Introduction

Distal renal tubular acidosis (dRTA) is a disorder characterized by the inability of the collecting duct system to secrete acids during metabolic acidosis¹. In addition to hyperchloremic metabolic acidosis, patients with this disease can present with hypokalemia, kidney stones, urinary sodium waste and difficulty to thrive. Expression of pathogenic variants in the ATP6V0A4, ATP6V1B1, FOXI1, WDR72 and SLC4A1 genes are the usual genetic aetiologies^2–4. The SLC4A1 gene encodes the anion exchanger 1 (AE1) protein, which is an electroneutral chloride/bicarbonate exchanger⁵. It exists in two forms: a 911 amino acid erythroid isoform known to interact with erythroid cytoskeletal proteins and participate in red cell respiration and integrity, and a 65 amino acid (NH₂-terminal) truncated isoform primarily found in the basolateral membrane of renal type A intercalated cells (A-IC)^6,7 and podocytes⁸. This isoform participates in bicarbonate reabsorption and through its physical and functional interaction with the cytosolic carbonic anhydrase II and apical H⁺-ATPase, it supports apical proton export and urine acidification⁹.

Pathogenic variants in the SLC4A1 gene can result in either red cell defects (such as Southeast Asian ovalocytosis^10,11 and hereditary spherocytosis¹²), renal cell defects (dRTA¹⁰) or both in patients with homozygous (Band 3 Coimbra and Band 3 Courcouronnes^13,14) or compound heterozygous variants^15,16. Renal SLC4A1 disease-causing variants have only been found in the transmembrane domain - where it could impact protein structure and its transport function- or in the short carboxyl domain – where it possibly affects protein-protein interactions. The pathophysiology of dominant or recessive SLC4A1 related dRTA (hereafter named dRTA) has originally been linked with the mis-trafficking defect of mutant kAE1 protein^10,17. However, recent in vivo studies in R607H (orthologous to human R589H dRTA variant) and L919X knockin mice and dRTA patients have revealed a complex pathophysiology where kAE1-expressing intercalated cells were lost, and in the remaining type-A intercalated cells, the H⁺-ATPase relocated intracellularly and accumulated autophagy marker p62 and ubiquitin-positive material¹⁸.

The highly active transport properties of collecting duct cells require the tight maintenance of cellular energy and homeostasis. The autophagy-mediated turnover of damaged organelles is necessary for protecting collecting duct cells as in most renal cells¹⁹. The chloride/bicarbonate exchange function of kAE1 in A-ICs confers a pivotal role in pH homeostasis and thus is a major contributor to cellular homeostasis. kAE1 protein interacts with several proteins such as integrin-like kinase (ILK), adaptor-related protein complex 1, 3 & 4 (AP-1, AP-3 & AP-4 mu1A), transmembrane protein 139 (TMEM139), kinesin family member 3B (KIF3B), and clathrin among others^7,20–22 that support protein stability and trafficking. It also interacts with homeostatic proteins including the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH)²³ and the antioxidant enzyme peroxiredoxin 6 (PRDX 6)²⁴, which play major roles in cellular energy metabolism and oxidative stress response, respectively.

In this study, we report the characterization of newly identified dRTA genetic variations and provide evidence of abnormal autophagy and degradative pathways in cells and kidneys from mice expressing dRTA mutant kAE1 proteins.

Concise Methods

Ethics Approval

This study was conducted in accordance with all national and institutional animal care guidelines and approved by the University of Alberta’s Animal Care and Use Committee (AUP #1277).

Newly identified SLC4A1 variations from dRTA patients

The patient carrying the R295H mutation was a boy carrying the variation in the homozygous state, whose genetic diagnosis was made at the age of 5, following growth retardation of −2 SD for both weight and height, with bicarbonate at 18 mmol/L, potassium at 2.8 mmol/L, chloridemia at 94 mmol/L, calcemia at 2.55 mmol/L, and a urinary pH of 7.5. He also had a history of a pyeloureteral junction syndrome that was surgically managed at the age of 3. The R295H dRTA variation is a nonsense homozygous substitution characterized by a replacement of guanine (G) on position 884 by adenine (A) in the coding sequence. It has an allelic frequency of 0.14 % in the European population.

