Knock‐out of dipeptidase CN2 in human proximal tubular cells disrupts dipeptide and amino acid homeostasis and para‐ and transcellular solute transport

Although of potential biomedical relevance, dipeptide metabolism has hardly been studied. We found the dipeptidase carnosinase‐2 (CN2) to be abundant in human proximal tubules, which regulate water and solute homeostasis. We therefore hypothesized, that CN2 has a key metabolic role, impacting proximal tubular transport function.


| INTRODUCTION
The role of dipeptide (DP) metabolism in health and disease has hardly been studied, and knowledge on its pathophysiological role is mainly limited to carnosine (β-Ala-His), even though significant biological functions have been demonstrated for other DPs such as Ala-Gln and Tyr-Asp. 1,2One of the major dipeptidases in humans is carnosinase 2 (CN2, EC 3.4.13.18) encoded by the CNDP2 gene. 3CN2 has a negligible role in carnosine metabolism in vivo 4 but degrades cysteinyl-glycine in yeast and mice 5,6 and thyreonyl-serine in the human kidney. 7CN2 forms Nlactoyl-amino-phenylalanine, 8 a signaling metabolite that suppresses feeding and obesity, 9 suggesting not only CN2 involvement in DP degradation but also extended regulatory functions within the body metabolism.3][14] Cndp2-KO mice exhibited aggravated progression of kidney damage after an acetaminophen overdose, probably related to increased ferroptosis. 6he human proximal tubule has a central role in water, ion and small nutrient transport, 15,16 is the primary location for renal gluconeogenesis 17 and plays a critical role in the progression of kidney disease. 18,19][22] The cellular AA pool is determined by a tight balance between uptake, biosynthesis and protein catabolism, and AA are used for energy production and macromolecules such as proteins and glutathione. 23In this context, the role of DP-degrading activity and its impact on AA availability in the kidney, however, has not yet been studied.We therefore investigated the relevance of CN2 for renal DP and AA metabolism.For this purpose, we generated a knockout of the CN2 gene (CNDP2-KO) in human proximal tubular cells, the main cell type of CN2 expression in the human kidney, and examined the impact on DP and AA concentrations and the effects on GSH metabolism, gluconeogenesis and energy production, protein biosynthesis and resulting impact on specific kidney cell function.

| CN2 is abundant in human kidney proximal tubules
First, we analyzed CN2 expression within different organ tissues of mouse and human.CN2 was most abundant in the kidneys of mouse and human, in particular in the proximal tubular segments, without differences between proximal tubular segments, and in smaller amounts also in distal tubules, parietal epithelial cells and glomerular cells (Figure 1).The most abundant DP in human kidneys were anserine, Asp-Gln, Gly-Asp, carnosine, and His-Ser, and the most abundant AA were glutamate, glycine, alanine and glutamine (Tables S1A,B).CNDP2-KO in HK-2 cells was established by CRISPR/Cas9 genome editing.After transfection, three independent CNDP2-KO clones (D10p1, H5p2 and H4p6) were identified with biallelic indel mutations leading to frameshifts and premature stop codons (Figure S1A).Western Blot analysis confirmed the absence of CN2 protein expression in all three clones compared to WT HK-2 cells and verified CNDP2 deficiency in CNDP2-KO clones (Figure S1B).Subsequent experiments were performed with clone H4p6, in which metabolic degradation activity for the substrate Ser-Gln was almost abolished (Figure S1C).Specific markers for proximal tubular epithelium AQP1, SGLT2 and NH3 + were present in WT and CNDP2-KO cells (Figure S2).

CNDP2-KO proximal tubular cells
To test then the extent to which CN2 plays a role in DP degradation in the cells, we compared the degradation rates of WT with those of CNDP2-KO (Table 1).Loss of CN2 function reduced degradation of 7 out of 21 DP studied and led to a marked (32%-86%) reduction of the concentration of all AA, which reached significance levels for >50% (11/19 AA).This effect was most marked for the highly abundant amino acids glutamate/glutamine (measured Glu may include des-aminated Gln), aspartate, serine, and alanine, but was also (highly) significant for cysteine and 5-oxoproline involved in the gammaglutamyl cycle (Table 2).In contrast, there was a marked increase in the cellular concentration of most DP, with an F I G U R E 1 CN2 in human and mice kidney, lung, liver and pancreas.Immunohistochemical staining of CN2 (scale bar 100 μm) demonstrates high CN2 abundance in proximal tubules (PT) and lower abundance in distal tubules (dt), glomeruli (G) and parietal epithelial cells (PEC), while minor amounts can be detected in the other organs.increase by 25%-101% for the highly abundant dipeptides (concentration >100 pmol/mg: Gly-Glu, Gly-Asp, anserine and Ala-Gln) and reached 168% for cysteinylglycine (Cys-Gly) which plays a major role in the gamma-glutamyl cycle.Cys-Gly, Gly-Glu and Ala-Glu were significant, twofold increased.The CN2-dependent DP accumulation is in line with the central role of CN2 in cellular DP metabolism.CNDP2-KO caused a marked reduction in the DP metabolic rate, which was most significant for Cys-Gly cleavage (Table 1).Although the DP degradation was reduced for several DPs in the CNDP2-KO, the degradation rate was not fully abolished, indicating the activity of further dipeptidases.Recombinant CN2 also has a broad DP substrate range and high DP degradation rates (Table S2); degradation efficiency (V max /K M ) was high for Cys-Gly and Ser-Gln, and 540-and 160-fold lower for carnosine (Table S3).The human kidney degradation rate for Ser-Gln was 19.4 ± 8.8 nmol/mg•h and for carnosine only 3.5 ± 2.4 nmol/mg•h, despite the presence of CN1 in the human kidney tissue.We then studied putative regulatory mechanisms of CN2 activity.Cys-Gly dose-dependently decreased Ser-Gln degradation by rCN2 and vice versa Ser-Gln reduced Cys-Gly degradation rate, suggesting an interacting inhibitory feedback loop on CN2 activity; GSH had a DP substrate-specific effect (Figure S3).

