%; P,0.05; riety of signals, including growth factors, amino acids, cellular n=5)aswellasasignificant increase in proteinuria at ap- D D energy content, and cellular stress, to regulate cellular growth proximately25kDinRap Tubule and RapRic Tubule animals D D and division as well as lipid and mitochondrial biogenesis.12,13 (Rap Tubule:+70%613%; RapRic Tubule:+44%66%; The wide range of mTORC1 phosphorylation targets is still P,0.05; n=5). In addition, a pronounced increase in urinary D incompletely defined but seems to vary in a cell-specific man- amino acid excretion was observed in Rap Tubule animals, and D ner. Conversely, mTORC2 seems to be activated by insulin and an even more pronounced increase was in RapRic Tubule an- D related pathways and controls several downstream AGC ki- imals but not in Ric Tubule mice (Figure 1M, Supplemental nases by phosphorylating their hydrophobic motif.14,15 Figure 2A). Amino acid plasma concentrations were similar Thereby, mTORC2 could play an important role in cell sur- in all experimental groups (Supplemental Figure 2B), vival and organization of the cytoskeleton. confirming a renal amino acid transport defect rather than Toinvestigate the effect of mTOR-controlled signaling in PT defects in hepatic or muscular metabolism. D D function, we generated inducible conditional Raptor, Rictor,or Compared with control mice, Rap Tubule and RapRic Tubule double-knockout mice in renal tubular cells along the nephron. animals showed significantly higher food and fluid intakes, which were associated with increased urinary volume and sodium excretion (Supplemental Table 1). No alterations D RESULTS were found in Ric Tubule animals. Together, these data show a significant mTORC1–dependent defect in tubular Loss of mTOR in PT Cells Results in a Fanconi-Like reabsorption of glucose, phosphate, amino acids, LMW pro- Phenotype teins, and albumin. Expression of the mTORC1 and mTORC2 complexes in the PT was verified by staining for mTOR, RAPTOR, S6K1, ribosomal mTOR Regulates PTC Endocytosis and Intracellular S6 protein (S6P), and N-Myc Downstream Regulated 1 Trafficking In Vivo (NDRG1) (Supplemental Figure 1). All components were Togain additional insights into the effects of gene knockout on strongly expressed in the S1 and S2 segments of the PT in either PT endocytosis, mice were injected with horseradish perox- overlay or close vicinity to MEGALIN. The proximal tubular S3 idase (taken up by fluid-phase endocytosis) and Alexa555- segment only showed minor signal intensity for mTOR, S6K1, coupled lactoglobulin (reabsorbed by receptor-mediated and S6P and was devoid of signals for RAPTOR and NDRG1. endocytosis) before perfusion fixation.16 Morphologic anal- To examine the effects of mTORC deletion on PT function, we ysis revealed a strong reduction of fluid-phase endocytosis in D D generated mice with doxycycline–inducible Pax8 promotor– Rap Tubule and RapRic Tubule animals, whereas no change was D driven tubular deletion of Raptor (Raptorfl/fl 3 Pax8rtTA 3 observed in Ric Tubule mice (Figure 2A). Similarly, confocal D TetOCre; subsequently termed Rap Tubule), Rictor (Rictorfl/fl 3 microscopy analysis showed a reduced receptor–mediated en- D D D Pax8rtTA 3 TetOCre; subsequently termed Ric Tubule), or both docytosis in Rap Tubule and RapRic Tubule mice in comparison

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Figure 1. Proximal tubular deletion of mTORC1 leads to a Fanconi like syndrome. (A–C) Schematic of recombination strategy and site of inducible Pax8rtTA 3 TetOCre–mediated (A) Raptor,(B)Rictor, and (C) double knockout within the tubular system. (D–F) Dem- D onstration of knockout efﬁcacy by Western blot of (D) RAPTOR and downstream target p-S6P/S6P in Rap Tubule, (E) RICTOR and the D downstream target p-NDRG1/NDRG1 in Ric Tubule, and (F) RAPTOR and RICTOR and downstream targets p-S6P/S6P and p-NDRG1/ DTubule 32 DTubule NDRG1inRapRic . (G) Urinary glucose (gluc) and phosphate (PO4 ) excretions in RapRic were increased. (H and I) Coomassie staining of SDS-PAGE prepared from urine samples loaded after normalization to urinary creatinine concentration from (H) D D D D Rap Tubule and (I) RapRic Tubule. (J) Urinary albumin excretion of Rap Tubule and RapRic Tubule. (K) Urinary neutral amino acid excretion D D D of Rap Tubule,RicTubule,andRapRicTubule. ACR, albumin-creatinine-ratio; Con, control; crea, creatinine. *P value versus control ,0.05; **P value versus control ,0.01.

232 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 230–241, 2017 www.jasn.org BASIC RESEARCH

Figure 2. Deletion of mTORC1 leads to a reduced fluid-phase as well as receptor-mediated endocytosis and blocks intracellular trafficking. (A) Fluid-phase and (B) receptor-mediated endocytosis is shown by the uptake of horseradish peroxidase or Alexa555- D coupled lactoglobulin (red stain), respectively, injected 5 minutes before perfusion-fixation. Reduced uptake is found in Rap Tubule and D D RapRic Tubule. (C) Cell by cell analysis of receptor-mediated endocytosis in Rap Tubule 3 mT/mG mice. Cre-positive cells (green stain) show reduced endocytic uptake of Alexa555-coupled lactoglobulin (red vesicular staining) in comparison with Cre-deficient cells. (D) D D D Quantification of Alexa555-coupled lactoglobulin of Rap Tubule,Ric Tubule, and RapRic Tubule normalized to the respective control mice. D D Significantly reduced uptake is shown for Rap Tubule and RapRic Tubule animals. (E) Assessment of endogenous albumin content in D D D Rap Tubule,Ric Tubule, and RapRic Tubule. Note the increased number and size of albumin-containing vesicles. In immunofluorescence images, kidney sections were counterstained with Alexa647 phalloidin (blue stain) to mark actin filaments of the BBM. Con, control. Scale bar, 20 mm. **P,0.01.

D D with respective control animals (Figure 2B). Introducing an mT/ Rap Tubule mice and even more RapRic Tubule mice showed D mG reporter allele into Rap Tubule animals revealed very few an accumulation of endogenous albumin with increases in ves- events of a mosaic Cre expression in PT cells. However, these icle size and number (Figure 2E). This is in agreement with few mosaic areas allowed a detailed cell to cell analysis of endo- reduced intracellular trafficking and protein processing. Be- cytosis. Cre–positive, Raptor–deficient cells in comparison with cause MEGALIN and CUBILIN are the main receptors for Cre–negative, Raptor–positive cells showed a highly impaired PT endocytosis, their expression pattern and abundance were receptor–mediated endocytosis as identified by reduced uptake analyzed. Immunoblotting and staining analyses revealed no of Alexa555 lactoglobulin (Figure 2C). To quantify receptor– change between groups in either subcellular localization or mediated endocytotic flux, fluorimetric measurements of expression level (Supplemental Figure 3). endocytosed Alexa555 lactoglobulin in tissue homogenates Taken together, these data show that mTOR function is were performed. Reduced endocytotic fluxes were observed in required for PT endocytosis and intracellular trafficking, D D Rap Tubule and RapRic Tubule animals, with no difference in whereas the abundance of the apical scavenger receptors D Ric Tubule mice (Figure 2D). Toexamine intracellular traffick- seemed unaffected. To analyze whether duration of kinase ing, we stained for cellular endogenous albumin distribution. deletion could play a role, we assessed histologic alterations in

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D D Rap Tubule and RapRic Tubule mice 3 months after induction. performed shRNA–mediated viral knockdown of the main D D In Rap Tubule and RapRic Tubule (much stronger) animals, the mTORC1–dependent translational regulator S6K1, RICTOR, renal cortex was reduced, which was mainly caused by shrink- or both genes together. Compared with mock transduction, all age of the convoluted part of the PT (Supplemental Figure virallytransducedOKCsshowedasigniﬁcant reduction in 4A). MEGALIN and CUBILIN expression levels remained un- their endocytosis rate, with the strongest decrease observed D D altered in Rap Tubule but were decreased in RapRic Tubule in S6K1 or S6K1/RICTOR shRNA knockdown (Supplemental (Supplemental Figure 4, B and C). We next analyzed the ex- Figure 5, C and D). mTORC1 and mTORC2 signaling showed pression pattern of the Na+/K+-ATPase (NKA), a basolateral no inﬂuence on lysosomal pH and enzyme activity (Supple- protein being expressed in a polarized fashion. Interestingly, mental Figure 5E). In addition, enzymatic activity of galac- D D in Rap Tubule and RapRic Tubule animals 3 months postinduc- tosidase, mannosidase, and a-glucuronidase remained tion, apical NKA–containing vesicles were observed, showing unchanged on viral knockdown of S6K1, RICTOR, or S6K1/ an apical mis-sorting of this protein. In addition, we RICTOR compared with mock transduction (data not discerned a striking reduction of basolateral NKA expression shown). Formation of clathrin-coated vesicles as a readout D in RapRic Tubule animals. These data show that apical scaven- of membrane formation was reduced in S6K1 and double D ger receptor expression remained intact in Rap Tubule mice, shRNA–transduced OKCs (Supplemental Figure 5, F and G). although signs of epithelial depolarization could be detected, This may affect the formation of endocytotic vesicles as de- D whereas PT cells dedifferentiated in RapRic Tubule animals, scribed in the electron microscopic analysis of the transgenic leading to reduced MEGALIN and CUBILIN expression. mouse models.

