Overall study design, workflow, and schematic overview. (A) Transcriptome profiling with gene microarray performed on cystinotic, normal, and CDME-treated RPTECs and fibroblasts (Step 1). Bioinformatic analysis allowed the identification of genes that are differentially regulated in cystinosis versus normal in both the cell types. Pathway analysis was performed with all the significantly regulated genes, and 11 pathways were found to be significantly affected in cystinotic RPTECs. Most of these pathways and the genes in these pathways are crucial for lysosomal acidic pH and mitochondrial ATP production. Dysfunctional mitochondria and compromised lysosomal pH were validated in step 2. Our CRISPR-mediated CTNS-/- immortalized renal cell line mimicked cystinosis patient primary cells isolated from their urine. Further, we rescued (Step 3) the disease phenotype of the cell by over-expressing ATP6V0A1, the most significantly downregulated gene among other V-ATPases. RNP, Ribonucleoprotein; V-ATPase, vacuolar ATPase; RPTEC, renal proximal tubular epithelial cells. (B) A schematic overview of normal and cystinotic RPTECs. We previously showed that loss of function of cystinosin in cystinotic cells inactivates the mTORC pathway, and induces autophagy, mitophagy, and clusterin protein expression. In this study, we show that downregulation of CTNS gene also downregulates V-ATPase expression resulting in the loss of lysosomal acidity (pH). The basic lysosomal pH thus blocks its clearance after autophagosome-lysosome fusion. Inhibited autophagy flux due to lysosomal acidity loss may explain why cystinotic cells have increased LC3B-II expression. Loss of acidic lysosomal pH also impairs proton- dependent import of amino acids and other metabolites (noted in our microarray data) into the lysosome lumen, and inhibits conversion of these imported large heterodimeric amino acids into usable form. This increases the presence of metabolites in the cytoplasm that may compromise mitochondrial function and increase ROS production through an unspecified mechanism.

Microarray study design and transcriptional changes for cystinotic, normal, and CDME- treated RPTECs or fibroblasts.

(A) Study design: Two types of cells were used- RPTECs and fibroblasts. For each cell type there were three groups – normal that serves as control - commercially obtained, cystinotic – obtained from individuals with cystinosis, and CDME-treated normal cells. (B) PCA plot further highlights the unique injury mediated gene profile in cystinotic RPTEC versus normal. Gene expression patterns of cystinotic RPTECs and fibroblasts were found to have no commonality, with cystinostic fibroblasts similar to normal. Gene expression patterns in cystinotic RPTECs are distinct and negatively correlated with normal RPTECs. Whereas gene expression in normal RPTECs laden with CDME are similar to the normal rather than disease phenotype, and hence have a positive correlation with each other. (C) Microarray data from both cell types are represented as a correlation heatmap. Cystinotic RPTECs showed markedly different expression profiles from both the normal and CDME loaded normal RPTEC, which was more similar to normal than diseased. Gene expression patterns in normal, cystinotic, and CDME-treated fibroblasts were found to be similar to each other depicting that nephropathic cystinosis-related changes are highly specific to renal cells.

Pathway analysis was performed with all the significantly regulated genes, and 11 pathways were found to be significantly affected in cystinotic RPTECs.

List of all the v-ATPases that are significantly downregulated in cystinosis RPTECs compared to normal.

Experimental overview for the development of immortalized CTNS-/- RPTECs using CRISPR- Cas9 RNPs.

(A) Outline of RNP CRISPR editing. The CTNS gene is located on chromosome 17p13.3 and consists of 12 exons, of which the first 2 are non-coding. Therefore, the guide RNA was targeted towards exon 3 to completely knockout the functional CTNS gene. CRISPR-Cas9 ribonucleoproteins (crRNPs) were synthesized in vitro to knockout CTNS gene and delivered to immortalize RPTECs by nucleofection. These cells were expanded for molecular validation of gene editing and downstream functional assays. (B) The guide RNA target sequence and associated PAM are highlighted on the right. We performed Sanger sequencing and the TIDE output calculating percent indels from chromatograms is depicted in the bar- graph. (C) Validation of CTNS knockout (-/-) in immortalized RPTEC by using two primers targeting different exons – CTNS#1 targets between exon 2-3 and CTNS#2 targets between exon 9-10. We have shown that in the CRISPR/Cas9 CTNS-/- RPTEC there is a significant reduced cystinosin RNA levels with both the primers. (D) Validation of CTNS -/- in immortalized RPTEC. Increased intracellular accumulation of cystine is shown by HPLC-MS/MS method in control and CTNS-/- RPTECs.