CNDP2-KO proximal tubular cells
In line with a reduced concentration of the important gamma-glutamyl cycle amino acids (Cys, Gly, 5-oxoproline, Glu) and increased Cys-Gly, CNDP2-KO also led to a significant reduction of glutathione concentrations both in the reduced (GSH) and oxidized (GSSG) state (by 60% and 80%, Figure 2).In the presence of redox stress, loss of CN2 function was not associated with a reduced GSH/GSSG ratio (87:1 in WT and 200:1 in CNDP2-KO cells).In line with this, the NAD/NADPH ratio-a measure of redox status-and malondialdehyde, a marker of lipid peroxidation, were not different in CNDP2-KO and WT cells (Table S4).Exogenous oxidative stress did not further reduce CNDP2-KO cell viability than in WT cells (Figure S4A).Cys-Gly, which accumulated in the CNDP2-KO cells and has a high antioxidative capacity (Figure S4B), may have compensated for the alterations in the glutathione pathway.CNDP2-KO-cells, however, were more sensitive to Cisplatin, which interferes with DNA replication, and cell viability was consistently reduced compared to WT cells (Figure S4C).20% of human gluconeogenesis occurs in the kidney.Among the reduced AA in CNDP2-KO cells, nine are glucogenic (Gln, Asp, Ser, Glu, Ala, Cys, Val, His, and Gly), together with a reduced pyruvate concentration in compared to WT cells, while glucose and glycerol-3P concentrations were not altered.Energy status was reduced, that is, AMP/ATP and ADP/ATP ratios were increased (Table S4).The concentration of myo-inositol, a carboxylic sugar derivate, was reduced in CNDP2-KO compared to WT cells.In downstream metabolic pathways, expression of myo-inositol oxygenase (MIOX) mRNA was doubled, while Inositol-3-phosphate (MIPS) and Inositol monophosphatase (IMPA 1) were not altered (Figure S5).Lipidomic analysis of CNDP2-KO cells demonstrated differences in the glycerophospholipid composition, total glycerophospholipid concentration was unchanged (p = 0.6).Glucosylceramides were decreased (Figure S6, Table S5).

| CN2-dependent alterations of proximal tubular cell viability
CNDP2-deficient cells were phenotypically altered, that is, were larger (forward scatter: 233 ± 3 vs.197 ± 2), and displayed higher granularity (side scatter 190 ± 1.1 vs. 215 ± 5.7; two-way ANOVA both p < 0.0001; Figure 3A).CNDP2-KO cell viability was 25% and 50% lower than that of WT cells after 24 and 48 h, proliferation rate was reduced, readily explaining the time-dependent differences in viable cell counts between WT and KO cells.In WT cells, the DP Cys-Gly and Gly-Glu, accumulating in CNDP2-KO cells dose-dependently reduced viable cell counts, demonstrating detrimental effects of the DP accumulation (Figure 3B,C).
In contrast, supplementation of the 11 most depleted AA and myo-inositol for 48 h did not restore viable CNDP2-KO cell counts (Table S6), indicating non-reversibility of the metabolic depletion-induced effects.Likewise, the activity of caspase-3 was 1.0 ± 0.1 relative to control in both WT and KO cells, excluding an impact of caspase-dependent cell apoptosis.We did not find evidence of ferroptosis in CNDP2-KO proximal tubular cells.Expression of ferroptosis-related prostaglandin-endoperoxide synthase 2 (PTGS2) was reduced to <1% (p < 0.0001) and the ferroptosis inhibitor ferrostatin (0.1-0.3 μM) did not improve CNDP2-KO cell viability.Iron stress (ammonium ferric citrate, 0.05-0.4mM) did not further reduce cell viability of KO cells than in WT cells.

