kAE1 R295H, Y413H, S525F and R589H dRTA mutants are either dysfunctional or prematurely degraded.

(A) Alpha Fold predicted structure of the kidney isoform of Band 3 anion exchanger 1 (kAE1) with core and gate domains highlighted. The dRTA kAE1 mutation sites are coloured in blue with line extensions detailing specific amino acids mutated. (B) Immunoblot showing expression of kAE1 WT, R295H, Y413H, S525F and R589H and corresponding actin band in mIMCD3 cells treated with and without doxycycline for 24 hrs. Mouse anti-HA antibody was used to detect kAE1-HA, top (open circle) and bottom bands (closed circle) correspond to kAE1 carrying complex and high mannose oligosaccharides, respectively. (C) Immunostaining of kAE1 WT or mutant (red) and β-catenin (green) in polarized mIMCD3 cells. Scale bar = 10 μm. Red = kAE1, Green = ß-Catenin. (D) Immunoblot of cycloheximide (CHX) chase assay with corresponding actin in kAE1 mIMCD3 WT and Y413H cells showing the degradation of kAE1 Y413H after 3hrs CHX incubation. (E) Cartoon depicting the transporter activity and expected changes in pHi in cells expressing kAE1 WT (left) or inactive mutant (right). (F) Graphical representation of intracellular pH (pHi) measurement of mIMCD3 kAE1 WT, R295H, Y413H, S525F and R589H cells. Error bars correspond to mean ± SEM, n = minimum 32. *P<0.05, **P<0.01 using one-way ANOVA followed by a Dunnett’s post-hoc test. (G) Rate of intracellular alkalinisation in WT or mutant mIMCD3 cells normalized to WT+ Dox. **** indicates P < 0.0001 using one-way ANOVA followed by a Dunnett’s post-hoc test. Error bars correspond to mean ± SEM, n= minimum 4

Autophagy is upregulated in dRTA kAE1 mutants in vitro and in vivo.

(A-C) Representative immunoblots of LC3B and p62 with corresponding actin abundance in kAE1 WT, R295H, S525F and R589H mIMCD3 cells at steady state, under starvation (Starv) or 400 nM Bafilomycin A1 (Baf) treatment. Note that p62 and LC3B were detected on the same blot for panel A and C, therefore the same actin blot is shown for both panels. (D-I) Quantification of all immunoblots showing the ratio of LC3B II to total LC3B and p62. Error bars correspond to mean ± SEM, n= 3-8. * P<0.05, ** P<0.01, *** P<0.005, ****P < 0.001 using one-way ANOVA followed by a Tukey’s post-hoc test. Immunoblots (J) and quantification (K, L) of LC3B and p62 abundance in kAE1 R607H KI mouse whole kidney lysates. Error bars correspond to mean ± SEM, n= minimum 5. ***P < 0.005 using one-way ANOVA followed by a Tukey’s post-hoc test.

dRTA kAE1 mutants have more alkaline steady-state intracellular pH and altered autophagy flux.

(A) Representative immunoblots of cell surface biotinylation experiments in mIMCD3 cells expressing kAE1 WT or mutants (top panels), with control staining of plasma membrane marker Na+/K+-ATPase (middle panel) and intracellular marker actin (bottom panel). (B) Quantification of 3 independent cell surface biotinylation experiments. Data are represented by a single representative blot for each variant. N.s., not significant using one-way ANOVA. Error bars correspond to mean ± SEM, n=3. (C) Immunofluorescence staining in eGFP-RFP-LC3 transfected mIMCD3 cells expressing kAE1. GFP = green, RFP = red, kAE1= cyan, nuclei = dark blue (merge only). Scale bar = 2 μm. Graphical representation of number of yellow (autophagosomes) (D) and red (autolysosomes) (E) puncta per cell expressing kAE1. Error bars correspond to mean ± SEM, n= minimum 32. ** P<0.01, *** P<0.005, ****P < 0.001 using one-way ANOVA followed by a Tukey’s post-hoc test. (F) Grouped graph of the number of yellow (autophagosomes) and red (autolysosomes) puncta per cell expressing kAE1, respectively. Note that the statistical analysis displayed only compared yellow and red groups for simplification. Error bars correspond to mean ± SEM, n= minimum 32. ** P<0.01, ****P < 0.001 using two-way ANOVA followed by a Sidak’s post-hoc test. (G-I) Immunoblot of LC3B, LAMP1 and actin in kAE1 WT, S525F and R589H mIMCD3 cells at steady state and under chemically reduced intracellular pH conditions. Graphical representation of the ratio of LC3B II to total LC3B ratio (J) or LAMP 1 (K) at steady state versus at low pHi in mIMCD3 kAE1 WT, S525F and R589H. Black circles indicate steady state cells and triangles indicate low pHi cells. Error bars correspond to mean ± SEM, n= 3. ** indicates P<0.01 using two-way ANOVA followed by a Sidak’s post-hoc test.

dRTA kAE1 mutants have bigger or more lysosomes than WT in vitro and in vivo.