Loss of mTOR Results in Altered Apical Plasma Proteomic and Phosphoproteomic Analyses of Kidney Membrane Topology with a Reduced Reabsorption Cortex Identify a Putative mTOR–Dependent Surface In Vivo Transport Regulatory Protein Network Light and electron microscopy analyses were performed to To further elucidate the molecular causes for the observed correlate reduced PT function with changes in morphology. reduction in PT transport function, we performed detailed Periodic acid–Schiff staining on paraffin sections revealed re- proteomic and phosphoproteomic analyses of renal cortex in D duced brush border membrane (BBM) microvilli in all trans- Rap Tubule and respective control animals. Although overall D D genic knockout mice, which was most evident in Rap Tubule differences between control and Rap Tubule animals were D and RapRic Tubule compared with control animals (Figure rather subtle (Figure 4A), several amino acid transporters, 3A). The overall gross morphology, however, remained un- such as SLC6A19 (B0AT1), SLC7A7 (y+LAT1), and SLC3A2 changed as shown in methylene blue–stained semithin sec- (4F2hc), as well as proteins related to polarity and endocytosis tions (Figure 3B). Subcellular electron microscopy analysis of (e.g.,DOCK8,Bcl2-like1[BCL-xL], Ras–related protein D D PT S1 segments in Rap Tubule and RapRic Tubule mice showed Rab-10 [RAB10], and sorting nexin 8 [SNX8]) were reduced reduced microvilli length and endosomal vesicle formation (Figure 4B, Supplemental Table 3). Making use of the mosaic D compared with control animals as well as reduced numbers expression pattern of RAPTOR-deficient cells in Rap Tubule, D of mitochondria in RapRic Tubule animals (Figure 3C, Supple- we could confirm by immunohistochemistry the reduced ex- 0 + mental Table 2). Additionally, largely inflated early and late pression of B AT1, y LAT1, 4F2hc, DOCK8, and BCL-xL in D endosomes and Golgi cisternae were observed in Rap Tubule RAPTOR-deficient cells in comparison with wild-type cells D and RapRic Tubule mice. (Figure 5, A–E). This was corroborated by Western blot analysis from isolated cortical kidney membranes (Figure 5F), Reduced Endocytosis Is Caused by Diminished which confirmed our proteomic findings. Formation of Clathrin-Coated Vesicles Interestingly, BBM-bound enzymes, such as 59 nucleotid- To gain additional mechanistic insights, we analyzed endocy- ase, glutamylaminopeptidase, and angiotensin-converting tosis in the well established opossum kidney proximal tu- enzyme 2, or mitochondrial proteins, such as cytochrome bule cell (OKC) line.17 OKCs were incubated with 100 nM P450 4A12A and cytochrome b-c1 complex subunit 8, were D rapamycin (mTORC1 inhibitor), 100 nM PP242 (mTORC1+2 upregulated, likely in a compensatory fashion in Rap Tubule mice inhibitor), or 250 nM torin (mTORC1+2 inhibitor) for 3 days (Figure 4B). We carried out gene ontology enrichment of the or 3 or 10 mM PF-4708671 (S6K1 inhibitor) overnight before changed protein population compared with the unchanged endocytosis assays followed by flow cytometric analysis of en- protein population. Several nutrient transport pathways in docytosed Alexa647 albumin. Similar to our in vivo findings, the PT were again found to be significantly downregulated in D treatment with rapamycin, PF-4708671, PP242, or torin led Rap Tubule mice (Figure 4C). Small filtered proteins taken up to a strong reduction of endocytosed Alexa647 albumin (Sup- by the PT (e.g., transthyretin, angiotensinogen, retinol binding plemental Figure 5A). Increasing concentrations of rapamycin protein, vitamin D binding protein, and b2-microglobulin) D reduced the endocytosis rate in a concentration-dependent were enriched in the renal cortexes of Rap Tubule animals com- manner (Supplemental Figure 5B). To allow specific differen- pared with controls (Figure 4D, Supplemental Table 3). Over- tiation between mTORC1 and mTORC2 signaling, we all analysis of phosphosites again only showed subtle

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D Figure 3. mTOR deletion leads to ultrastructural alterations in PT cells. (A) Periodic acid–Schiff–stained sections of control (Con), Rap Tubule, D D Ric Tubule, and RapRic Tubule showing reduced BBM length in mutants compared with control mice. (B) Semithin sections counterstained D D D with methylene blue from Con, Rap Tubule,RicTubule,andRapRicTubule showing intact morphology of epithelial cells. (C) Electron mi- D D D croscopy images of Con, Rap Tubule,RicTubule,andRapRic Tubule showing reduced number and length of BBM microvilli and reduced number of clathrin-coated vesicles as well as recycling endosomes in otherwise normal cellular structures in mutant compared with control animals. Note the increase in early and late endosomes in mutant cells compared with Con in C. Scale bar, 20 mm and 2 mm. differences between the two genotypes (Figure 4E). However, Drosophila melanogaster system to perform an siRNA knock- detailed analysis revealed reduced phosphorylation of several down in nephrocytes, the highly endocytotic storage kidney transport proteins, especially amino acid transporter SLC7A9 cells of the fly. Interestingly, knockdown of GBF-1 led to a (b0,+AT) and the sodium–dependent glucose transporter 2 markedreductioninendogenousuptakeofafluorescently (SGLT2; SLC5A2) providing additional potential molecular ex- labeled marker protein (Figure 5G). In line, TEM showed a planation for the observed aminoaciduria and glucosuria (Fig- phenotype consistent with a severely deranged endocytotic ure 4F, Supplemental Table 4). Additionally, an up to eight machinery (Figure 5H). times reduction on three so far unknown phosphosites of the In contrast, several mTORC2 targets, especially NDRG1, 2 Na+/HCO3 cotransporter (NBC1; T3, S232, and S1060) and were found to have an increased phosphorylation status (T328 the ArfGEF GBF-1 (S179) were identified. To further evaluate and S342). Similar to the gene ontology enrichment analysis on the functional importance of the later protein, we used the protein level, transmembrane transport activity as well as

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D Figure 4. Proteomic analyses of Rap Tubule mice shows reduced abundance and phosphorylation of both tubular transport proteins and regulators of endocytosis. (A) Hierarchical clustering of identified proteins and the samples on the basis of the protein LFQ intensities. D (B) Volcano plot of the proteomic analysis of Rap Tubule mice compared with the control mice. Proteins beyond the curved lines were considered to be significantly changed (red, increased; blue, decreased). (C) Analysis of over–represented gene ontology (GO) terms in the decreased and increased protein populations compared with the unchanged protein population (Fisher exact test; size of the squares represents number of condensed terms). (D) Cumulative histogram of all quantified proteins (black) and proteins with the uniprot keyword secreted (red). Subgroups of this protein population with small molecular weight are depicted in magenta. (E) Hi- erarchical clustering of identified phosphorylation site intensity. (F) Volcano plot of the intensity of phosphorylation sites in the D Rap Tubule mice compared with the control mice. Phosphorylation sites beyond the curved lines were considered to be significantly changed. (G) Analysis of over–represented GO terms in the significantly changed phosphorylation site population compared with the D unchanged population (Fisher exact test). (H) Comparison of logarithmized expression ratios (Rap Tubule to control) for proteins and their respective phosphorylation sites. Density is color coded. (I) Position-weighted matrix of phosphorylation motifs present in the increased and decreased phosphorylation site populations. GOCC, go-term cellular component; GOMF: go-term molecular function; LFQ, label free quantification; TTA: trans-tubular-absorption. membrane components were most significantly regulated DISCUSSION (Figure 4G). Interestingly, when integrating both proteomic and phosphoproteomic datasets, it became evident that Although the pleiotropic functions of mTOR kinases have changes in phosphorylation were not a mere consequence of recently become evident, very little is known about their changes in protein abundance (Figure 4H). We finally analyzed complex role in regulating epithelial protein transport and phosphomotifs being up- or downregulated. Downregulated endocytosis. Interestingly, registry datasets after renal trans- phosphorylation sites mainly contained a proline-directed mo- plantation document reduced serum potassium and phos- tif ([S/T]–P), a motif consistent with phosphorylation by phorus levels in sirolimus-treated patients,19–21 pointing mTOR but also, other proline–directed kinases (Figure 4I).18 toward an mTOR regulatory function for electrolyte transport Phosphorylation sites containing the AGC Kinase motif RxxS and homeostasis. Using inducible, gene–targeted, specificdis- were increased. ruption of mTORC1, mTORC2, or both mTOR complexes in

236 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 230–241, 2017 www.jasn.org BASIC RESEARCH

D Figure 5. Reduced expression of transport proteins and regulators of endocytosis can be found in proximal tubular cells of Rap Tubule D mice. For better illustration, Rap Tubule 3 mT/mG mice were used for analysis of expression levels of various proteins by cell to cell 0 + analysis of Cre-positive (green stain) compared with wild-type cells. (A) B AT1, (B) y Lat1, (D) DOCK8, and (E) BCL-xL showed strongly reduced expression levels in Cre-positive cells compared with wild-type cells. Borders between cells types are marked with white bars. (C) In the case of 4F2hc, Alexa555 lactoglobulin was used to distinguish between Cre-positive cells and wild-type cells, and borders are marked by white bars. Cre-positive cells with reduced uptake of Alexa555 lactoglobulin also showed reduced basolateral ex- 0 + DTubule pression level of 4F2hc. (F) Western blots of B AT1, y Lat1, 4F2hc, DOCK8, and BCL-xL from control (Con) and Rap (left panel) and densitometric evaluation (right panel) confirm immunohistochemical results. (G) RNAi-mediated knockdown of Gbf-1 (CG8487; gartenzwerg) was generated in Drosophila, and RFP-ANP (red) fluorescence uptake was measured in nephrocytes (green). Quantifi- cation is presented in right panel. (H and I) Transmission electron microscopy images of Drosophila from (H) control and (I) Gbf-1 knockdown are shown. In comparison with control nephrocytes with a proper formation of the endocytotic machinery, Gbf-1 knockdown shows loss of clathrin vesicles and early, late, and recycling endolysomes, whereas the formation of aberrant vesicular structures is increased. Scale bar, 20 mminA–E; 2 mminH;0.2mminH,inset;0.5mminI;0.25mm in I, inset. *P value versus control ,0.05; **P value versus control ,0.01. RFP-ANP, red-fluorescent-protein atrial natriuretic peptide; RNAi, RNA interference.