CNDP2-KO human proximal tubular cells
We analyzed how the KO of CN2 affected biological processes by RNA-seq.GO term analysis of differentially regulated genes demonstrated upregulation of the biological processes' ion transport, positive regulation of cell communication, cell adhesion and cell junction organization, while regulation of localization, cell development, locomotion and regulation of ion transport were downregulated (Figure 4).KEGG pathway analysis indicated alterations in pathways related to "protein digestion and absorption" (hsa04974) and "amino acids" (hsa00330, hsa00380).We therefore also analyzed other dipeptidases and DP transporters.Expression of dipeptidyl peptidase 3 (DPP3) and peptidase D (PEPD) were reduced by 40% (both p < 0.0001) in CNDP2-KO cells, but not of dipeptidase 1 (DPEP1) and DPP 4, 7, 8, 9. Expression of the dipeptide transporter PHT2 (SLC15A3) was 1.6-fold upregulated in KO cells, PEPT2 (SLC15A2), PHT1 (SLC15A4) and Cubilin (CUBN) were similar in CNDP2-KO and WT cells.DPEP2 and 3 and PEPT1 (SLC15A1) were not expressed in HK-2 cells.

| CN2-dependent trans-and paracellular ion and solute transport
To understand the consequence of CNDP2-KO-induced metabolic changes, we studied key transport functions of proximal tubular cells.General ion permeability, assessed by transepithelial resistance (TER) measurement, was higher in CNDP2-KO HK-2 cells over time and reached a two-fold higher plateau level than WT cells after 9 days (39.9 ± 6.9 vs. 22.5 ± 2.9 Ω•cm 2 in WT cells, p < 0.0001, Figure 5A).Incubation of WT cells with the three DP accumulating most in the CNDP2-KO cells (Ala-Glu, Cys-Gly and Gly-Glu) at 0.33-and 1-mM concentrations increased TER (Figure 5B), suggesting that this accumulation due to disturbed DP degradation in the CNDP2-KO increased TER.
As TER reflects ion permeability, we measured transand paracellular main ion permeabilities across key transcellular ion transporters.Cystic Fibrosis Transmembrane Conductance Regulator (CFTR; chloride-transport) and SGLT1/2 (sodium-glucose transporter 1 and 2) function were unaffected by the CNDP2-KO (Figure 6A).In contrast, the activity of the sodium-proton-exchangers (NHE) was increased (Figure 6B), suggesting also alterations of the acid-base regulations and activity of proton-driven DP transport via peptide transporters like PepT2.Dilution potential measurements demonstrated higher cation selectivity in CNDP2-KO HK-2 cells than in WT cells, as the ratio P Na /P Cl increased (1.17 ± 0.09 vs. 2.00 ± 0.20, n = 11;16, p < 0.01, Figure 6C).This change was based on a reduced permeability for chloride, while the permeability for sodium was unaffected (Figure 6D).Analyzing permeability for macromolecules in CNDP2-KO cells, we found reduced permeability coefficients for the paracellular flux markers 10 kDa dextran and 66 kDa albumin (71.1 ± 6.0% and 37.9 ± 6.6%; p < 0.05, <0.0001) in comparison to WT cells (Figure 6E).Paracellular transport of ions, solutes and water is determined by the composition of tight junction (TJ) proteins.We therefore analyzed TJ expression in the proximal tubular cells.CLDN2, CLDN8, and ILDR2 were reduced, while CLDN1 and CLDN3 protein expression were increased  in CNDP2-KO versus WT HK-2 cells (Figure 7A).ZO-1, a scaffolding protein of the TJ, was not changed in abundance, but increasingly localized in the membrane of the CNDP2-KO cells suggesting a higher TJ network integrity (Figure 7B).