(A) Immunofluorescence images of kAE1 WT, S525F and R589H mIMCD3 cells incubated with Magic Red substrate for 1hr at 37°C in the dark. Green = kAE1, magenta = active lysosomes, blue = nuclei. Scale bar =16 µm. (B) Graphical representation of number and size of active lysosomes per cell. Error bars correspond to mean ± SEM, n= minimum 30. **** P<0.0001 using one-way ANOVA followed by Tukey’s post-hoc test. Immunofluorescence images of LAMP 1 and ß1 ATPase in kidney cortex (C) or medulla (E) from kAE1 WT and R607H KI mice. Blue = nuclei, magenta = LAMP 1 (lysosomes), Yellow = ß1 ATPase, light blue “G” indicates the location of a glomerulus. Scale bar = 8μm. Graphical representation of the number and volume of LAMP1 vesicles in ß1 ATPase positive cells in the kidney cortex (D) or medulla (F) of WT or R607H KI mice. Error bars correspond to mean ± SEM, n= 60. **** P<0.001 using Student’s t-test.

dRTA kAE1 mutant cells have lower ATP production rate and abnormal mitochondrial content.

Oxygen consumption rate (OCR) (A) and Extra Cellular Acidification Rate (ECAR) (B) of empty vector (EV) transfected cells, kAE1 WT, S525F or R589H mIMCD3 cells analyzed in a Seahorse XFe96 Extracellular Flux Analyzer with the ATP Rate Assay Test Kit. All cell lines, including EV transfected cells, were incubated with doxycycline, to eliminate a potential effect of doxycycline on measurements. (C) Graphical representation of the combination of ATP production rates from mitochondrial respiration (mitoATP) and glycolysis (glycoATP) of kAE1 WT, S525F and R589H mIMCD3 cells measured in real-time following sequential injections of oligomycin and Rotenone +Antimycin A. Error bars correspond to mean ± SEM, n= minimum. * p < 0.05, ****p < 0.0001 using one-way ANOVA followed by Tukey’s post-hoc test. Graphical representations of mitochondrial respiration (D) and glycolytic ATP production (E) in kAE1 WT, S525F and R589H mIMCD3 cells. Error bars correspond to mean ± SEM, n= minimum 8. **P<0.01, ****p < 0.0001 using one-way ANOVA followed by Tukey’s post-hoc test. (F) Immunofluorescence staining of TOM 20 and kAE1 in kAE1 WT, S525F and R589H mIMCD3 cells. Blue = nuclei, Magenta = TOM 20, Green = kAE1. Scale bar = 8 µm. (G) Graphical representation of total TOM 20 fluorescence intensity per cell expressing kAE1. Error bars correspond to mean ± SEM, n= minimum 40. ***p < 0.001, ****p < 0.0001 using one-way ANOVA followed by Tukey’s post-hoc test. Immunofluorescence images of TOM 20 and ß1 ATPase in kidney cortex (H) or medulla (J) of kAE1 R607H WT and KI mice exposed to a salt depleted diet with an acid challenge26. Blue = nuclei, magenta = TOM 20 (mitochondria), Yellow = ß1 ATPase, light blue “G” indicates the location of a glomerulus. Scale bar = 8μm. Graphical representation of the total TOM 20 fluorescence intensity in ß1 ATPase positive cells in the cortex (I) or medulla (K) of the kidney. Error bars correspond to mean ± SEM, n= 90. * p < 0.05, ****p < 0.0001 using Student’s t-test.

Summary of findings in dRTA kAE1 variant expressing cells compared to WT.

Summary of findings in intercalated cells from dRTA R607H knock-in (KI) relative to WT mice.

Two-hour incubation in pH 6.6 media with 0.033 μM nigericin reduces cytosolic pH to 6.9.

(A) pHi of mIMCD3 cells incubated in either normal pH media of pH 6.6 media with 0.033 μM nigericin. Error bars correspond to mean ± SEM, n= minimum 4. ** P<0.01, using Student’s t test. (B) Immunoblots of p53 with corresponding actin abundance in mIMCD3 cells at steady state and under chemically reduced intracellular pH conditions. (C) Graphical representation of ratio of p53 at steady state versus at low pHi in mIMCD3 kAE1 WT, S525F and R589H cells. Black circles indicate steady state cells and triangles indicate low pHi cells. Error bars correspond to mean ± SEM, n= 3. ** indicate P<0.01 using two-way ANOVA.

dRTA kAE1 R607H knockin mice have lower abundance of intercalated cell marker B1 H+-ATPase, lysosomal protease Cathepsin D and antioxidant protein PRDX 6 in intercalated cells-enriched primary cells.

(A) Immunoblot of primary cells showing protein abundance of immature, intermediate and mature Cathepsin D (CTSD), antioxidant protein PRDX6 and intercalated cell marker B1 H+-ATPase with corresponding GAPDH and actin abundance. The first two panels are from the same blot but the second one is cropped on the region around 48 kDa and lower, and exposed longer. Primary cells were obtained by magnetic sorting and lysed upon collection. (B-F) Graphical representation of protein abundance of immature CTSD, intermediate CTSD, mature CTSD, B1 H+-ATPase and PRDX6 respectively in WT and knockin intercalated cell-enriched primary cells. Error bars correspond to mean ± SEM, n= minimum 4. * P<0.05, *** P<0.005 using Student’s t test.

Original immunoblots used for generating Figure 1.

Original immunoblots used for generating Figure 2.

Original immunoblots used for generating Figure 3.