For the Y413H variant, the patient was a female diagnosed at 1 month old, with an urinary pH of 7.5, evidence of nephrocalcinosis and failure to thrive. The S525F variant has been previously reported²⁵, but in brief, the patient was a 13 year old female with plasma pH of 7.25, plasma bicarbonate at 15.3 mmol/L who also presented with polyurethral junction syndrome, nephrocalcinosis and nephrolithiasis since childhood. The Y413H and S525F dRTA variations are nonsense heterozygous substitutions characterized by a replacement of thymine (T) at position 1237 by cytosine (C) and a replacement of cytosine (C) at position 1574 by thymine (T), respectively. The R589H dRTA variation has been previously described¹⁸. No follow up data were available for all patients.

Mice

Transgenic mice carrying the R607H knockin (KI) mutation (murine equivalent to human R589H mutation) were previously described¹⁸. Homozygous mice used throughout the study display incomplete dRTA with alkaline urine without metabolic acidosis at baseline as previously reported¹⁸. Homozygous mice or wild-type (WT) littermates were fed a standard rodent chow (Picolab® Rodent Diet 20 # 5053, LabDiet, ST. Louis, MO, USA) or for Figure 5 (H-K), a salt-depleted diet with acid challenge as previously reported²⁶ with adequate and constant water supply, and maintained on a 12-hour light and dark cycle throughout their lifespan.

Statistical analysis

All the experiments were independently repeated a minimum of three times. Experimental results were analyzed using the GraphPad Prism software and are summarized as mean ± SEM. All statistical comparisons were made using unpaired Student’s t test or one/two-way ANOVA followed by a post-hoc test as indicated in figure legends. A P-value less than 0.05 was considered statistically significant. All datasets were assessed for normality, and outliers identified by Prism were excluded.

Detailed Material and Methods are listed in the Supplementary Material.

Results

The dRTA kAE1 variants traffic to the basolateral membrane but have reduced transport activity in mIMCD3 cells

We first characterized three newly identified dRTA mutations and compared them with kAE1 WT or previously characterized kAE1 R589H mutant. Figure 1A depicts the alpha fold predicted structure of kAE1 showing amino acids mutated in each kAE1 mutant. kAE1 R295H is a recessively inherited substitution in the N-terminal cytosolic domain of the protein. kAE1 Y413H is a dominantly inherited substitution in transmembrane domain (TM) 1 of the core domain. In the gate domain, dominantly inherited S525F and R589H substitutions occur in TM 5 and TM 6, respectively. We generated the newly identified dRTA variant cDNAs and expressed them or kAE1 WT in mIMCD3 cells. As seen on Figure 1B, kAE1 R295H, S525F and R589H variants display 2 typical bands similar to kAE1 WT. The top band (open circle) encompasses kAE1 proteins carrying a complex oligosaccharide that have reached the Golgi and beyond, while the bottom band (black circle) corresponds to high mannose-carrying kAE1 proteins located in the endoplasmic reticulum. However, kAE1 Y413H mutant bands intensity was overall weaker than WT and displayed a predominant single band aligned with high mannose-carrying kAE1 proteins. These results indicate that the 3 newly described dRTA mutants are successfully expressed in mIMCD3 cells. We next localized these mutants by immunofluorescence in polarized mIMCD3 cells. Both kAE1 WT and mutants appropriately co-localized with basolateral membrane marker beta-catenin in polarized mIMCD3 cells (Figure 1C). kAE1 R589H location has previously been reported at the basolateral membrane in polarized mIMCD3 cells¹⁸. However, staining for kAE1 Y413H was again weaker and seemed more intracellular than other mutants. To address a possible premature degradation of this variant, we measured its lifetime and observed that its degradation begins 6 hours post-synthesis while kAE1 WT abundance remained stable for 24 hours (Figure 1D). Finally, to assess the transport activity of the mutants, we examined the steady-state cytosolic pH (pHi) and rate of intracellular alkalinisation of mIMCD3 cells expressing kAE1 WT or mutants (Figure 1E-G). Using BCECF-AM, we observed that the steady state intracellular pH (pHi) of kAE1 mutant cells was more alkaline than WT cells (except for kAE1 R295H cell pHi, which has a similar trend but is not significantly different from WT) (Figure 1F), in agreement with rate of intracellular alkalinisation (Figure 1G). Note that transport activity, measured as rate of alkalinisation, is measured by reverting kAE1 exchange activity from bicarbonate export to import (see Methods, protocol for transport assay), hence a reduced alkalinisation rate is observed in cells expressing defective kAE1 protein compared to WT (panel G). Overall, these results indicate that except for kAE1 Y413H mutant, the other newly described variants are expressed and traffic to the basolateral membrane of polarized mIMCD3 cells, similar to the previously published kAE1 R589H mutant. Given that the premature degradation of kAE1 Y413H mutant likely explains the dRTA phenotype, we did not perform further assays on cells expressing this protein.