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PT cells, we can now delineate the influence of mTOR signal- DOCK8, a member of the evolutionarily conserved DOCK ing on PT physiology, protein transport, and endocytosis with family proteins,28 was shown to bind to nucleotide-free unprecedented detail (Figure 6). In fact, disruption of CDC42 to mediate the GTP-GDP exchange reaction. Thereby, mTORC1 signaling leads to a Fanconi-like syndrome, which CDC42 is spatially activated at the plasma membrane,29 where presents with phosphaturia, glucosuria, aminoaciduria, and it plays a crucial role in apical differentiation and BBM for- LMW proteinuria. On a molecular level, both fluid-phase mation.30 Another GEF, the ArfGEF GBF-1, presented a D and receptor–mediated endocytosis are impaired and coincide strongly diminished phosphorylation status in Rap Tubule with an altered BBM length and vesicle formation in the PT. animals. Arf-GEFs are nucleotide exchange factors for Arf GTPases, which have diverse functions in vesicle coat forma- mTOR Regulates the Endocytosis Machinery in PT tion and vesicle-cytoskeleton interactions.31 SNX8 was found Epithelial Cells to localize at early endosomes, and its knockdown was shown Tubular albuminuria and LMW proteinuria are well estab- to affect intracellular vesicle trafficking.32 RAB10 has been lished features of Fanconi syndrome and known to occur in localized to many organelles, such as endoplasmic reticulum patients treated with mTOR inhibitors after renal transplan- (ER), Golgi, early endosomes, and recycling endosomes, and tation.22,23 Our study now highlights a significantly reduced mediates membrane trafficking within the cell partly by reg- D D tubular internalization in Rap Tubule and RapRic Tubule mice, ulating endosomal phosphatidylinositol-4,5-bisphosphate which was consistent with endocytosis assays performed in levels.33,34 Together, these data reveal a novel theme of mTOR vitro. Surprisingly, MEGALIN and CUBILIN, which are the function by regulating a series of proteins being specifically in- main renal scavenger receptors for filtered urinary proteins, volved in endocytosis and vesicle transport. were not directly regulated by mTOR in our functional studies (short–term follow-up after induced mTOR deletion).9 This is mTOR Regulates Specific Apical Tubular Transport in contrast to recently published data, in which long–term (6 Proteins months) rapamycin treatment was shown to lead to a reduced Many BBM transporters were affected by Raptor deletion, re- MEGALIN expression in mice.24 However, it has been recog- sulting in a Fanconi-like syndrome. SGLT2 (SLC5A2) is the nized that long-term administration of rapamycin in high main apical glucose transporter in the PT S1 and S2 segments, doses additionally inhibits mTORC2 and exerts cytotoxic ef- with an exclusive localization to the BBM.35 The transporter fects.25 In agreement, long–term follow-up studies (3 months) activity is decisively regulated by phosphorylation.36 Phos- D D in our double–knockout RapRic Tubule mice showed a reduced phoproteomics of Rap Tubule mice pointed to a reduced phos- D MEGALIN expression, whereas Rap Tubule mice maintained a phorylation of SGLT2 at S623 in comparison with control stable MEGALIN expression. Additionally, we detected a animals. This residue is a highly conserved phosphosite strong reduction in the overall renal cortex volume because between species, including human SGLT2 (S624).36 Aug- D of shortening of the S1/2 segment in Rap Tubule mice, and it mented phosphorylation at this site was shown to enhance D was more pronounced in RapRic Tubule mice, confirming the glucose transport by increasing membrane insertion of importance of mTOR signaling for general cellular programs, SGLT2.36 Loss of mTORC1-induced phosphorylation at D such as cell growth, proliferation, and metabolism. S623 could, hence, contribute to glucosuria in Rap Tubule D In addition to the impairment of the initiating steps of and RapRic Tubule animals. D D endocytosis, we also observed a disturbed endocytic trafficking Rap Tubule and even more so, RapRic Tubule mice dis- as indicated by increased amounts of endocytosed albumin in played a highly increased excretion of neutral, basic, and D D Rap Tubule and RapRic Tubule animals. Furthermore, proteo- acidic amino acids compared with control mice (Figure 1, D mic analysis of Rap Tubule identified increased intracel- Supplemental Figure 2). Proteomic and phosphoproteomic D lular amounts of LMW proteins, such as angiotensinogen, analyses of Rap Tubule animals identified reduced expression retinol binding protein, vitamin D binding protein, and b2- or phosphorylation states of many amino acid transporters microglobulin. These proteins are physiologically endocy- known to be highly expressed in the PT.37 Among these, tosed by PT cells and further processed for degradation or SLC6A19 (B0AT1), SLC7A7 (y+LAT1), and SLC3A2 (4F2hc) transcytosis.26 Intracellular trafficking is a prerequisite for had reduced expression levels, whereas diminished phosphor- both processes, and its disturbances may, hence, lead to in- ylation was found in SLC7A9 (b0,+AT) at position S15 and S18. tracellular accumulations as observed in our animal models B0AT1 mediates neutral amino acid transport from the lumi- (Figure 2). nal compartment into the cellular lumen.37 b0,+AT, a member D Proteomic data of Rap Tubule kidney cortex did identify re- of the heterodimeric amino acid transporter family, is the light duced expression or phosphorylation level of interesting can- subunit of SLC3A1 (rBAT), facilitating dibasic amino acid and + didate proteins, such as BCL-xL, GBF-1, SNX8, RAB10, and cysteine transport into the cell. y LAT1, another member of DOCK8, to explain the reduced endocytosis rate, altered api- the heterodimeric amino acid transporter family, is exclusively cal membrane differentiation, and impaired intracellular traf- expressed in the PT, whereas its companion 4F2hc is ubiqui- + ficking. BCL-xL is an antiapoptotic protein with a role in the tously expressed. y LAT1 and 4F2hc both assemble to regulation of membrane dynamics and endocytosis.27 form a heterodimeric amino acid transporter localizing to

238 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 230–241, 2017 www.jasn.org BASIC RESEARCH

Figure 6. Putative mTORC1–dependent regulation of proximal tubular transport and endocytosis. (A) mTORC1 seems to regulate apical and basolateral PT transport proteins. mTORC1 could directly phosphorylates SGLT2 and NBC1 to increase reabsorption of glucose and sodium and excretion of sodium and bicarbonate. mTORC1 might positively influence the expression level of the amino acid transporter B0AT1 and y+LAT-4F2hc and the phosphorylation status of b0,+AT1-rBAT to reabsorb filtered amino acids and secrete them into the bloodstream. (B) mTORC1 could regulate PT endocytosis, membrane biogenesis, vesicle trafficking, and cell polarity. mTORC1 likely controls apical membrane biogenesis and cell polarity by affecting expression level of DOCK8, apical clathrin–coated pit and vesicle formation, and endocytosis by affecting the expression levels of BCL-xL, SNX8, and RAB10 and phosphorylation level of GBF-1. Also, it controls proper endoplasmic reticulum (ER) function and Golgi integrity again by influencing the expressions and phosphorylation levels of RAB10 and GBF-1, respectively. the basolateral membrane of the PT predominantly in the S1 syndrome (Figure 6). Our in-depth analysis reveals probable segment. Interestingly, this transporter mediates efflux of di- molecular pathways being affected in an organ-specificfash- basic amino acids in exchange for neutral amino acids and ion. Additionally, we have identified a novel main kinase reg- sodium together with b0,+AT and its subunit rBAT. Loss of ulator of PT function, opening new avenues for subsequent function mutations result in an inefficient urea cycle with studies to unravel the kinase networks governing PT transport consecutive hyperammonemia,37 which could, at least in in health and disease. D part, explain increased urea values of RapRic Tubule animals. D Phosphoproteomic analysis of Rap Tubule renal cortexes revealed a strong reduction in the phosphorylation level of CONCISE METHODS three phosphorylation sites on SLC4A4 (NBC1). Inactivating mutations of this gene were reported to lead to renal PT aci- Animals and Treatments dosis, another commonly observed feature of Fanconi syn- All animal experiments were conducted according to the National drome.6 Whether the identified phosphosites influence Institutes of Health guide for the care and use of laboratory animals as NBC1 transport needs additional determination on a molec- well as the German law for the welfare of animals, and they were ular level.38,39 In accordance with other reports using rapamy- approved by local authorities (G-13/07, M-9/11, and M-4/13). Mice cin, despite similar expression of the major PT phosphate were housed in an SPF facility with free access to chow and water and a transporters (NaPi-IIa, NaPi-IIc, and Pit-2), we detected 12-hour day-night cycle. Breeding and genotyping were done D D phosphaturia in Rap Tubule and RapRic Tubule animals.40,41 according to standard procedures. Raptorfl/fl and Rictorfl/fl mice Influence of mTORC1 on so far unidentified interaction part- have been described previously42 and were crossed to Pax8rtTA43 ners might be responsible for this enigmatic finding. and TetOCre44 animals. At 5 weeks of age, inducible animals and In summary, mTORC1 deletion leads to a generalized PT respective controls (lacking either TetOCre or Pax8rtTA) received dysfunction, mimicking the clinical diagnosis of Fanconi doxycycline hydrochloride (Fagron, Barsbuettel, Germany) via the

J Am Soc Nephrol 28: 230–241, 2017 mTOR Regulates PT Transport 239 BASIC RESEARCH www.jasn.org drinking water (2 mg/ml with 5% sucrose protected from light) for a 8. Christensen EI, Birn H, Storm T, Weyer K, Nielsen R: Endocytic recep- total of 14 days. Toestimate recombination efficacy and identify cellular tors in the renal proximal tubule. Physiology (Bethesda) 27: 223–236, mosaicisms, we crossed the reporter mouse strain mT/mG with some 2012 9. Christensen EI, Verroust PJ, Nielsen R: Receptor-mediated endocytosis fl fl 3 3 45 Raptor / Pax8rtTA TetOCre mice (Supplemental Material). in renal proximal tubule. Pflugers Arch 458: 1039–1048, 2009 10. Grahammer F, Wanner N, Huber TB: mTOR controls kidney epithelia in Presentation of Data and Statistical Analyses health and disease. Nephrol Dial Transplant 29[Suppl 1]: i9–i18, 2014 Quantitative data are presented as means6SEM. Statistical compar- 11. Laplante M, Sabatini DM: mTOR signaling in growth control and dis- – isons were performed using the GraphPad Prism Software Package 6 ease. Cell 149: 274 293, 2012 12. Wullschleger S, Loewith R, Hall MN: TOR signaling in growth and (GraphPad Software, La Jolla, CA). For statistical comparison, the metabolism. Cell 124: 471–484, 2006 – – Mann Whitney U test, the two tailed t test, and where appropriate, 13. Peterson TR, Sengupta SS, Harris TE, Carmack AE, Kang SA, Balderas E, ANOVA were used. P values of ,0.05 were considered statistically Guertin DA, Madden KL, Carpenter AE, Finck BN, Sabatini DM: mTOR significant. complex 1 regulates lipin 1 localization to control the SREBP pathway. Cell 146: 408–420, 2011 14. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM: Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307: 1098– ACKNOWLEDGMENTS 1101, 2005 15. Ikenoue T, Inoki K, Yang Q, Zhou X, Guan KL: Essential function of TORC2 in PKC and Akt turn motif phosphorylation, maturation and We thank Temel Kilic, Barbara Joch, Benjamin Klormann, Anahita signalling. EMBO J 27: 1919–1931, 2008 Rassi, Brigitte Scolari, Patricia Matthey, Danièla Grand-Habegger, 16. Rickheit G, Wartosch L, Schaffer S, Stobrawa SM, Novarino G, Weinert Mélanie Bousquenaud, and Ruth Herzog for expert technical assis- S, Jentsch TJ: Role of ClC-5 in renal endocytosis is unique among ClC tance. We also thank all members of our laboratories for helpful exchangers and does not require PY-motif-dependent ubiquitylation. J – discussions and support. Biol Chem 285: 17595 17603, 2010 17. Sorribas V, Markovich D, Hayes G, Stange G, Forgo J, Biber J, Murer H: This study was supported by German Research Foundation grants Cloning of a Na/Pi cotransporter from opossum kidney cells. JBiol CRC 1140 (to F.G. and T.B.H.) and CRC 992 (to T.B.H.), the Hei- Chem 269: 6615–6621, 1994 senberg Program (T.B.H.), European Research Council grant 616891 18. Miller ML, Jensen LJ, Diella F, Jørgensen C, Tinti M, Li L, Hsiung M, (to T.B.H.), BMBF–Joint Transnational grants 01KU1215 (to T.B.H.) Parker SA, Bordeaux J, Sicheritz-Ponten T, Olhovsky M, Pasculescu A, and STOP-FSGS 01GM1518C (to T.B.H.), Excellence Initiative of the Alexander J, Knapp S, Blom N, Bork P, Li S, Cesareni G, Pawson T, Turk BE, German Federal and State Governments EXC294, BIOSS II (T.B.H.), Yaffe MB, Brunak S, Linding R: Linear motif atlas for phosphorylation- dependent signaling. Sci Signal 1: ra2, 2008 and the Swiss National Foundation (F.T.). 19. Golbaekdal K, Nielsen CB, Djurhuus JC, Pedersen EB: Effects of rapamycin on renal hemodynamics, water and sodium excretion, and plasma levels of angiotensin II, aldosterone, atrial natriuretic peptide, DISCLOSURES and vasopressin in pigs. 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J Am Soc Nephrol 28: 230–241, 2017 mTOR Regulates PT Transport 241 Supplemental material

Functional Measurements

To study renal excretion, mice were placed in metabolic cages for two days on control diet

(C1000, Altromin, Lage, Germany) and tap water. Urinary creatinine and serum urea were measured using enzymatic colorimetric creatinine and urea kits (Lehmann, Berlin, Germany),

Urinary albumin was measured using a fluorimetric albumin test kit (Progen, PR2005,

Heidelberg, Germany) following the manufacturer´s instructions. Evaluation of albuminuria

(expressed as the albumin to creatinine ratio) was performed as previously described 1.

