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 kAE1 expression and corresponding actin band of mIMCD kAE1 WT, R295H, Y413H, S525F and R589H cells treated with and without doxycycline for 24 hrs. Mouse anti-HA antibody was used to detect kAE1-HA, top and bottom bands correspond to kAE1 carrying complex and high mannose oligosaccharides, respectively. Immunostaining of kAE1 WT or mutant (red) and calnexin (green) in non-polarized mIMCD3 cells (C), and kAE1 WT or mutant (red) and b-catenin (green) in polarized (D) mIMCD cells. Scale bar = 10 μm. Red = kAE1, Green = ß-Catenin. (E) 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. (F) Immunoblot of cycloheximide (CHX) chase assay with corresponding actin in kAE1 mIMCD WT and Y413H cells showing the degradation of kAE1 Y413H after 3hrs CHX incubation.

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

(A-C) Immunoblots of LC3 B 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. (D-I) Quantification of immunoblots shown in A-C showing the ratio of LC3B II to total LC3B and p62. Error bars correspond to mean ± SEM, n= minimum 3. * 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) Immunofluorescence staining of kAE1 in eGFP-RFP-LC3 transfected mIMCD3 cells expressing kAE1. GFP = green, RFP = red, kAE1= cyan. Scale bar = 8 μm. Graphical representation of number of yellow (autophagosomes) (B) and red (autolysosomes) (C) 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. (D) 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. (E) Graphical representation of intracellular pH measurement of mIMCD 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. (F-H) 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 (I) or LAMP 1 (J) 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. Scale bar = 8um. 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.

(A - B) Oxygen consumption rate (OCR) and Extra Cellular Acidification Rate (ECAR) of empty vector transfected cells, kAE1 WT, S525F or R589H mIMCD3 cells analyzed in a Seahorse XFe96 Extracellular Flux Analyzer with the ATP Rate Assay Test Kit. The empty vector transfected cells provided a control for 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 (I) 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. Scale bar = 8um. (I) Graphical representation of the total TOM 20 fluorescence intensity in ß1 ATPase positive cells in the cortex (J) 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.