Figure 1.

Autophagy processes are altered in mIMC3 cells expressing the kAE1 R295H, S525F and R589H dRTA mutants and in R607H knockin kidney lysates

In mice expressing kAE1 R607H (the murine equivalent to human dRTA R589H substitution), a striking reduction in type-A intercalated cells was noted and in the remaining cells, autophagy marker p62 and ubiquitin accumulated in these abnormally enlarged cells¹⁸. We therefore investigated the autophagy machinery in dRTA mutant mIMCD3 cells and in R607H knockin (KI) mice. We first examined the ratio of autophagosome marker LC3BII protein relative to the total intensities of LC3BI and LC3BII as well as p62 levels. These experiments were performed at steady state, upon autophagy induction by starvation, or after autophagy inhibition by Bafilomycin (Baf) A1 (Figure 2A-C). Figure 2A – I shows a consistent increase in the ratio of LC3B II to total LC3B (I + II) in the mutant cells at steady state (panel D), with starvation (panel F) and with Baf A1 (panel H) except for the kAE1 R295H mutant which was not significantly different from WT. This suggests an altered autophagy process in the mutant cells, in agreement with preliminary findings from Mumtaz and colleagues¹⁸. There was no significant difference in p62 abundance in mutant cells compared to WT at steady state and with starvation (Figure 2E&G). However, with Baf A1, R589H mutant cells had significantly lower p62 abundance compared to WT (Figure 2I). To confirm these findings, we next assessed the abundance of these markers in whole kidney lysates from the kAE1 R607H KI mice. We observed that the total LC3B abundance was significantly higher in homozygous KI mice (KI/KI) compared to WT, with no difference in p62 (Figure 2J-L). Overall, these results suggest an abnormal autophagy in mutant mIMCD3 cells and in kidneys of R607H KI mice. Given the lack of difference in phenotype between the recessive kAE1 R295H and kAE1 WT mIMCD3 cells, we focused the subsequent experiments on dominant kAE1 S525F and R589H mutant cells. Further investigations will be needed to understand the pathophysiology associated with the kAE1 R295H novel variant.

Figure 2.

Late autophagy steps are blocked in dRTA kAE1 mutant-expressing cells due to their alkaline intracellular pH

Given these preliminary findings of abnormal autophagy in dRTA mutant cells, we examined in more detail their autophagy machinery by transiently transfecting them with the eGFP-RFP-LC3 construct and monitoring the rate of autophagosome and autolysosome formation. In this assay, the green fluorescent protein (eGFP) fused to LC3 is quenched in the acidic environment of the autolysosome, while both eGFP and red fluorescent protein (RFP) fluoresce in vesicles in the neutral lumen of the autophagosome^27,28. To avoid the poor efficiency of transient transfection in polarized cells, experiments were conducted in sub-confluent cells pending that the mutant proteins are present at the plasma membrane. We therefore performed cell surface biotinylations on 70-80 % sub-confluent cells, which confirmed a robust plasma membrane abundance of both kAE1 R589H and S525F that was not significantly different from the WT protein (Figure 3A&B). Therefore, we next assessed the efficiency of the autophagy machinery in WT or dRTA mutant-expressing cells (Figure 3C-F). Focusing on cells expressing either kAE1 WT, kAE1 S525F or R589H, we quantified the number RFP+ (red, acidic autolysosomes) and double eGFP+/RFP+ (yellow, not acidic autophagosomes) vesicles. kAE1 S525F mutant cells have significantly more autophagosomes than WT counterparts (Figure 3D), and both kAE1 S525F and R589H have significantly more autolysosomes than WT (Figure 3E). Figure 3F shows that both mutants had significantly more autophagosomes (yellow) than autolysosomes (red). This finding suggests an upregulation of autophagy and inhibition in the downstream steps of the process that involves the fusion of autophagosomes with the lysosome²⁹. As autolysosomes require luminal v-H⁺-ATPase-dependent acidification to efficiently clear cell debris ³⁰, the higher intracellular pH seen in mutant cells (Figure 1F) may be detrimental to v-H⁺-ATPase full activity and impair proper autolysosomal acidification³¹. We therefore wondered whether chemically acidifying the pHi in mutant cells would rescue the autophagy machinery (Figure 3G-K). We first determined that incubation of mIMCD3 cells in 0.033 μM nigericin in cell culture medium at pH 6.6 for 2 hours acidified cytosolic pH to 6.9 without causing cell death (Supplementary Figure 1A-C). Next, we observed that chemically reducing pHi to 6.9 in mutant expressing cells reduced the ratio of LC3B II to total LC3B (Figure 3J) and the abundance of lysosomal-associated membrane protein 1 (LAMP1) in R589H cells (Figure 3 K) to levels similar to WT cells at steady state. These findings suggest that abnormal autophagy in the mutant cells may be caused by their alkaline pHi, resulting from a reduced anion exchange activity of the mutant kAE1 protein.