| DISCUSSION
Knowledge on the DP metabolism in health and disease is mainly limited to carnosine and CN1, even though significant biological functions have been demonstrated for other DPs such as Ala-Gln and Tyr-Asp. 1,2We now demonstrate that CN2 degrades carnosine only to a minor extent, in line with previous findings. 24In contrast, both recombinant and proximal tubular cell CN2 have a broad specificity for degradation of a variety of DP, with high degradation rates.Depending on the DP, degradation rate was 4-to 300-fold higher than the degradation of carnosine by recombinant human CN1, 25 by CN1 in human proximal tubular epithelial cells and by human kidney tissue. 26This underlines the metabolic importance of CN2 for the kidney and its potential as a key candidate regulating DP-dependent cellular functions.
CN2 is ubiquitously expressed with high levels found within the kidney. 6We show that within the human kidney, CN2 is present in parietal glomerular epithelial cells that are essentially involved in glomerular regeneration and scarring, 27 and to be highly abundant in human proximal tubules, that reabsorb the vast majority of freely filtered solutes. 15We did not observe differences in CN2 abundance across the proximal tubular segments.Recent kidney single-cell studies confirm CNDP2 expression in the proximal tubules in different clusters (human protein atlas), but mRNA levels do not necessarily reflect protein abundance.Besides most ions and water, nutrients are completely reabsorbed here; carbohydrates like glucose, also AA and DP, and the highly abundant CN2 may play a critical metabolic role within the proximal tubular cells.In line with this notion, CNDP2-KO in human proximal tubular cells increased cellular concentrations of the majority of DP, but without a complete breakdown of DP degradation, which suggests partial compensation by other dipeptidases.We identified seven further dipeptidases beyond CN1 and CN2, of which DPP3 and PEPD were downregulated in CNDP2-KO, which may explain the observed discrepancies in the accumulation of several DP and their CN2-dependent metabolic rate.
Together with the altered cellular DP profile, the AA profile was markedly affected in CNDP2-KO cells, with 11 of 19 measured AA being reduced.This indicates a strong impact of CN2 on cellular AA availability, by high rate degradation of the different DP, present at about 1000-fold lower cellular concentrations than AA.These findings are also in line with previously reported correlations of the organ-specific DP profiles in mice with their respective AA concentrations. 28The cellular AA pool is determined by a tight balance between uptake, biosynthesis and protein catabolism and plays a central role in energy production, macromolecule formation, and for glutathione synthesis and thus in the regulation of fundamental cellular processes. 29,30Cellular uptake of dipeptides occurs by proton-coupled oligopeptide transporters (POTs) via an inwardly-directed proton gradient and negative membrane potential.At present, four members of the POT family, namely PEPT1 (SLC15A1), PEPT2 (SLC15A2), PHT1 (SLC15A4) and PHT2 (SLC15A3), have been identified in mammals. 31,324][35] In the proximal tubule, megalin and the cubilin/amnionless receptor mediate the uptake of filtered proteins that escape the glomerular filtration barrier; 36 however, the role of megalin and cubilin in renal dipeptide transport needs further investigations.According to our RNA-seq data, SLC15A2 (PEPT2), SLC15A3 (PHT2)) and SLC15A4 (PHT1) and cubilin (CUBN) are expressed in the proximal tubular cells, with a higher expression of SLC15A3 in KO compared to WT cells.
Additional metabolic alterations secondary to CNDP2-KO relate to glucose and energy metabolism.20% of human gluconeogenesis occur in the kidney, the proximal tubule is the primary location for renal gluconeogenesis. 37Nine glucogenic AA were reduced, as was the pyruvate concentration and the energy status in CNDP2-KO compared to WT cells.Cellular glucose and glycerol-3P concentrations were not altered, which, however, may be due to a lack of gluconeogenic activity in the HK2 cells and greater effects might occur in vivo.Proximal tubular cells are highly energy-dependent. 19hese findings, together with the alterations in downstream metabolites and in the lipid profiles deserve further studies.The AA cysteine, glycine and glutamate, composing glutathione and 5-oxoproline, another component of the glutathione cycle, were reduced, readily explaining the lower cellular concentrations of GSSG and GSH.In vitro studies support the finding that CN2 is involved in cysteine recycling and the redox homeostasis of tubular cells. 6The reduced glutathione levels of CNDP2-KO cells, however, did not result in a higher sensitivity to oxidative stress, possibly due to the intracellular accumulation of Cys-Gly, which we demonstrate to have high antioxidative action, similar to N-acetylcysteine.However, cell viability reduction by cisplatin, a chemotherapeutic agent interfering with DNA replication, was more pronounced in CNDP2-KO than in WT cells, a finding of potential relevance in cancer treatment.The therapeutic use of GSH in tumor diseases under cisplatin treatment is debated, as the conjunction of GSH with cisplatin may reduce the efficacy of Cisplatin. 38The previously suggested link of the CNDP2-KO and altered glutathione status to ferroptosis 6 could not be reconfirmed in the human proximal tubular CNDP2-KO cells.Of note, the immortalized HK-2 cells, albeit widely used, may only partially reflect metabolic functions, different results may be obtained with primary human proximal tubular epithelial cells in vitro, and in kidneys in vivo.
Knock-out of CNDP2 resulted not only in major metabolic alterations but also deteriorated essential proximal tubular cell functions, including para-and transcellular solute transport.RNA-seq analyses revealed several biological functions being affected by the CNDP2-KO, including cell adhesion, communication, junction organization and ion transport.In addition, the viability and proliferation rate of the CNDP2-KO cells were reduced, which we were able to link to the accumulation of DP.In WT cells, Cys-Gly, accumulating in CNDP2-KO cells, dosedependently reduced cell viability, and supplementation of depleted AAs, or myo-inositol did not restore the viability of the CNDP2-KO cells, pointing to a more complex metabolic imbalance and irreversible alterations, respectively.Consistent with the notion of Cys-Gly induced cytotoxicity, in hepatoma-derived cells, CN2 knock-down reduced Cys-Gly turnover and cell viability. 6he metabolic shifts in DP and AA concentrations in CN2 deficient proximal tubular cells also affected the transport for ion and macromolecular solutes as suggested by the KEGG analysis.General ion permeability is reflected by the TER, which we found to be increased in the CNDP2-KO, also indicating a tightening effect with enhanced barrier properties.Again, a DP-dependent effect is suggested by the increased TER of WT cells incubated with three DPs accumulating in the CNDP2-KO Permeability for macromolecules (10 and 66 kDa) was reduced.Within the nephron under physiological conditions, 10 kDa molecules pass the intact glomerular filter and undergo tubular reabsorption.The much bigger albumin (66 kDa) only passes the filter in disease settings representing an early sign and pathomechanisms of nephropathy in the majority of patients. 39The decreased permeability for macromolecules suggests a tightening of the epithelium, lowering uptake in response to the DP accumulation.
As the proximal tubule is a leaky epithelium, high transport rates not only occur via the large number of transporters facilitating transcellular uptake, but take place to even higher extent via the paracellular pathway, which is determined by tight junction proteins, mainly by the composition of members of the claudin family. 40e found CLDN2 and CLDN8 to be markedly reduced and CLDN1 and 3 to be increased, which could lead to the observed changes in ion permeability.While CLDN1 and CLDN3 are barrier-enhancing claudins without any charge selectivity, 41,42 CLDN2 is forming paracellular channels for cations and water. 43Its downregulation should enhance the barrier against cations but we rather cation-selective permeability in the CNDP2-KO cells.As CLDN8 is forming selective paracellular barriers against cations, 44,45 its downregulation could counterbalance the effects that were expected due to CLDN2 downregulation, leading to an apparent permeability reduction only for anions, although both were affected on paracellular level.Other tight junction proteins of relevance for ion permeability within the proximal tubule were not affected.
However, though not changed in expression, we observed an increased abundance of the scaffolding protein ZO-1 at the membrane of the CNDP2-KO cells, suggesting higher tight junction network integrity and stability, which again supports the finding of an enhanced barrier.Furthermore, ZO-1 is an important factor for osmotic pressure regulation of cell volume 46 and has tensiondependent functions for epithelial stability. 47Changes in ZO-1 enrichment together with the observed changes in ion, DP and AA concentrations might also affect intracellular osmotic pressure.This assumption is supported by the observed upregulation of MIOX, an enzyme which is exclusively expressed in the kidney proximal tubule 48 and up-regulated by hyperosmotic stress. 49Altered osmotic pressure may in turn further affect ion and solute transport.On the transcellular level, we did not find changes in CFTR and SGLT 1/2 transport activities but observed higher activity of the NHEs.In the proximal tubule, NHE1, NHE3, NHE4 and NHE8 are expressed within the apical or basolateral cell membrane and function as cation-proton antiporters. 50They are involved in cell volume and cellular pH regulation, again pointing to altered osmotic pressure.In addition, the proton transport by NHEs links these transporters to di-and tripeptide uptake via for example PepT2, possibly contributing to the alterations in DP and AA profiles observed with the lack of CN2.The increased NHE activity may represent a regulatory response to the altered peptide metabolism and the assumed hyperosmotic pressure.