Vacuolar-ATPase, ATP6V0A1, is downregulated in both, cystinotic and CTNS -/- RPTECs, further confirming that our in vitro cellular model behaves like primary cystinotic renal cells. This highlights that in addition to the loss of lysosomal cystinosin there are also other key biological changes in the lysosomes in nephropathic cystinosis. (A-B) Immunoblot analysis of the expression of ATP6V0A1 in cystinotic and CTNS -/- RPTECs with their respective controls. (4C) Immunoblot analysis of the expression of ATP6V0A1 in lysosomal fraction isolated from control and CTNS -/- RPTECs. Absence of GAPDH expression depicts successful isolation of pure lysosomes from RPTECs. Results are represented as mean ± SD. The statistically significant differences between the two groups are indicated in the figure. Student’s t-test (* P≤0.05; **P≤0.01; ***P≤0.001). There was no difference between LAMP2 expression in control and cystinotic RPTECs, making this marker a good control for the experiment. (D) Immunofluorescence and confocal microscopy showing endogenous LAMP2 and ATP6V0A1 distribution in normal and cystinotic RPTECs. Colocalization of ATP6V0A1 and LAMP2 is shown in the right panel. Results are representative of at least 3 separate experiments. Cystinotic and CTNS -/- RPTECs have an intracellular basic pH compared to their respective controls. Cells were stained with BCECF-AM and measured with a microplate fluorimeter in triplicate wells. The pH-dependent spectral shifts exhibited by BCECF allow calibration of the pH response in terms of the ratio of fluorescence intensities measured at two different excitation wavelengths (490, 440 nm). (E) RPTECs isolated from cystinosis patient urine had a more basic pH than normal individuals (**P≤0.01). (F) Similarly, to the primary cells, our in vitro cellular model, CTNS-KO RPTEC, also had a more basic pH than control. The unpaired t-test indicated significant differences (***P≤0.001) between control and diseased cells. Data are representative of three biological replicates. Values represent mean ±SD for three independent experiments.

Increased immunopositivity to LC3B-II, or autophagy, and decreased immunopositivity to phospho-p70S6 kinase, or mTORC1 activity.

(A) Immunofluorescence and confocal microscopy showing endogenous LC3B and LAMP2 distribution in normal and cystinotic RPTECs. Colocalization of LC3B and LAMP2 is shown in the right panel. (B) Immunofluorescence and confocal microscopy showing endogenous LC3B and phospho-p70S6 kinase (marker of mTOR activity) distribution in normal and cystinotic RPTECs. Colocalization of LC3B and phosphor-p70S6 kinase is shown in the right panel. (C-D) Immunoblot analysis of the expression of phosphorylated-p70S6 kinase and total p70S6 kinase in cystinotic and CTNS -/- RPTECs with their respective controls. Since there is a change in total p70S6K expression in with and without the CTNS, we normalized both, the phosphorylated and total protein, to GAPDH. mTORC1 plays a central role in cell growth, proliferation, survival, and autophagy inhibition via AMPK, therefore, the presence of lower activity of mTORC1 in cystinosis supports the observations of increased cell death and autophagy as shown by increased LC3B puncta. Results are representative of at least 3 separate experiments (biological replicates). Results are represented as mean ± SD. The statistically significant differences between the two groups are indicated in the figure. Student’s t-test (* P≤0.05; **P≤0.01; ***P≤0.001).

The Agilent Seahorse Mitochondria Stress Test detected mitochondrial defects in primary cystinotic and CTNS-/- RPTECs compared to their respective controls. Top Center) XF Cell Mito Stress Test assay design and standard output parameters. (A) XF Cell Mito Stress Test shows that primary cystinotic RPTECs have diminished basal and maximal respiration, ATP-linked respiration, spare respiratory capacity, non-mitochondrial oxygen consumption, and proton leak compared with its control (normal RPTEC) when both were treated with specific electron transport chain (ETC) inhibitors. (B) Similar to the primary diseased cells, the CTNS-/- also demonstrated diminished mitochondrial activity. Both cystinotic and CTNS-/- RPTECs exhibited similar patterns of compromised mitochondrial function, with significantly (Normal vs. Cystinosis: p<0.025, p<0.0019, p<0.023; human immortalized RPTEC (HuIm) vs. CTNS-/-: p=0.006, p=0.001, p=0.0002, respectively) decreased basal and maximal respiration and ATP-linked respiration compared to controls. The OCR linked to proton leak and cells spare respiratory capacity was significantly (Normal vs. Cystinosis: p<0.007, p<0.0007; HuIm vs. CTNS-/-: p<0.0001, p=0.002 respectively) lost in cystinotic cells. Non-mitochondrial oxygen consumption did not significantly change in cystinotic or CTNS-/- RPTECs. There was a substantial (Normal vs. Cystinosis: p<0.007; HuIm vs. CTNS-/-: p<0.0001) increase in OCR linked to proton leak. Values represent mean ± SD for three independent experiments (* P≤0.05; **P≤0.01; ***P≤0.001). OCR, oxygen consumption rate.