Figure 3.

mIMCD3 cells expressing dRTA kAE1 mutants and R607H KI kidney tissues have abnormal lysosome number and size

The accumulation of autophagosomes and autolysosomes as seen above may suggest one or a combination of the following: an inability of autophagosomes to fuse with lysosomes and/ or a defect in lysosomal degradative activity in the mutant cells^19,32. We first examined the lysosomal degradative activity by assessing lysosomal protease Cathepsin B activity using Magic Red staining, a probe that fluoresces upon lysosomal protease cleavage (Figure 4A-B). In agreement with increased RFP+ vesicles (Figure 3E), the kAE1 S525F mutant cells had a significantly higher number of Magic Red positive vesicles than WT whereas the kAE1 R589H mutant cells had significantly larger Magic Red positive vesicles, suggesting an accumulation of undigested material³³. To validate this finding in vivo, we performed immunostaining and quantified LAMP1 positive staining in ß1 ATPase positive cells (a marker of ICs) in WT and R607H KI mouse kidney sections. The KI mice showed significantly more and larger LAMP1 positive vesicles compared to WT mice in both cortex and medulla (Figure 4 C-F). We probed further into the lysosomal activity by quantifying lysosomal protease Cathepsin D (immature, intermediate and mature) protein abundance by immunoblot in isolated primary murine A-ICs. Although the abundance of immature and intermediate cathepsin D did not differ between genotypes, the KI mice showed a significantly decreased abundance of mature cathepsin D (Supplementary Figure 2). Thus, in line with in vitro findings, A-IC from homozygous R607H KI mice display relatively more and larger lysosomes with reduced active protease abundance than WT littermates, suggesting a lysosomal defect in the dRTA kAE1 mutant cells.

Figure 4.

Figure 5.

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

Lysosomal degradation is highly dependent on a low luminal pH generated in part by the vacuolar-type H⁺-ATPase³⁴ whose activity depends on ATP hydrolysis. We therefore analysed the ATP production rate in mIMCD3 cells, specifically glycolysis and oxidative phosphorylation. We measured the oxygen consumption rate (OCR) (Figure 5A) and extracellular acidification rate (ECAR) (Figure 5B) in empty vector transfected, kAE1 WT or mutant cells. Both kAE1 S525F and R589H mutant cells had a lower ATP production rate compared to WT (Figure 5C). More specifically, the R589H mutant cells had a lower mitochondrial ATP production rate (Figure 5D) whereas the kAE1 S525F mutant cells exhibited a lower glycolytic ATP production rate (Figure 5E). With the mitochondria being the major ATP-producing organelles³⁵, we assessed mitochondrial content by immunostaining of translocase of the outer membrane 20 (TOM20) both in vitro and in vivo. Both kAE1 S525F and R589H mutant cells have higher mitochondrial content compared to WT as determined by total overall intensity of TOM20 positive puncta (Figure 5F&G). In line with this result, although a decreased fluorescence intensity was observed in the cortex, there was a significantly higher TOM20 fluorescence intensity in medullary kidneys of homozygous R607H KI mutant mice compared to WT littermates (Figure 5H-K).

Discussion

In this study, we characterized three newly identified dRTA-causing kAE1 variations. Combining in vivo and in vitro studies, we demonstrated that reduced transport activity of the kAE1 mutants correlated with increased cytosolic pH, reduced ATP synthesis, attenuated downstream autophagic pathways and lysosomal dysfunction, pertaining to the fusion of autophagosomes and lysosomes and/ or lysosomal degradative activity (See summary in Supplementary Tables 1 and 2).