| Implications for pathological significance and outlook
Despite the limitations of in vitro cell models studied in limited number of independent experiments, the consistent and major alterations of DP and AA abundances and of associated metabolic pathways in CN2 deficient tubular cells together with markedly comprised cell functions including ion and large solute transports, suggest far reaching impact of CN2, which deserve subsequent in vivo studies.In complex In vivo systems, the CN2 activity may have a very different impact than in vitro in the human proximal tubular epithelial cells.In context of the nephron, dysregulation of CN2 might affect the whole body, as the kidney has important regulatory functions not only in reabsorption of ions and nutrients or excretion of metabolic end products and toxins, but also in regulation of acid-base-homeostasis and of blood pressure.Ion transport along the nephron regulates extracellular volume and blood pressure. 51The proximal tubule plays a critical role in the progression of kidney disease, 18 CN2-related dysfunction may impact outcomes in patients with hypertension, diabetes and chronic kidney disease, where genetic variants of CNDP2 already have been linked to outcome. 10,11Also, the role of CN2 present in human parietal glomerular epithelial cells, which play a key role in glomerular regeneration, 27 deserves in-depth analyses.Additionally, the association of CN2 expression to tumors [12][13][14] might be well-connected to altered regulations in metabolism, which we here observed on cell culture level, but which in organs might influence tumor development and progression.

| MATERIALS AND METHODS
Chemicals were purchased from Sigma-Aldrich (Schnelldorf, Germany) unless indicated otherwise.

| Cell culture
Immortalized human tubular cells (HK-2; American Type Culture Collection CRL-2190) were grown in RPMI GlutaMAX medium (Thermo Fisher Scientific, Waltham, MA) with 0.1 or 10% fetal calf serum (v/v) and 1% penicillin and streptomycin (v/v) at 37°C with 5% CO 2 .Experimental settings were performed in 0.1% fetal calf serum media, whereas 10% fetal calf serum media was used to adhere the cells after splitting.Cells were splitted using 0.25% trypsin (Thermo Fisher Scientific, Waltham, MA) and were used from passage 1 to 16.

| Enzyme activity
CN2 activity was assayed for recombinant enzyme, in cell culture and human kidney tissue according to a method described before. 53In brief, the reaction was initiated by the addition of 1 mM individual dipeptide to the recombinant enzyme (human CNDP2 protein, GeneTex, Irvin, CA), to cell homogenate in optimized RIPA buffer (50 mM Tris/HCl, 0.1% TritonX-100 (v/v), 0.5% Sodiumdeoxycholat (w/v), 50 mM NaCl and 20 μM MnCl 2 , pH 7.5) or human kidney homogenate in specific buffer (20 mM Hepes pH 7.2, 210 mM mannitol, 70 mM sucrose, 50 mM NaCl, 20 μM MnCl 2 , pH 7.5).The reaction was terminated at pre-determined intervals in liquid nitrogen or by adding 1% trichloroacetic acid depending on the method of amino acid detection.