Correction of ATP6V0A1, by transfecting the CTNS-/- RPTECs with a Myc-DDK-tagged human ATP6V0A1-plasmid, partially rescued the disease cell phenotype. (A) Immunoblot showing successful correction of ATP6V0A1 protein in the CTNS-/- cells. Correction of ATP6V0A1 significantly reduced the LC3-II protein expression. (B) The XF Cell Mito Stress Test shows that correction of ATP6V0A1 in CTNS-/- cells significantly improved basal respiration, and increased maximal respiration and ATP-linked respiration. Overall, ATP6V0A1 correction in CTNS-/- cells most closely resemble the normal human immortalized (HuIm) RPTECs compared to CTNS-/- or CTNS-/- with control plasmid. Electron microscopic evaluation of CTNS-/- RPTECs, and its partial recovery by overexpressing ATP6V0A1. (C) Correction of ATP6V0A1 in CTNS-/- cells had no effect on its cystine level. (D) TEM of control, CTNS-/- , and ATP6V0A1 overexpressed CTNS-/- (CTNS-/- +V0A1) RPTECs at low magnification. Asterisks indicate autophagic vacuoles (AV). Number of AV per cell from 8 different sections of each sample was counted and depicted as a bar graph. Significant increase in AV (p=0.006) is observed in CTNS-/- compared to that of RPTECs and CTNS-/- +V0A1 groups. (E) TEM of control, CTNS-/- , and CTNS-/- +V0A1 RPTECs at low magnification. Asterisks indicate mitochondria. Number of mitochondria per cell from 8 different sections of each sample was counted and depicted as a bar graph. Significant decrease (p=0.0005) in mitochondria is observed in CTNS-/- compared to that of RPTECs and CTNS-/- +V0A1 groups. Both figures A and B indicates rescue of the diseased phenotype by the correction of ATP6V0A1 (p=0.019; p=0.02, respectively). (F) Structurally abnormal ER-wrapped mitochondria with lesser cristae was observed in CTNS-/- compared to that of RPTECs. Though, structurally normal mitochondria with cristae was observed in CTNS-/- +V0A1 group, but majority of the mitochondria are wrapped by ER. Scale = 0.5um. (G) Intracellular LD was observed in more number of cells in CTNS-/- and CTNS-/- +V0A1 samples than control RPTECs. LDs have a unique structure, which is delimited by a monolayer of phospholipids differing from the classical bilayer structural organization. Arrows indicate endoplasmic reticulum (ER). Scale = 0.2um. One way ANOVA or t-test as suitable. Values represent mean ± SD for at least three independent experiments (* P≤0.05; **P≤0.01; ***P≤0.001).

Astaxanthin (ATX) but not Cysteamine or Vitamin E upregulates ATP6V0A1 and rescued the cystinosis RPTEC phenotype.

(A) Immunoblot showing that 100uM of cysteamine treatment for 24 hours did not increase the expression of ATP6V0A1 in CTNS-/- RPTECs. (B) Immunoblots showing VitaminE has no effect on ATP6V0A1 expression in CTNS-/- RPTECs. (C) Immunoblots showing ATX upregulates ATP6V0A1, reduced the LC3-II (p=0.04) protein expression. Values represent mean ± SD for three independent experiments (* P≤0.05; **P≤0.01; ***P≤0.001). (D) ATX reduced (p=0.0002) mitochondrial ROS normalized to cell viability measured by WST-1. (E) ATX improved mitochondrial membrane potential (p=0.002, p<0.0001 at both 10uM and 20uM, respectively) in CTNS-/- as shown by the JC1 ratio. Values represent mean ± SD for atleast three independent experiments (* P≤0.05; **P≤0.01; ***P≤0.001).

List of the 10 v-ATPases showing no significant changes in its expression in cystinosis fibroblasts, CDME-treated fibroblasts and CDME-treated RPTECs compared to their respective controls.

Amino Acids & Sialic Acid Transporters. Microarray identified additional transcriptional changes in amino acid and sialic acid transporters in cystinotic RPTEC. Significant downregulation of SLC17A1, SLC17A3, SLC17A5, SLC3A1, and SLC7A7. (* P≤0.05; **P≤0.01; ***P≤0.001).

Immunofluorescence and confocal microscopy showing endogenous LAMP2 and ATP6V0A1 distribution in human immortalized RPTEC and CTNS-/- RPTECs. Colocalization of ATP6V0A1 and LAMP2 is shown in the right panel. Decreased immunopositivity to ATP6V0A1 is noted in CTNS-/- RPTECs compared to its control. The pattern is similar to primary RPTECs isolated from individuals with cystinosis.

ATX Dose Response Curve.

We used three concentrations of ATX 10, 20, 50 uM out of which 50 uM was toxic to RPTECs as it killed the cells. However, ATX improved mitochondrial membrane potential (p=0.002, p<0.0001 at both 10uM and 20uM, respectively) in CTNS-/- as shown by the JC1 ratio. Values represent mean ± SD for at least three independent experiments (* P≤0.05; **P≤0.01; ***P≤0.001).