Plasma concentrations of Na+, K+ and Ca2+ were measured by flame photometry (efux 5057,

Eppendorf, Hamburg, Germany). Plasma and urinary magnesium and phosphorus concentration were determined by colorimetric methods (Lehmann, Berlin, Germany).

Urinary and plasma HPLC measurements

Amino acid concentrations in plasma and urine were determined on Biochrom 30+ amino acid analyzers (Laborservice Onken, Gründau, Germany), using cation exchange chromatography and post-column derivatization with ninhydrin. Prior to injection, samples were deproteinized with sulfosalicylic acid. External control was performed using the

ERNDIM quality control scheme (www.erndimqa.nl) "Amino Acids".

Fixation and tissue processing for immunohistochemistry and immunoblotting

Kidneys were shock-frozen for biochemical evaluation or perfused retrogradely using 4%

PFA in PBS and either postfixed o/n and subsequently transfered into 0.1% PFA in PBS for paraffin embedding or processed for electronmicroscopy or transferred into 800 mOsm

Sucrose in PBS overnight and subsequently frozen in OCT. Paraffin embedded sections were further processed for PAS-staining. To assess fluid phase and receptor mediated endocytosis HRP (Sigma, Schnelldorf, Germany) was given at a concentration of 120μg/g

BW and Alexa555 (Invitrogen, Karlsruhe, Germany) labeled ß-lactoglobulin (Sigma) at 4μg/g

BW i.v. into the retroorbital plexus under light isoflurane anesthesia. Kidneys were harvested

5 min later and immediately perfused with 4% PFA in PBS and processed as described above. For electron microscopy tissue specimens were post-fixed in 1.5% glutaraldehyde and embedded in Epon 812 (Serva, Heidelberg, Deutschland) followed by contrasting ultrathin sections with osmium and uranylacetate. For isolation of membrane fractions kidneys were homogenized in isolation buffer and processed as described 2. Total protein concentration was measured using the Pierce BCA Protein Assay reagent kit (Pierce,

Schwerte, Germany) and controlled by Coomassie staining.

SDS-PAGE and Immunoblotting

Proteins were solubilized and SDS gel electrophoresis was performed on 8-10% polyacrylamide gels. After electrophoretic transfer of the proteins to nitrocellulose membranes, equity in protein loading and blotting was verified by membrane staining using

0.1% Ponceau red. Membranes were probed with primary antibodies and then exposed to

HRP-conjugated secondary antibodies (Dianova, Hamburg, Germany). Immunoreactive bands were detected by chemiluminescence (Amersham Pharmacia, Glattbrugg,

Switzerland). Densitometric evaluation was performed by BIO-PROFIL Bio-1D image software (Vilber Lourmat, Eberhardzell, Germany).

Immunohistochemistry

Cryosections were blocked with 5% skim milk/PBS, incubated with the respective primary antibody followed by the suitable cy-2 or cy-3-coupled secondary antibody (Dianova).

Double-antibody staining procedure was controlled by parallel incubation of consecutive sections, each probed only with one single antibody. Sections were analyzed using a multilaser confocal scanning microscope (SP5, Leica, Heerbrugg, Switzerland).

Antibodies

The following antibodies were used: rabbit anti-raptor (24C12), rabbit anti-rictor (53A2), rabbit anti-p70S6K (49D7), rabbit anti-phospho-p70S6K (108D2), rabbit anti-S6P (5G10), rabbit anti-phospho-S6P (D57.2.2E), rabbit anti-4E-BP1 (53H11), rabbit anti-p-4E-BP1

(T37/46, 236B4); all purchased from Cell Signaling Technologies and sheep anti-NDRG1, sheep anti-phospho-NDRG1 (kind gift of D. Alessi, University of Dundee, UK), guinea pig anti-megalin 3, goat anti-cubilin (Santa Cruz Biotechnology, Heidelberg, Germany, A-

20),rabbit anti-clathrin (Abcam, Cambridge UK, ab 21679), rabbit- anti B0AT1 4, rabbit- anti y+LAT1 5, 4F2hc (CD98 M-20, Santa Cruz Biotechnology, Heidelberg, Germany), DOCK8

(ab121177 Abcam), BCL-xL (54H6 Cell Signaling Technologies) and Alexa647-coubled phalloidin (ThermoFischer Scientific, Zug, Switzerland).

Electron microscopic evaluation

Ultrathin sections were analyzed using a transmission electron microscope Philips CM12.

Images were taken from transverse sectioned S1 segments near the urinary pole and ultrastructural parameters such as microvilli length, number of microvilli per inner diameter length, number of total endocytic vesicles per cell area, number of early endosomes and late endosomes per cell area and mitochondria per cell area were evaluated morphometrically using Fiji software (1.49k). To avoid evaluation of tangentially sectioned epithelia, parallel alignment of BBM microvilli was used as a criterion. Only patent tubules were examined. Cell culture, viral transfection and treatment

Opossum kidney cells (OKC) were kindly provided by H. Murer, University of Zürich,

Switzerland. Cells were cultured at 37°C in 95% air/ 5% CO2 in high-glucose (450 mg/dl)

DMEM supplemented with 5% fetal bovine serum, penicillin (100 U/ml), and streptomycin

(100 μg/ml). OKC were transduced with recombinant adenovirus expressing shRNA targeting human p70S6K (S6K1) and rictor driven by the U6 promoter (kindly provided by Z. Yang,

Fribourg, Switzerland 6). Targeting sequences was in congruent with the respective opossum cDNA sequences. Viral titer was determined by QuickTiter™ Adenovirus Titer Immunoassay

Kit (LuBio Science, Luzern, Switzerland). The control recombinant adenoviruses expressing shRNA targeting LacZ was from Invitrogen life Technologies, Zug, Switzerland. S6K1 targeting sequence: GGACATGGCAGGAGTGTTTGA; Rictor targeting sequence:

ACTTGTGAAGAATCGTATCTT. At a confluence of 70-90% OKC were transduced with the recombinant adenovirus at titers of 100 MOI and cultured in complete medium for 3–5 days.

Degree of knockdown was verified by TaqMan of S6K1 mRNA (Hs00177357_m1) compared to the housekeeping mRNA Tata-box binding protein (Mm00446973_m1) and the protein expression by western blot analysis of S6K1 and rictor. In comparison to MOCK transduction, OKC transduced with either S6K1 shRNA alone or S6K1 and rictor shRNA revealed a knockdown of S6K1 mRNA of -68 ± 12% and -67 ± 11%, *p < 0.05 respectively.

S6K1 protein expression was reduced by -69 ± 15% and -63 ± 12%, respectively. Rictor protein expression was in comparison to MOCK transduction reduced by -66 ± 17% and by –

87 ± 20% for OKC treated either with rictor shRNA alone or S6K1 and rictor shRNA, respectively.

In the case of endocytosis assay, cells were serum-starved overnight. Confluent OKC were incubated either for 3 days with various mTOR kinase inhibitors or overnight with various concentrations of rapamycin. Additionally, p70S6K specific inhibitor PF-4708671 incubated for 16-18h was used. If not otherwise indicated the following concentrations were used; 100 nM rapamycin, 250 nM torin, 100 nM PP242 and 3 μM and 10 μM PF-4708671 (Sigma Aldrich) in FCS-free medium. 10 μM colchicine (Sigma Aldrich), a microtubule inhibitor, was always used overnight. Subsequently, the endocytosis assay was performed by incubating

OKC with 0.1 mg/ml Alexa647-coupled albumin (Molecular Probes, Zug, Switzerland) for 30 minutes, followed by extensive washing, fixing with 3%PFA in PBS and flow cytometry measurements. The downstream effects of various concentrations of rapamycin incubated overnight was tested by immunoprecipitation of 4E-BP1 followed by western blot analysis of p-4E-BP1. Afterwards membranes were stripped and revealed with anti-4E-BP1 antibody.

The ratios of p-4E-BP1/4E-BP1 with increasing concentrations of rapamycin were: 0 nM: 100

± 31%, 5 nM: 79 ± 17%, 10 nM: 75 ± 7%, 100 nM 62 ± 13% and 500 nM: 63 ± 21 %.

Measurement of lysosomal pH and enzyme activity

The pH of lysosomes was measured with LysoSensor Yellow/Blue DND-160 (Molecular

Probes, Zug, Switzerland) according to the manufacturer’s protocol. In brief, OKC were collected by trypsin/EDTA and stained with LysoSensor at 1:200 concentrations in standard culture medium for 10 minutes followed by washing with PBS. Fluorescence of cell suspension in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 7.0) was measured by a microtiter plate at excitation 365 nm/emission 450 nm (for blue) and 485 nm/emission 550 nm (for yellow). Standard curves were obtained in each sample using pH-fixed MES-buffer

(pH 4.0 - 6.5) with 10 μM nigericin (Sigma Aldrich, St. Gallen, Switzerland) and 10 μM monensin (Sigma Aldrich).