| RNA-seq
Total RNA was extracted from HK-2 human kidney tubular cells wild type or subjected to CRISPR-Cas9 mediated CNDP2-KO (5 vs. 5 samples) using the total RNA Kit from peqGOLD VWR (VWR, Radnor, PA, USA) with incolumn DNase treatment, following the manufacturer's standard protocol.RNA concentration was quantified with a Nanodrop 2000 and the RNA quality was assessed using the Bioanalyzer 2100 with the RNA 6000 nano kit (Agilent).Only samples with RIN (RNA integrity number) >9 were used for RNA-seq library preparation in multiplexing using the Illumina Truseq Stranded mRNA kit.The library has been run on an Illumina Nextseq 500 sequencer.Demultiplexed Fastq files were checked for quality with FastQC program and mapped against the GRCh38 human genome with the GENCODE 27 (Ensembl 90) annotation using the STAR (Spliced Transcripts Alignment to a Reference) package.Differentially expressed genes were identified with the DESeq2 package, 54 using the Benjamini-Hochberg (BH) adjustment for false discovery rate (FDR) calculation.Principal Component Analysis (PCA) plot was generated using the plotPCA function of the DESeq2 package.Volcano plot has been obtained using the default scatterplot function of R. Protein coding genes with a FDR < 10% and a log2 (fold change) >2 and <−2 were selected for subsequent analysis.GOterms relative to biological processes (BP) and molecular function (MF) were calculated using the Classic Fisher statistics of the topGO package and were plotted using the ggplot2 package (Bioconductor).Similarly, KEGGS-mapped pathways were calculated on differentially expressed genes using the Fisher statistics.Heatmaps were generated on significant or custom GOterms based on scaled rlog (regularized log transformation) expression values (z-score).RNAseq fastq files can be obtained from the Arrayexpress platform (www.ebi.ac.uk/ array expre ss/ ) under the accession number E-MTAB-11436.

| Stress conditions
Experiments simulating stress conditions were all performed in 0.1% fetal calf serum media with 1% penicillin and streptomycin (v/v).Oxidative stress was induced by incubation with 0.3, 3 and 6 nM FeSO 4 and 0.05, 0.5 and 1 mM H 2 O 2 (Fenton reagent) for 24 h.Treatment with 0.1, 0.2, 0.3 or 0.4 μM Ferrostatin-1 was performed over 48 h.Ammonium ferric citrate (FAC) was used to simulate ferroptosis; glucose stress was simulated by increasing glucose concentrations; treatment duration was over 24 h.

| Cell proliferation assay (BrdU)
Cell proliferation rate was determined with the Assay Cell proliferation Elisa BrdU (Roche, Basel, Swiss) following precisely manufacturing protocol.5000 to 313 HK-2 WT and CNDP2-KO cells per well were seeded into a 96 well plate following 2-fold serial dilution and cells were adhered overnight under 10% FCS media condition.Cells were washed with DPBS following incubation with media containing 0.1% FCS for 24 h.Incubation with BrdU labelling solution was performed for additional 24 h, and measurements were performed photometrically at 370 nm 5 min after adding the substrate solution to the wells.For evaluation, proliferation rate of 1250 cells seeded per well was used.

| FACS
The size and granularity in HK-2 WT and CNDP2-KO cells was analyzed by flow cytometry.Cells were centrifuged for min at 300 g, 4°C and in PBS with 1% FCS resuspended for analysis.Measurements were performed using an LSR II cytometer (BD Biosciences, Heidelberg, Germany) and analyzed by FACS Diva (Becton Dickinson, San Jose, CA, USA) and FlowJo software version 10.1r5 (Ashland, OR, USA).Cell size and granularity were determined by forward Scatter (FSC) and side scatter (SSC).
For GC/MS analysis, a GC-ToF system was used consisting of an Agilent 7890 Gas Chromatograph (Agilent, Santa Clara) fitted with a Rxi-5Sil MS column (30 m × 0.25 mm × 0.25 μm; Restek) coupled to a Pegasus BT Mass Spectrometer (LECO).The GC was operated with an injection temperature of 250°C and 1 μL sample was injected with a split ratio of 10.The GC temperature program started with a 1 min.hold at 40°C followed by a 6°C/ min ramp up to 210°C, a 20°C/min ramp up to 330°C and a bake-out at 330°C for 5 min.using Helium as carrier gas with constant linear velocity.The ToF mass spectrometer was operated with ion source and interface temperatures of 250°C, a solvent cut time of 9 min and a scan range (m/z) of 50-600 with an acquisition rate of 17 spectra/s.The ChromaTof v5.50 software (LECO Corporation, Michigan) was used for data processing.
To analyze total fatty acids, 80 μL of the lower organic phase after extraction were transferred to a glass vial and dried in a speed-vac without heating.For transmethylation reactions, pellets were re-dissolved in 40 μL TBME (tert-Butyl methyl ether, Sigma) and 20 μL TMSH (Trimethylsulfoniumhydroxid, Sigma), incubated for 45 min.at 50°C and analyzed using a GC/MS-QP2010 Plus (Shimadzu®) fitted with a Zebron ZB 5MS column (Phenomenex®; 30 m × 0.25 mm × 0.25 μm) for fatty acid methyl esters (FAME).The GC was operated with an injection temperature of 230°C, 1 μL sample was injected with split mode (1:10).The GC temperature started with 40°C (1 min) followed by a 6°C/min ramp to 210°C, a 20°C/min ramp to 330°C and a bake-out for 5 min.at 330°C using Helium as carrier gas with constant linear velocity.The MS operated with ion source and interface temperatures of 250°C, a solvent cut time of 7 min and a scan range (m/z) of 40-700 with an event time of 0.2 s.The "GCMS solution" software (Shimadzu®) was used for data processing.