In line with previous observations in mIMCD3 cells¹⁸, the kAE1 R295H, Y413H and S525F mutants were properly localized to the basolateral membrane after polarization. When expressed in Madin-Darby canine kidney (MDCK) cells, other dRTA-causing kAE1 mutants such as dRTA R602H, G701D, V488M, deltaV850 variants exhibited a plasma membrane trafficking defect^10,36,37. In contrast, the kAE1 R589H mutant was correctly targeted to the plasma membrane¹⁸. Functionally, cells expressing the Y413H and S525F mutants exhibit about 60 % reduction in chloride/bicarbonate exchange activity compared to kAE1 WT, similar to the previously characterized recessive G701D mutant but unlike the R295H mutant³⁸. Therefore, the mechanism causing dRTA remains unclear in the case of the newly identified recessive R295H variant. Additionally, the kAE1 Y413H variant exhibited a shorter half-life than WT counterpart, likely explaining dRTA, and thus was not further investigated. These findings add to the growing list of SLC4A1 gene variations causing dRTA.

We next investigated the roots of the altered autophagy briefly reported in R607H KI mice¹⁸ using mIMCD3 cells and whole kidney lysates. In kidneys of the KI mice, a decrease in the number of A-IC, accumulation of p62 and ubiquitinylated proteins and enlarged remaining A-ICs, suggested altered autophagy processes in dRTA mutant cells and in homozygous R607H KI mice^18,38. During the autophagy process, LC3B I (a marker for autophagosomes) is converted to lipidated LC3B II³⁹ and p62 aggregates to facilitate the degradation of ubiquitinated proteins within the autophagosome complex⁴⁰. In mIMCD3 cells expressing dRTA kAE1 S525F and R589H variants, LC3B lipidation was increased compared to WT, an increase that persisted with both Baf A1 treatment and starvation. Although opposite effects were expected under inhibition or induction of autophagy, such similar effect has been previously described. In the proximal tubule of obese mice, LC3B accumulation indicating a stagnated autophagy flux, was observed with both chloroquine treatment and 24 hour starvation⁴¹. Similarly, in NRK-52E cells, a disruption of the autophagy machinery in high cadmium-stressed cells resulted in LC3B II accumulation under either Baf A1 or rapamycin (an autophagy inducer) treatment⁴². Although LC3B II is elevated during both increased autophagy flux and disrupted autophagy, we did not observe significant accumulation of p62 in mIMCD3 cells expressing dRTA mutants. p62 is specifically a marker of autophagy-mediated protein clearance^43–45. Therefore, p62 accumulation in R607H KI mouse kidney sections strongly suggests a compromised autophagy-mediated clearance while the increased LC3B lipidation without significant changes in p62 in mIMCD3 cells points towards either an increased autophagic flux and/or a disrupted autophagy.

To obtain a clearer picture of the precise autophagic pathway altered in the dRTA mutants, we probed further into the different stages of autophagy and autophagy flux. We noted an accumulation of autophagosomes and autolysosomes in the S525F and R589H mutant cells. This was recapitulated in the R607H KI mice which showed significantly more and larger LAMP 1 positive vesicles in both kidney cortex and medulla, suggesting a blockage in late steps of autophagy flux in both dRTA mutant cells and KI mice. Such blockage has been implicated in the pathophysiology of several diseases. In lysosomal storage disease, lysosome accumulation in proximal tubule cells is a key component in the pathways mediating epithelial dysfunction¹⁹. In this study, restoring autophagy flux attenuated disease progression. Another study in SK-N-SH, RT4-D6P2T and HeLa cells implicated autophagosome and lysosome accumulation in cellular toxicity associated with neurodegenerative diseases³². In agreement with altered lysosomal function, we also noted a greater abundance and size of active cathepsin B lysosomal protease vesicles in mIMCD3 cells. Increased cathepsin B activity affects lysosomal biogenesis, autophagy initiation and cellular homeostasis⁴⁶. In the renal context, cathepsin B knockout mice demonstrated a higher resistance and quicker recovery from glomerular damage⁴⁷, suggesting that cathepsin B accumulation may be detrimental to the cells. Increased cathepsin B abundance in the dRTA mutant cells also correlates with the accumulation of lysosomes. Overall, these findings suggest that the pathogenesis of dRTA in our in vitro and in vivo models involves an inhibition of autophagy flux at the downstream steps involving autophagosome-lysosome fusion and lysosomal protein clearance ^29,33,48,49.