The activity of lysosomal enzymes was measured by incubating homogenate of virally transduced OK cells with the appropriate substrate. The reaction was allowed to proceed for

30 minutes at 37°C and then stopped with 5 mL of 85 mM glycine-carbonate buffer (pH

10.5). 200μL of homogenate was incubated with 100μL of 0.5 mM 4-methylumbelliferyl (MU)-

ȕ-galactoside in 100 mM citrate-SKRVSKDWH EXIIHU S+ ZLWK 0 1D&O ȕ-

Glucuronidase was measured by incubating 200μL of homogenate with of 100μL of 1 mM 4-

MU-ȕ-d-glucuronLGH LQ P0 DFHWDWH EXIIHU S+ Į-Mannosidase was measured by incubating 50μL of homogenate with 100 μL of 4 mM 4-MU-Į- d-mannopyranoside in 100 mM citrate-phosphate buffer (pH 4.0). The substrates were purchased from Sigma Aldrich,

St. Gallen, Switzerland. The released 4-MU was measured against blank and standard 4-MU solutions. Fluorescence was determined with a Perkin Elmer 2030 Multilabel Reader Victor

X3 at 450 nm after excitation at 360 nm.

D. melanogaster nephrocyte model system

D. melanogaster stocks were cultured on standard cornmeal molasses agar food and maintained at 25°C. RNAi-Based Nephrocyte Functional Screen Procedure: Virgins from

MHC-ANF-RFP, HandGFP, and Dot-Gal4 transgenic lines (Gift from Zhe Han, University of

Michigan, Ann Arbor, USA) were crossed to UAS-CG8487-RNAi (VDRC TID 42140/GD) males at 25°C; 2 days after crossing, flies were transferred to small collection cages with grape juice agar plates to collect the embryos for 24 hours at 25°C. Collected embryos were aged for 48 hours at 29° C and then subjected to examination of the RFP accumulation in pericardial nephrocytes using a confocal microscope. The RFP mean fluorescence intensity of GFP positive areas was measured to quantify the uptake efficiency.

Virgins of prospero-Gal4 (gift from Barry Denholm, University of Edinburgh, Edinburgh, UK) were crossed to UAS-CG8487-RNAi males (VDRC TID 42140/GD) for a GCN-specific knockdown of Gartenzwerg. GCN preparations for TEM were fixed in 4% PFA and 1 %

Glutaraldehyde and embedded in low melting Agarose. Tissue samples were then embedded in epoxy resin (Durcopan, Plano, Germany). Ultrathin sections of 40nm thickness were analyzed using a Zeiss TEM 906 (Zeiss, Oberkochen, Germany). Sample preparation for MS/MS

Snap frozen kidney cortex were homogenized on ice and solubilized in 8M cold urea buffer containing 50mM ammonium bicarbonate and 1x Protease and phosphatase inhibitor cocktail (Pierce, ThermoFischer Scientific, Rockford, USA). Lysates were cleared with a 20 min spin of 20.000 g in a benchtop centrifuge. Next, the supernatant was recovered and proteins were reduced, alkylated and digested at a 1:100 w/w trypsin/total protein ratio as previously described 7. Protein abundance was measured using a commercial BCA assay

(Pierce). 5% of the digest was retained for proteomic analysis. 800 μg of the remaining peptides was subjected to phosphopeptide enrichment using Fe-NTA immobilized metal affinity chromatography columns (Pierce) without further fractionation as previously described 7, 8. All samples were cleaned up using C18 resin in stage tips as previously described 9.

Nano-liquid chromatography and mass spectrometry

The cleaned peptides were separated using nLC on a house-packed 50cm C18 column in a column oven. The particle size was ௗȝm C18 beads (Dr Maisch GmbH, Ammerbuch-

Entringen, Germany). We analyzed the peptides using the following solvent gradient with ascending concentrations of buffer B as compared to buffer A.: t=0min; 4% [Buffer B], 05 min, 6%; 125 min, 23%; 132 min, 54%; 138 min, 85% ; 143 min, 85%; 145 min 5%). Buffer B was 80% ACN, 0.1% FA and buffer A was 0.1% FA. The flow rate was constant with 250 nl/min. The phosphopeptides were separated using nLC with the following gradient (0min,

10% [Buffer B]; 5min, 10%; 125 min; 38%, 132 min 60%; 138, 95; 143, 95%; 148 min; 95% and 148 min 5%) Peptides were directly sprayed into a quadrupole-orbitrap based mass spectrometer QExactive Plus (Thermo) run in positive ion mode as previously described 10.

The acquisition parameters were as follows: A MS1 precursor scan was followed by 10 MS2 scans of the most intense ions (Top10 method). The resolution was 70000 for MS1 scans and 17500 for MS2 scans. AGC target was 3e6 for MS1 scans, and 5e5 for MS2 scans. Mass range was 300-1750 m/z for MS1 scans, and 200-2000 m/z for MS2 scans. Isolation window was 2.1 m/z, dynamic exclusion was enabled (20s).

Bioinformatic analysis

Raw files were searched using the MaxQuant software package, v 1.5.1.0 11, including LFQ algorithm 12. The raw data were searched against a recent uniprot reference proteome database for mouse including common potential contaminants (Decoy mode: revert). The parameters were mainly default with a few modifications: Carbamidomethylation of cysteins as fixed modification, deisotoping enabled, mass accuracy for first search 20ppm, for second search 4.5 ppm. PSM, protein and site FDR was set to 0.01, minimal peptide length was 7, score for unmodified peptides was 0, and modified was 16. At least one peptide was necessary for protein identification. Variable modifications included N-terminal acetylation and methionine oxidation. In addition, phosphorylation (mass shift approximately +80) on

STY residue was added as a variable modification for samples enriched for phosphorylated peptides. Match between runs (matching time 0.7min, matching window 20min) and label free quantification was enabled. At least 2 counts were necessary for a ratio. The MaxQuant output .txt files (PhosphoSTY.txt and proteingroups.txt) were then analyzed, visualized and normalized using the Perseus software, v. 1.5.1.0. For LFQ, only 5 missing values were tolerated, and for phosphorylation sites no missing value was tolerated. All other proteins or sites were filtered from the dataset. Two samples, for which identification count and/or

MS/MS run was not sufficient (protein identification number less than -1SD from the mean) were removed entirely from the analysis. Missing values were imputed (SD downshift 1.8, width 0.3) after logarithmizing intensities and checking for normal distribution. Hierarchical clustering was performed based on the Euclidean distance. Vulcano plots were generated after a paired two-sample t-test. To determine significantly changed proteins, and to circumvent the multiple testing problem, a statistical approach similar to significance analysis of microarrays (SAM) 13 was utilized. This approach can also be applied to proteomics data.

The threshold for significance was 0.3 after 250 randomizations (s0=1). Proteins and phosphorylation sites considered significant were exported and depicted in the respective tables. GO terms, keywords (uniprot) as well as PFAM domains and KEGG pathways were annotated using the Perseus annotation packages. Next, a fishers exact test was performed to assess whether there were special categories overrepresented in the changed protein and site populations as compared to the non-changed entities (for proteins, this test was performed separately for the significantly increased and the significantly decreased proteins).

The significantly changed GO terms were reanalyzed and condensed using Revigo 14 and visualized in Cytoscape 15. The cumulative histogram was generated using the Prism

(Graphpad) software. Phosphorylation motifs of the upregulated or downregulated phosphopeptide population were generated using the phosphologo software 16.Known phosphosites, known substrates and regulatory sites were added manually to the Table S4.

Using a binary decision tree we separated the phosphorylation motifs in “basophilic”,

“proline-directed”, “acidophilic” and “others”. 17

All MS raw files, including search results and metadata, are deposited at the

ProteomeXchange repository 18. Project accession: PXD002422

(http://www.ebi.ac.uk/pride/archive/projects/PXD002422). Supplemental Figure Legends

Supplementary Fig. 1. Triple labeling of either mTOR, RAPTOR, S6K1, S6P or NDRG1

(red); megalin (green) and phalloidin to stain actin filaments (blue) on control mouse renal sections. (A) mTOR is localized to the subapical membrane region, (A’) merged image demonstrating proximal tubular expression. Insert of (A’) is depicted in (A’’) showing mTOR localized in vicinity to megalin. (B) RAPTOR localizes to vesicles of the subapical membrane region of S1 and S2 portions of the proximal tubule, (B’) merged image demonstrating proximal tubular expression. For more detailed analysis insert of (B’) is depicted in (B’’) presenting a partial overlap between RAPTOR and megalin. (C) S6K1 is found in vesicular structures of the subapical membrane region and throughout the cytoplasm, (C’) merged image demonstrating proximal tubular expression. Note, S3 segments marked by an asterisk are devoid of vesicular expression and show only intracellular staining. Insert of (C’) is depicted in (C’’) showing the vesicular structures positive for S6K1 and megalin in the S1 segment. (D) Ribosomal S6 protein (S6P) is found in the apical membrane region of the proximal tubule; (D’) merged image is verifying proximal tubular expression. Insert of (D’) is depicted in (D’’) showing ribosomal S6P expression in close vicinity to megalin with occasional overlap. (E) NDRG1 is found to be expressed in the apical membrane region of the convoluted part of the proximal tubule; (E’) merged image is verifying proximal tubular expression. Asterisk marks the S3 segment of the proximal tubule which lacks a NDRG1 signal. Insert of (E’) is depicted in (E’’) showing partial coexpression of NDRG1 and megalin.

Magnification as indicated by scale bar.

Supplementary Fig. 2. (A) Urinary acidic and basic amino acid excretion of Rap'Tubule,

Ric'Tubule and RapRic'Tubule. (B) Serum concentration of neutral, acidic and basic amino acids of Rap'Tubule, Ric'Tubule and RapRic'Tubule. *P-value vs. control <0.05; ** P-value vs. control

<0.01. Supplementary Fig.3. Immunohistochemical staining of MEGALIN (A) and CUBILIN (B) on

Rap'Tubule, Ric'Tubule and RapRic'Tubule in comparison to their respective control. (C) Western blot analysis of MEGALIN from Rap'Tubule, Ric'Tubule and RapRic'Tubule. Densitometric analysis of MEGALIN western blots from Rap'Tubule, Ric'Tubule and RapRic'Tubule. No significant difference in expression intensity or subcellular distribution were observed.

Supplementary Fig. 4. Histological alterations of Rap'Tubule and RapRic'Tubule 3 month after induction. (A) PAS-stained paraffin sections of Con, Rap'Tubule and RapRic'Tubule. Black stippled line indicates boundary between cortex and outer medulla and yellow stippled line boundary between outer stripe and inner stripe. (B) Expression of MEGALIN from Con,

Rap'Tubule and RapRic'Tubule. (C) Expression of CUBILIN from Con, Rap'Tubule and

RapRic'Tubule. (D) Expression of Na+/K+-ATPase (NKA) from Con, Rap'Tubule and

RapRic'Tubule. Distal tubules are indicated by an asterisk. Missorting of NKA containing vesicles towards the apical membrane in Rap'Tubule and RapRic'Tubule and reduced basolateral expression in RapRic'Tubule is shown. Magnification as indicated by scale bar.