| Transepithelial electric resistance (TER)
Tubular cell monolayer integrity and barrier function was assessed as described previously. 58In brief, a cell suspension (5 × 10 4 cells/cm 2 ) was seeded and cultured on a polyester mesh transwell filter (0.4 μm pore size, 12-well type; Costar.MA.USA) under normal culture conditions.The inner and outer chambers of the Transwell were filled with 0.2 mL and 1 mL culture medium, respectively.TER was measured daily using an EVOM volt/ohm meter with STX-2 electrodes (World Precision Instruments, Sarasota, FL, USA).To calculate the normalized TER of each monolayer.Background TER of a blank filter was subtracted from the TER of the respective cell monolayer.To compare baseline TER of WT HK-2 and CNDP2-KO HK-2 cells, TER was measured daily for up to 21 days, and the medium was changed every 3-4 days.The treatment of WT HK-2 with dipeptides was initiated.when the monolayer was formed and differentiated as demonstrated by a plateau in the TER (around 5 days post-seeding).

| Paracellular permeability analysis of HK-2 monolayers
The paracellular permeability of HK-2 monolayers was determined by measuring concentrations changes of fluorescein isothiocyanate (FITC) labeled 10 kDa dextran and 66 kDa albumin (Sigma-Aldrich, St. Louis, MO, USA) in the lower transwell compartment, following their addition to the upper compartment at a concentration 1 mg/mL.An equimolar amount of unlabeled dextran was added to the lower compartment of the Transwell to maintain isotonic conditions.Calibration curves were established from FITC dextran stock solutions and sample fluorescence was determined using a fluorescence spectrophotometer (F-2000; Hitachi, Tokyo, Japan) at an excitation wavelength of 490 nm and an emission wavelength of 520 nm in one black 96 well plate (Greiner, Nürtingen, Germany).Solute permeability coefficients (SPC; m/s) were calculated using the following formula: where V is the volume of the lower compartment (m 3 ), A the membrane area (m 2 ), ∆t the time interval (s), and C the concentration of the molecular marker (Cu0 = concentration at the upper side at time 0; Cl4 = concentration at the lower side at 4 h; Cl2 = concentration at the lower side at 2 h).

| Determination of NHE activity
To measure the transport activity of NHEs, the pH-dependent fluorescence of acridine orange was used.Cells were seeded in same amounts on 96-well plates and grown to confluence.Medium was replaced by sodium-free preincubation buffer (280 mM Mannitol, 5 mM MES, 2 mM MgCl 2 , pH 5.5) with or without 10 μM EIPA (5-(N-Ethyl-N-isopropyl)amiloride), which inhibits several NHEs completely or in parts, 59 for 2 h.The buffer was then replaced by sodium-free measuring buffer (240 mM Mannitol, 20 mM HEPES, 2 mM MgCl 2 , 6 μM acridine orange, pH 7.5) with or without the 10 μM EIPA.Emission shifts at 530 nm were detected for 1 min after addition of Na-Gluconate (final concentration 100 mM) in a plate reader (Tecan Infinite M200, Tecan, Switzerland, 493 nm excitation and 530 nm emission), and slopes of intensity changes were compared between wildtype and CN2-KO cells in the absence or presence of the NHE3 inhibitor EIPA.

| Dilution potentials
Cells grown on transwell filters were mounted into Ussing chambers with an area of 0.6 cm 2 .Resistance of bathing solutions was measured prior to each experiment and subtracted.Ussing chambers and water-jacketed gas lifts were filled with 10 mL standard Ringer's solution (in mM: Na + 140; Cl − 149.8;K + 5.4; Ca 2+ 1.2; Mg 2+ 1; HEPES 10; D(+)glucose 10, D(+)-mannose 10, beta-hydroxybutyric acid 0.5, and L-glutamine 2.5 mmol/L, pH 7.4).The solution was equilibrated with 5% CO 2 and 95% O 2 at 37°C.Potential changes were recorded by switching the solution of one hemi chamber to a solution containing a reduced concentration of NaCl and all other components identical to standard Ringer's.Osmolality was balanced by mannitol.The resulting dilution potentials were used for calculations using the Goldman-Hodgkin-Katz equation as reported before. 43,60,  of transporter activity depending on ions.CFTR activity was determined by stimulating Cl − secretion by 10 μmol/L forskolin, inhibiting CFTR then with 10 μmol/L 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and stimulating again to derive noninhibitable fractions.For glucose transport, transport via SGLT1/2 was stimulated by addition of glucose (100 mM 1 mL/5 mL apical bathing solution).After 20 min, phlorizin was added apically, and after further 20 min, again glucose was added to derive non-inhibitable fractions.

| Mice and human tissues
Wildtype mice (C57BL/6J) were housed in the Interfaculty Biomedical Facility (IBF) at Heidelberg University.Food and water were supplied ad libitum.At sacrifice (age 40 weeks), kidney, lung, liver and pancreas were harvested, formalin fixed and paraffin embedded for histopathology.The studies were approved by the respective authorities (Regierungspräsidium Karlsruhe, Germany, 35-9165 81/G-209/16).
Human tissue samples were provided by the Tissue Bank of the National Center for Tumor Diseases (NCT), Heidelberg, Germany, in accordance with the regulations of the tissue bank and the approval of the ethics committee of Heidelberg University (S-284/2018).Immunohistochemical staining against CN2 was performed in tissue microarrays (TMAs).
Cryo-conserved samples were used for dipeptides and amino acid analysis, mRNA und protein expression analysis via qPCR and western blotting enzymatic activity.Analysis were performed as described in the respective paragraphs.

| Statistics
All experiments were performed at least in three biological replicates, including 3 to 16 technical replicates.All data are presented as mean ± SD, if not stated otherwise.Student's t-test or if three or more groups were tested against each other one-way ANOVA were performed.Outliers for experiments with human kidney tissue were identified using robust regression and outlier removal (ROUT) method with Q = 1.Statistical analysis was performed using GraphPad Prism 9 software.Evaluation of RNA-seq data see corresponding paragraph.Two sided tests were used, and p value <0.05 was considered significant.

| CONCLUSION
In conclusion, CN2-mediated DP degradation has major impact on the metabolic profile of human proximal tubular cells.CN2 deficiency substantially compromises DP, AA and associated metabolic pathways, reduces energy status, cell viability and proliferation, and alters essential tubular cell transport functions, altogether indicating a key role of CN2 enzyme, that already can be connected to known kidney pathologies.Our findings implicate the necessity for in vivo modulation of CN2, which will elucidate the functional role of CN2 under physiological and pathophysiological conditions.Understanding of CN2 regulation bears promising therapeutic potential.

F I G U R E 3
CNDP2-KO and WT human proximal tubular cells morphology, viability and proliferation rate.(A) Forward and side scatter (FSC/SSC) profiles demonstrate increased size and granularity of CNDP2-KO HK-2 cells compared to WT cells (FACS, n = 3); representative phase contrast light microscopy images are given on the right (5-, 20-, 40-fold magnification).(B) Cell viability was decreased in CNDP2-KO (white bar) compared to WT cells (black bars, 0.1% FCS, MTT assay; n = 16), and in WT cells dose dependently reduced with two of the three DP most accumulated in CNDP2-KO cells, n = 3. (C) Proliferation rates were reduced by the CNDP2-KO (BrdU-assay), n = 5.Data are mean and SD, *p < 0.05, †p < 0.01, ‡p < 0.001 (unpaired t-test).

F I G U R E 4
Gene and biological pathway regulation in CNDP2-KO human proximal epithelial cells.(A) Principal component analysis revealed difference between CNDP2-KO and WT HK-2 cells with a clustering of the individual replicates.(B) Up-and down-regulated genes in CNDP2-KO HK-2 vs. WT cells (n = 5).The vertical dashed lines indicate 4-fold increased, the horizontal dashed line displays -log10(0,1) = 1.(C) Upregulated and (D) downregulated biological processes are displayed regarding their GeneRatio, significances as -log(pvalue) and the number of significant altered genes.

F I G U R E 5
Transepithelial resistance and paracellular macromolecule transport in CNDP2-KO and WT human proximal tubular cells.(A) Transepithelial resistance, a measure of ion permeability, was increased in CNDP2-KO HK-2 cells compared to WT cells (n = 10-11) and (B) increased in WT cells when exposed to the combination of the three DP most accumulated in the KO cells.Data are mean and SD.*p < 0.05, †p < 0.01, ‡p < 0.001 (unpaired t-test).

F I G U R E 7
Tight junction protein expression and location in CNDP2-KO and wild type human proximal tubular cells.(A) Expression of tight junction proteins Claudin 2 and 8 (CLDN2 and 8) and of Immunoglobulin Like Domain Containing Receptor 2 (ILDR2) was decreased and of CLDN 1 and 3 was increased in CNDP2-KO cells compared to WT. Representative western blots and quantifications of 4 blots are given.Full blots are deposited in supplementary file.(B) Immunofluorescence staining of scaffolding protein zonula occludens-1 (ZO-1) demonstrates increased membrane ZO-1 abundance in CNDP2-KO cells (n = 4).Data are mean and SEM.*p < 0.05, †p < 0.01 (unpaired Student's t-test).

4. 18 |
Ion secretion/transport via cystic fibrosis transmembrane conductance regulator (CFTR) and glucose transport via sodium-glucose linked transporters 1 and 2 (SGLT1/2) Cells grown on transwell filters were mounted into Ussing chambers in glucose-free Ringer's solution.Changes in short-circuit current (I SC ) were recorded and used for SPC = − 4V ΔtA ln Cu0 − 4Cl4 Cu0 − 4Cl2 DP concentrations and DP metabolic rates in WT and CNDP2-KO human proximal tubular cells.
T A B L E 1

T A B L E 2 AA
concentrations in WT and CNDP2-KO human proximal tubular epithelial cells.