Supplementary Fig. 5. Pharmacological inhibition of mTORC1, mTORC1/2 or P70S6K1 and knock-down of mTORC2 and/or P70S6K1-induced signaling in proximal tubule cells. (A) Quantitative evaluation of flow cytometrical analysis of endocytosis assay performed in opossum kidney cells (OKC) treated for 3 days with 100 nM rapamycin, 100 nM

PP242 and 250 nM torin or overnight with 10 μM colchicine. Representative image of flow cytometrical analysis (right). (B) Quantitative evaluation of flow cytometrical analysis of endocytosis assay performed on OKC treated with increasing concentrations of rapamycin or colchicine overnight. Representative image of flow cytometrical analysis (right). (C)

Quantitative evaluation of flow cytometrical analysis of endocytosis assay performed on OKC treated with 3μM and 10 μM PF-4708671 (PF) and 100nM rapamycin for 16-18 hours.

Representative image of flow cytometrical analysis (right). (D) Quantitative evaluation of flow cytometrical analysis of endocytosis assay performed on viral transduced OKC with knockdown of S6K1, RICTOR or both proteins. Representative image of flow cytometrical analysis (right). (E) Lysosomal pH of virally transduced OKC with knockdown of S6K1,

RICTOR or both proteins. (F) Quantitative assessment of clathrin-coated vesicles per Pm3 of virally transduced OKC with knockdown of S6K1, RICTOR or both proteins. (G)

Representative images of merged z-scans in a 3D illustration are depicted. Scale bar = 40

Pm. *P-value vs. control <0.05; ** P-value vs. control <0.01; #P-value vs. control <0.001. Supplemental tables

Supplemental Table 1. Physiological parameters of Con, Rap'Tubule, Ric'Tubule and

RapRic'Tubule. All values are rounded means given with min-max and n-number. *P-value vs. control <0.05; ** P-value vs. control <0.01.

Supplemental Table 2. Quantitative electron microscopy analysis. Microvilli length, number of microvilli per length cell surface, number of vesicles per cell area and mitochondrial area per cell area given in % of Rap'Tubule, Ric'Tubule and RapRic'Tubule in comparison to their respective controls. *P-value vs. control <0.05; **P-value vs. control <0.01; #P-value vs control <0.001.

Supplemental Table 3. Identified proteins with significant changes in expression level of

Rap'Tubule in comparison to control. (A) Proteins which were significantly increased, (B) proteins which were significantly reduced.

Supplemental Table 4. Identified changes of phosphorylation level of proteins of Rap'Tubule in comparison to control. (A) Phosphorylation sites of proteins which were significantly increased, (B) phosphorylation sites of proteins which were significantly reduced. In addition known phosphosites, substrates and regulatory sites are indicated. Supplemental Information References

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and dissemination. Nature Biotechnology, 32: 223-226, 2014. Supplement al Figure 1

A mTOR B RAPTOR C S6K1 D S6P E NDRG1

20m m 20m m 20m m 20m m 20m m A' B' C' D' E' * * * *

20m m 20m m 20m m 20m m 20m m A'' B'' C'' D'' E''

10m m 10m m 10m m 10m m 10m m Supplemental Figure 2

Con Rap Tubule Ric Tubule RapRic Tubule

Con RapRic Tubule Supplement al Figure 3

A MEGALIN B CUBILIN

Con Rap Tubule Con Rap Tubule

20 m 20 m 20 m 20 m Con Ric Tubule Con Ric Tubule

20 m 20 m 20 m 20 m Con RapRic Tubule Con RapRic Tubule

20 m 20 m 20 m 20 m C D 200 Con Rap Tubule

150

Con Ric Tubule 100

50 Con RapRic Tubule

0 Rap Tubule Ric Tubule RapRic Tubule Supplemental Figure 4

Con Rap Tubule RapRic Tubule A

100 m 100 m 100 m B

20 m 20 m 20 m C *

20 m 20 m 20 m D * * * *

10 m* 10 m * 10 m Supplemental Figure 5

A B 120 con con % 100

% 100

5 nM Rapa 80 PP242 i n 80

t m 10 nM Rapa

rapamycin b

u 60 ** l 60 *

a l *

7 - 100 nM Rapa C torin *

** 4 7 40 40

endocytosed

a 6 ** 500 nM Rapa endocytosed **

x a 20 colchicine

e 20 # x #

l A colchicine

A 0 0 colchi- con rapa- PP torin colchi- con 5 10 100 500 cine mycin 242 cine nM rapamycin

C 120 D 120 100 mock mock

% % 100

n 3 M PF n

i t Rictor

t ** n

n u 80

m # rapamycin m

o S6K1

u 60 C *

l 10 M PF l 60

a a S6K1/

- # -

7 40 # 7

4 4 40 Rictor

endocytosed colchicine endocytosed

a a ** colchicine

x 20 x **

e e 20 #

A 0 A 0 colchi- con 3M10 M rapa- colchi- mock S6K1 Rictor S6K1/ cine PF PF mycin cine Rictor E F G

6 mock S6K1 S6K1/Rictor

3 2.5

m / H 2

5.5 s

l l **

a c #

i 1.5 s

m e

o 5 v

n 1 i

s r

l h

4.5 t a

l 0.5 40 m 40 m 40 m c 4 0 mock S6K1 Rictor S6K1/ mock S6K1 Rictor S6K1/ Rictor Rictor Con Rap'Tubule- Ric'Tubule RapRic'Tubule

body weight 28,9 [27,5-30,3], (n=50) 27,7 [25,9-29,5], (n=19) 25,2 [23,1-27,2], (n=12) 24,0 [22,3-25,7] **, (n=19) (g)

food intake 99 [91-108], (n=42) 118 [102-134] *, (n=12) 109 [98-120], (n=12) 127 [111-143] **, (n=15) (mg/ g body weight)

fluid intake 218 [166-270], (n=39) 675 [458-892] **, (n=12) 233 [135-331], (n=11) 407 [329-484] **, (n=15) (μl/ g body weight)

serum urea 53 [42-64], (n=17) 62 [47-78], (n=6) 61 [40-81], (n=6) 105 [84-126] **, (n=6) (mg/dl)

urine volume 50 [39-60], (n=42) 387 [206-568] **, (n=12) 58 [29-87], (n=12) 140 [109-172] *, (n=15) (μl/ g body weight)

urinary sodium 148 [134-162], (n=41) 195 [127-263] *, (n=12) 152 [115-190], (n=12) 208 [173-243] **, (n=15) (μmol/ mg crea/ g food)

urinary potassium 241 [216-162], (n=41) 291 [211-370], (n=12) 211 [196-225], (n=12) 315 [262-368], (n=15) (μmol/ mg crea/ g food)

urinary calcium 1,5 [1,1-1,8], (n=38) 2,5 [1,4-3,6], (n=12) 1,0 [0,7-1,4], (n=12) 1,9 [1,4-2,4], (n=15) (μmol/ mg crea/ g food) Table 1 - Physiologic parameter Values are rounded means + 95%CI, * = p-value vs Con < 0,05, ** = p-value vs Con < 0,01 Table 2

number of Number of microvilli lenght in Mitochondria in microvilli per μm vesicles per cell μm % lenght cell surface area Con 1.71 ± 0.23 4.91 ± 0.41 1.01 ± 0.14 40.53 ± 5.13

'Tubule Rap # 1.25 ± 0.04** 3.42 ± 0.49 0.71 ± 0.04** 37.42 ± 2.35 Con 1.52 ± 0.19 5.05 ± 0.61 1.05 ± 0.12 41.45 ± 5.77 Ric'Tubulue 1.31 ± 0.17* 3.38 ± 0.65* 0.83 ± 0.15* 37.52 ± 3.57 Con 1.55 ± 0.16 4.81 ± 0.44 1.00 ± 0.12 39.58 ± 2.71

'Tubule RapRic # # # 1.06 ± 0.05 2.98 ± 0.17 0.58 ± 0.06 32.12 ± 3.47*

A. Proteins increased in Rap 'Tubule as compared to control

Gene log2 intensity names Uniprot Protein names (Rap/control) -log(P-value) Obp1a Q9D3H2 MCG117626 3,02 2,50 Rplp1 P47955 60S acidic ribosomal protein P1 2,93 1,49 Ipo4 Q8VI75 Importin-4 2,65 1,58 Reg3g O09049 Regenerating islet-derived protein 3-gamma 2,64 1,47 Parpbp Q6IRT3 PCNA-interacting partner 2,62 0,67 Ppp2r4 P58389 Serine/threonine-protein phosphatase 2A activator 2,55 2,64 Retn Q99P87 Resistin 2,54 3,80 Ahsg P29699 Alpha-2-HS-glycoprotein 2,51 3,55 Gm14743 A2BHD2 Protein Gm14743 2,38 2,78 Reg1 P43137 Lithostathine-1 2,33 2,12 Guca2b O09051 Guanylate cyclase activator 2B 2,26 4,00 Ambp Q07456 Protein AMBP 2,24 3,56 Napsa O09043 Napsin-A 2,23 1,75 Cops7a Q9CZ04 COP9 signalosome complex subunit 7a 2,20 1,93 Scgb1a1 Q06318 Uteroglobin 2,16 1,42 Apoa4 P06728 Apolipoprotein A-IV 2,11 3,39 Ttr P07309 Transthyretin 2,10 2,25 Agt P11859 Angiotensinogen 2,04 3,98 Serpinf1 P97298 Pigment epithelium-derived factor 2,03 3,12 Gm2a Q60648 Ganglioside GM2 activator 2,00 4,89 Lyz2 P08905 Lysozyme C-2 1,99 4,12 Rbp4 Q00724 Retinol-binding protein 4 1,93 2,00 S100a6 P14069 Protein S100-A6 1,90 1,24 Apoc3 P33622 Apolipoprotein C-III 1,89 2,41 Cst3 P21460 Cystatin-C 1,87 4,07 Rnase4 Q8C7E4 Ribonuclease 4 1,87 3,36 Cpq Q9WVJ3 Carboxypeptidase Q 1,86 1,37 Gc P21614 Vitamin D-binding protein 1,82 3,40 Cox6b1 P56391 Cytochrome c oxidase subunit 6B1 1,75 1,17 Ctbs Q8R242 Di-N-acetylchitobiase 1,74 2,73 Chi3l3 O35744 Chitinase-3-like protein 3 1,66 3,28 Slc37a4 Q9D1F9 Protein Slc37a4 1,63 0,83 Chchd2 Q9D1L0 Coiled-coil-helix-coiled-coil-helix domain-containing protein 2, mitochondrial 1,62 1,68 Nup155 Q99P88 Nuclear pore complex protein Nup155 1,59 1,39 Dsg4 Q7TMD7 Desmoglein-4 1,55 0,70 Igfbp2 D3YU40 Insulin-like growth factor-binding protein 2 1,55 1,50 C4b P01029 Complement C4-B 1,54 3,38 Arl2 Q9D0J4 ADP-ribosylation factor-like protein 2 1,54 2,18 Tgfb1i1 E9PYQ1 Transforming growth factor beta-1-induced transcript 1 protein 1,54 1,76 Cd5l Q9QWK4 CD5 antigen-like 1,52 4,55 Cfb P04186 Complement factor B 1,52 1,68 Kng2 Q6S9I0 Kng2 protein 1,49 1,82 Eva1a D3Z511 Protein eva-1 homolog A 1,48 2,18 N/A P01837 Ig kappa chain C region 1,47 1,99 S100a10 P08207 Protein S100-A10 1,47 1,47 Snrpb P27048 Small nuclear ribonucleoprotein-associated protein B 1,46 1,18 Abhd11 Q8K4F5 Alpha/beta hydrolase domain-containing protein 11 1,44 0,84 Sept6 Q9R1T4 Septin-6 1,44 1,70 Prelp Q9JK53 Prolargin 1,43 1,47 Mtx2 O88441 Metaxin-2 1,42 0,90 Ctbp1 O88712 C-terminal-binding protein 1 1,40 0,71 Retnlg Q8K426 Myeloid cysteine-rich protein 1,39 1,59 Lsm14a Q8K2F8 Protein LSM14 homolog A 1,39 1,32 Cyp4a12a Q91WL5 Cytochrome P450 4A12A 1,37 0,76 Nek7 Q9ES74 Serine/threonine-protein kinase Nek7 1,37 1,59 Uqcrq Q9CQ69 Cytochrome b-c1 complex subunit 8 1,36 0,78 Amy1 P00687 Alpha-amylase 1 1,36 0,83 Hao2 Q9NYQ2 Hydroxyacid oxidase 2 1,34 2,07 Glyctk Q8QZY2 Glycerate kinase 1,33 1,77 Snrnp40 Q6PE01 U5 small nuclear ribonucleoprotein 40 kDa protein 1,32 1,09 Hpx Q91X72 Hemopexin 1,29 2,53 Tstd1 E9PY03 Protein Tstd1 1,26 1,09 Slc10a2 P70172 Ileal sodium/bile acid cotransporter 1,24 1,97 B2m P01887 Beta-2-microglobulin 1,23 2,45 Erp29 P57759 Endoplasmic reticulum resident protein 29 1,22 1,45 Apoa1 Q00623 Apolipoprotein A-I 1,21 1,92 Spink3 P09036 Serine protease inhibitor Kazal-type 3 1,19 2,68 Snrpc Q62241 U1 small nuclear ribonucleoprotein C 1,19 0,97 Myg1 F8WGG3 UPF0160 protein MYG1, mitochondrial 1,16 1,93 Hamp Q9EQ21 Hepcidin 1,16 1,78 Nt5e Q61503 5-nucleotidase 1,13 1,63 Them7 Q9DCP4 Novel Thioesterase superfamily domain and Saposin A-type domain contain 1,13 3,68 Eefsec Q9JHW4 Selenocysteine-specific elongation factor 1,12 1,57 Man2b1 O09159 Lysosomal alpha-mannosidase 1,12 1,55 Fnbp1l E9PUK3 Formin-binding protein 1-like 1,11 1,34 N/A P01843 Ig lambda-1 chain C region 1,11 1,49 Tpm1 B7ZNL3 ropomyosin alpha-1 chain 1,09 1,62 Enpep P16406 Glutamyl aminopeptidase 1,04 2,18 Ace2 Q8R0I0 Angiotensin-converting enzyme 2 1,04 3,46 Blvrb Q923D2 Flavin reductase (NADPH) 1,02 2,18 Ren1 P06281 Renin-1 1,01 2,01 Fdx1l Q9CPW2 Adrenodoxin-like protein, mitochondrial 0,98 1,53 Cpsf6 Q6NVF9 Cleavage and polyadenylation specificity factor subunit 6 0,97 1,63

B. Proteins decreased in Rap 'Tubule as compared to control

Gene log2 intensity names Uniprot Protein names (Rap/control) -log(P-value) Thsd4 Q3UTY6 Thrombospondin type-1 domain-containing protein 4 -2,79 0,98 4933436I01RQ9D3T1 Protein 4933436I01Rik -2,73 1,18 Dock8 Q8C147 Dedicator of cytokinesis protein 8 -2,70 0,82 Dnah10 F7ABZ6 Protein Dnah10 -2,50 1,89 Slc26a1 P58735 Sulfate anion transporter 1 -2,22 1,30 Slc7a7 Q9Z1K8 Y+L amino acid transporter 1 -2,08 1,55 Bloc1s5 Q8R015 Biogenesis of lysosome-related organelles complex 1 subunit 5 -1,89 0,64 Rtn3 Q9ES97 Reticulon-3 -1,89 1,17 Ppp6r1 Q7TSI3 Serine/threonine-protein phosphatase 6 regulatory subunit 1 -1,73 2,61 Dnajc12 Q9R022 DnaJ homolog subfamily C member 12 -1,57 3,45 Bcl2l1 A2AHX9 Bcl-2-like protein 1 -1,55 1,03 Fbxo22 D6REV6 F-box only protein 22 -1,55 2,20 Slc6a19 D6RJ80 Sodium-dependent neutral amino acid transporter B(0)AT1 -1,54 1,22 Slc5a12 Q49B93 Sodium-coupled monocarboxylate transporter 2 -1,51 4,94 Rab10 P61027 Ras-related protein Rab-10 -1,48 1,25 Uros Q3TPL3 Uroporphyrinogen-III synthase -1,44 0,82 Cbx1 P83917 Chromobox protein homolog 1 -1,39 0,95 Sdc4 O35988 Syndecan-4 -1,39 1,39 Cyp51a1 Q8K0C4 Lanosterol 14-alpha demethylase -1,38 1,20 Ankrd44 J3QK13 Serine/threonine-protein phosphatase 6 regulatory ankyrin repeat subunit B -1,35 0,94 Snx8 Q8CFD4 Sorting nexin-8 -1,24 1,05 Phgdh Q61753 D-3-phosphoglycerate dehydrogenase -1,21 3,57 Mrpl13 Q9D1P0 39S ribosomal protein L13, mitochondrial -1,20 1,26 Larp1 Z4YJT3 La-related protein 1 -1,13 1,68 Nccrp1 G3X9C2 F-box only protein 50 -1,12 1,06 G6pc P35576 Glucose-6-phosphatase -1,11 1,04 Slc16a1 P53986 Monocarboxylate transporter 1 -1,10 3,58 Hspa14 Q99M31 Heat shock 70 kDa protein 14 -1,09 1,22 Glul P15105 Glutamine synthetase -1,08 4,29 Chd4 E9QAS4 Chromodomain-helicase-DNA-binding protein 4 -1,02 1,47 Rhot1 Q8BG51 Mitochondrial Rho GTPase 1 -1,00 1,45 Slc43a2 Q8CGA3 Large neutral amino acids transporter small subunit 4 -1,00 2,96 Rpl6 P47911 60S ribosomal protein L6 -0,99 2,74 Rpl7 P14148 60S ribosomal protein L7 -0,93 1,79 Rpl26 P61255 60S ribosomal protein L26 -0,91 3,96 Ca14 Q9WVT6 Carbonic anhydrase 14 -0,91 2,50 Aacs Q9D2R0 Acetoacetyl-CoA synthetase -0,89 4,05 Slc3a2 P10852 4F2 cell-surface antigen heavy chain -0,88 4,26 Rpl19 A2A547 60S ribosomal protein L19 -0,88 2,53 Slc22a8 O88909 Solute carrier family 22 member 8 -0,82 3,07 log2 intensity -log(p MOTIF Gene names Uniprot Protein names Amino acid (Rap/control) value) Sequence window Known site (FOrigin PhosphoSitePRegulatory sitRegulatory sitRegulatory sitRegulatory sitRegulatory sitRegulatory sitCLASS Pdzk1 Q9JIL4 Na(+)/H(+) exchange regulatory cofactor NHE-RF3 S148 2,57 5,86 PRLCYLVKEGNSFGFSLKTIQGKKGVYLTDI +HTP BASOPHILIC Abcc2 Q8VI47 Canalicular multispecific organic anion transporter 1 S906 2,56 1,19 EIPDDAASLTMRRENSLRRTLSRSSRSGSRR +HTP BASOPHILIC Carhsp1 Q9CR86 Calcium-regulated heat stable protein 1 S53 2,34 0,51 NVVPSPLPTRRTRTFSATVRASQGPVYKGVC +CST;HTP BASOPHILIC Srrm2 Q8BTI8 Serine/arginine repetitive matrix protein 2 S454 2,28 1,28 LKPTPAPGSRREISSSPTSKNRSHGRAKRDK +CST;HTP PROLINE_DIRE Ndrg1 Q62433 Protein NDRG1 S342 1,69 1,44 RTASGSSVTSLEGTRSRSHTSEGPRSRSHTS +CST;HTP OTHER Gm8991 E9Q7H5 Heterogeneous nuclear ribonucleoprotein A3 S280 1,49 0,67 YGPMKGGSFGGRSSGSPYGGGYGSGGGSGGY PROLINE_DIRE Ndrg1 Q62433 Protein NDRG1 T328 1,38 1,40 GYMPSASMTRLMRSRTASGSSVTSLEGTRSR +CST;HTP BASOPHILIC Dync1h1 Q9JHU4 Cytoplasmic dynein 1 heavy chain 1 S4366 1,38 1,08 DDLAYAETEKKARTDSTSDGRPAWMRTLHTT +CST;HTP BASOPHILIC Nedd4l E9PXB7;Q8CFI0 E3 ubiquitin-protein ligase NEDD4-like;E3 ubiquitin-protein ligase S449 1,12 2,86 NNHLVEPQIRRPRSLSSPTVTLSAPLEGAKD + CST;HTP;LTP SGK1 + molecular association, regul14-3-3 beta(INDUCES) BASOPHILIC Apon G3X9D6 Apolipoprotein N, isoform CRA_a S114 1,12 2,54 GGKDSAETLVLQSQKSSQEEGIGNNEVILRH +HTP ACIDOPHILIC Apon G3X9D6 Apolipoprotein N, isoform CRA_a S115 1,12 2,54 GKDSAETLVLQSQKSSQEEGIGNNEVILRHL +HTP ACIDOPHILIC Stx7 Q8BH40 Syntaxin-7 S129 1,09 1,85 REKEFVARVRASSRVSGGFPEDSSKEKNLVS BASOPHILIC Eps8l2 Q99K30 Epidermal growth factor receptor kinase substrate 8-like protein 2 S17 1,08 1,04 SQSASMSCCPGAANGSLGRSDGVPRMSAKDL BASOPHILIC Tns1 A0A087WQS0 Protein Tns1 S1054 1,04 2,31 RQPAEPPGPLRRRAASDGQYENQSPEATSPR BASOPHILIC Mme Q61391 Neprilysin S4 1,01 2,11 ______MGRSESQMDITDINAPKPK + CST;HTP;LTP ACIDOPHILIC Aldob Q91Y97 Fructose-bisphosphate aldolase B S36 0,99 1,32 QRIVANGKGILAADESVGTMGNRLQRIKVEN +HTP BASOPHILIC Eif4b Q8BGD9 Eukaryotic translation initiation factor 4B S406 0,97 1,85 LDEPKLDRRPRERHPSWRSEETQERERSRTG + CST;HTP;LTP + translation, altered BASOPHILIC Cobll1 B1AZ15 Cordon-bleu protein-like 1 S363 0,97 1,19 VRTGSLQLSSTSIGTSSLKRTKRKAPAPPSK BASOPHILIC Ndrg2 Q9QYG0 Protein NDRG2 S350 0,91 1,41 SAASIDGSRSRSRTLSQSSESGTLPSGPPGH +CST;HTP BASOPHILIC Lmna P48678 Prelamin-A/C;Lamin-A/C S404 0,91 2,13 LSPSPTSQRSRGRASSHSSQSQGGGSVTKKR + CST;HTP;LTP BASOPHILIC Lmna P48678 Prelamin-A/C;Lamin-A/C S407 0,91 2,13 SPTSQRSRGRASSHSSQSQGGGSVTKKRKLE + CST;HTP;LTP OTHER Abcc2 Q8VI47 Canalicular multispecific organic anion transporter 1 S927 0,90 2,36 SRSSRSGSRRGKSLKSSLKIKSVNALNKKEE +HTP BASOPHILIC Srsf2 Q62093 Serine/arginine-rich splicing factor 2 S26 0,89 3,07 EGMTSLKVDNLTYRTSPDTLRRVFEKYGRVG +CST;HTP PROLINE_DIRE Clmn Q8C5W0 Calmin S419 0,88 1,42 ESSILSTRKDGRRSNSLPVKKTVHFEADLHK +HTP BASOPHILIC Prkd3 Q8K1Y2 Serine/threonine-protein kinase D3;Serine/threonine-protein kinase D S41 0,84 3,29 PSPCSSPKTGLSARLSNGSFSAPSLTNSRGS +HTP OTHER Fxyd1 Q9Z239 Phospholemman S83 0,79 3,88 QRTGEPDEEEGTFRSSIRRLSSRRR______+ CST;HTP;LTP + intracellular localization After stimulatOTHER Tgfb1i1 E9PYQ1 Transforming growth factor beta-1-induced transcript 1 protein S341 0,78 2,73 EGRPLCENHFHAQRGSLCATCGLPVTGRCVS OTHER Slc4a4 D3Z7G8 Electrogenic sodium bicarbonate cotransporter 1 T3 -2,99 1,15 ______MSTENVEGKPNNLGERGR ACIDOPHILIC Srrm2 Q8BTI8 Serine/arginine repetitive matrix protein 2 S1630 -2,58 1,07 PEHAPKSRTARRGSRSSIEPKTKSHTPPRRR +HTP ACIDOPHILIC Slc7a9 Q9QXA6 B(0,+)-type amino acid transporter 1 S15 -2,25 3,66 _MEETSLRRRREDEKSTHSTELKTTSLQKEV BASOPHILIC Slc16a1 P53986 Monocarboxylate transporter 1 S213 -1,86 3,11 IGPEQVKLEKLKSKESLQEAGKSDANTDLIG +HTP ACIDOPHILIC Slc7a9 Q9QXA6 B(0,+)-type amino acid transporter 1 S18 -1,77 1,71 ETSLRRRREDEKSTHSTELKTTSLQKEVGLL ACIDOPHILIC Srrm2 Q8BTI8 Serine/arginine repetitive matrix protein 2 S1631 -1,65 0,95 EHAPKSRTARRGSRSSIEPKTKSHTPPRRRS +CST;HTP ACIDOPHILIC Slc4a4 A7E1Z5 Electrogenic sodium bicarbonate cotransporter 1 S1060 -1,65 0,62 QQPFLSDNKPLDRERSSTFLERHTSC_____ BASOPHILIC Slc5a2 Q923I7 Sodium/glucose cotransporter 2 S623 * -1,39 0,97 RCLLWFCGMSKSGSGSPPPTTEEVAATTRRL +HTP PROLINE_DIRE Rptor Q8K4Q0 Regulatory-associated protein of mTOR S722 -1,34 4,04 PVRDSPCTPRLRSVSSYGNIRAVTTARNLNK + CST;HTP;LTP BASOPHILIC Gbf1 Q6A099 Gbf1 protein S179 -1,21 2,90 AGGMSDSSKWKKQKRSPRPPRHTTKVTPGSE PROLINE_DIRE Pdzk1 Q9JIL4 Na(+)/H(+) exchange regulatory cofactor NHE-RF3 S492 -1,20 2,62 PLVAGPDEKGETSAESEHDAHPAKDRTLSTA +HTP ACIDOPHILIC Wnk4 Q80UE6 Serine/threonine-protein kinase WNK4 S1196 -1,19 2,47 LQRSDLPGPGIMRRNSLSGSSTGSQEQRASK + CST;HTP;LTP SGK1;WNK4 BASOPHILIC Dcaf8 Q8N7N5 DDB1- and CUL4-associated factor 8 S100 -1,17 5,70 TGHYSINDESRGHGHSDEEDEEQPRHRGQRK +CST;HTP ACIDOPHILIC Ndrg1 Q62433 Protein NDRG1 S330 -1,15 0,86 MPSASMTRLMRSRTASGSSVTSLEGTRSRSH +CST;HTP BASOPHILIC Rptor Q8K4Q0 Regulatory-associated protein of mTOR S863 -1,13 2,81 QRILDTSSLTQSAPASPTNKGMHMHQVGGSP + HTP;LTP PROLINE_DIRE Tns1 A0A087WQS0 Protein Tns1 S1067 -1,12 1,45 AASDGQYENQSPEATSPRSPGVRSPVQCVSP PROLINE_DIRE Tra2b P62996 Transformer-2 protein homolog beta S95 -1,12 0,78 RSRSRSYSRDYRRRHSHSHSPMSTRRRHVGN +CST;HTP BASOPHILIC Tra2b P62996 Transformer-2 protein homolog beta S99 -1,12 0,78 RSYSRDYRRRHSHSHSPMSTRRRHVGNRANP +CST;HTP PROLINE_DIRE 4833439L19Rik D3Z1F7 Putative monooxygenase p33MONOX S164 -1,11 1,24 HPASAQSTPSSTPHASPKQKSRGWFPSGSST PROLINE_DIRE Lrp2 A2ARV4 Low-density lipoprotein receptor-related protein 2 S4577 -1,09 2,95 RSIDPSEIVPEPKPASPGADETQGTKWNIFK +HTP PROLINE_DIRE Dennd5b A2RSQ0 DENN domain-containing protein 5B S1068 -1,08 2,87 DEDLGKQCRTPPQQKSPTTTRRLSITSLTGK +HTP PROLINE_DIRE Dennd5b A2RSQ0 DENN domain-containing protein 5B T1062 -1,08 2,87 LMTSASDEDLGKQCRTPPQQKSPTTTRRLSI +HTP PROLINE_DIRE Hsp90aa1 P07901 Heat shock protein HSP 90-alpha S231 -1,07 3,00 YPITLFVEKERDKEVSDDEAEEKEEKEEEKE +CST;HTP ACIDOPHILIC Erc1 V9GWT6 ELKS/Rab6-interacting/CAST family member 1 S21 -1,05 1,40 RSVGKVEPSSQSPGRSPRLPRSPRLGHRRTN PROLINE_DIRE Smarcc1 P97496 SWI/SNF complex subunit SMARCC1 S309 -1,04 3,30 SFRQRISTKNEEPVRSPERRDRKASANSRKR +HTP PROLINE_DIRE Slc4a4 A7E1Z5 Electrogenic sodium bicarbonate cotransporter 1 S232 -0,98 1,44 KKSNLRSLADIGKTVSSASSPAMTHRNLTSS +CST;HTP BASOPHILIC Matr3 Q8K310 Matrin-3 S604 -0,93 1,17 KDKSRKRSYSPDGKESPSDKKSKTDAQKTES +CST;HTP PROLINE_DIRE Rbm14 Q8C2Q3 RNA-binding protein 14;RNA-binding protein 4B;RNA-binding protein 4 S215 -0,92 3,54 GQARQPTPPFFGRDRSPLRRSPPRASYVAPL +HTP PROLINE_DIRE Srsf2 Q62093 Serine/arginine-rich splicing factor 2 S189 -0,91 3,54 SSVSRSRSRSRSRSRSRSPPPVSKRESKSRS +HTP BASOPHILIC Srsf2 Q62093 Serine/arginine-rich splicing factor 2 S191 -0,91 3,54 VSRSRSRSRSRSRSRSPPPVSKRESKSRSRS +HTP PROLINE_DIRE Kiaa1468 A0A087WSS1 LisH domain and HEAT repeat-containing protein KIAA1468 S193 -0,90 2,29 AGSISTLDSLDFARYSDDGNRETDERVAEHE ACIDOPHILIC Ybx1 P62960 Nuclease-sensitive element-binding protein 1 S172 -0,84 2,87 QQNYQNSESGEKNEGSESAPEGQAQQRRPYR +CST;HTP ACIDOPHILIC Thrap3 Q569Z6 Thyroid hormone receptor-associated protein 3 S669 -0,82 2,16 YLKRGNEQEAAKNKKSPEIHRRIDISPSTFR +CST;HTP PROLINE_DIRE Mapt P10637 Microtubule-associated protein tau S527 -0,79 2,33 TREPKKVAVVRTPPKSPSASKSRLQTAPVPM + CST;HTP;LTP PROLINE_DIRE Arvcf P98203 Armadillo repeat protein deleted in velo-cardio-facial syndrome homolog S345 -0,78 2,29 PERGSLGSLDRVVRRSPSVDSTRKEPRWRDP +HTP PROLINE_DIRE Epb4.1l1 A2AUK8;E9PV14 Band 4.1-like protein 1 S461 -0,78 2,60 HDAGPDGDKREDDAESGGRRSEAEEGEVRTP +HTP OTHERH Epb4.1l1 A2AUK8;E9PV14 Band 4.1-like protein 1 S466 -0,77 2,86 DGDKREDDAESGGRRSEAEEGEVRTPTKIKE ACIDOPHILIC Dennd5b A2RSQ0 DENN domain-containing protein 5B S178 -0,76 3,19 DEGDATSLLKLQRYNSYDINRDTLYVSKSIC +CST;HTP BASOPHILIC

* Valid intensity values for this site only in 5 control and 4 Rap animals (missing value in 1 Rap animal)