The question remained as to how these dRTA variants altered autophagy. The SLC4A1-3 gene family that includes AE1 are regulators of intracellular pH in different cell types^50–52. The reduced anion exchange activity in mIMCD3 cells expressing the R589H and S525F variants expectedly correlated with an increased pHi compared to WT counterparts. Given that pHi variations can impact autophagy^{31,34,53–55}, we wondered whether this was the case for cells expressing kAE1 dRTA mutants. We observed that an acidic pHi in mutant cells restored autophagy levels similar to WT. Previous studies reported more perinuclear localization of lysosomes and autophagosome-lysosome fusion in cells with an increased pHi^53,54. Although not examined in our study, an increase in intracellular pH due to starvation decreased the levels of lysosomal kinesin superfamily member KIF2 and ADP-ribosylation factor-like 8B (ARL8), which are responsible for redistributing lysosomes to the cell periphery. This reduction subsequently inhibited mTORC1, resulting in increased autophagosome synthesis and autophagosome-lysosome fusion⁵³.

While an alkaline cytosolic pH partially explains the impairment in autophagy flux, a close relationship also exists between intracellular pH and cellular energy dynamics and metabolic stress, all of which are key regulators of autophagy⁵⁶. Therefore, it was plausible that kAE1 variant-induced autophagy dysregulation occurs through a signalling pathway akin to that of energy deprivation-induced autophagy. This hypothesis is supported by our findings of reduced ATP production rate in dRTA kAE1 S525F and R589H mutant cells compared to kAE1 WT cells. We also found that both kAE1 S525F and R589H mutant cells have higher mitochondrial content compared to kAE1 WT cells. The increased mitochondrial content coupled with low ATP point towards improper mitochondrial function in dRTA variant cells. Low ATP levels as seen in dRTA mutant cells may impair autophagy as was shown in human RPE cells⁵⁷. ATP reduction in RPE cells led to complex mitochondrial changes such as structural disorganization, enzyme activity decline, and oxidative damage to mitochondrial components and DNA⁵⁷. Similarly, in pancreatic islet cells, an alkaline pHi led to increased uptake of phosphate by mitochondria, accelerating the production of superoxide, promoting mitochondrial permeability transition, and inducing translational attenuation due to endoplasmic reticulum stress, ultimately impairing insulin secretion⁵⁸. Overall, our data support that expression of the kAE1 variants increases pHi which alters mitochondrial function and leads to reduced cellular energy levels that eventually attenuates energy-dependent autophagic pathways including autophagosome-lysosome fusion and lysosomal protein clearance.

In light of these observations, we postulated that correcting the alkaline pHi of dRTA mutant-expressing mIMCD3 cells will alleviate this blockage in autophagy flux. We observed that a chemically engineered pHi of 6.9⁵⁹ reduced LC3B II accumulation and LAMP1 abundance in mIMCD3 mutant cells to expression levels similar to that of WT cells at baseline. This suggests that a chemically reduced pHi facilitated protein clearance in the two dRTA mutant cells^31,53 as noted in other studies. In one such study, treatment of SH-SY5Y cells with FCCP and nigericin also acidified intracellular pH and triggered autophagy and mitophagy³¹. Similarly, acid loading in proximal tubular cells under chronic metabolic acidosis showed increased autophagic flux and mitophagy⁶⁰. These findings are in line with our results and establish a link between altered autophagy flux and the alkaline pHi of dRTA variant cells. Overall, our study provides one pathway (altered pHi) by which dRTA may arise. However, different variants induce different degree of functional defects as seen in Figure 1F & G. The kAE1 R295H, the only reported amino acid substitution in the amino-terminal cytosol causing dRTA, does not affect the transporter’s function or pHi. Therefore, this variant may cause dRTA via a different pathway, for example defective protein-protein interactions, than transport-defective S525F or partially inactive R589H variants.

In conclusion, our study established a strong relationship between the expression of defective kAE1 proteins, reduced mitochondrial activity, decreased autophagy and impaired protein degradative flux. Whether this abnormal degradative pathway explains the premature loss of A-IC will need to be elucidated in further studies.

Supplementary Material

Material and Methods

Antibodies and chemicals

Mouse anti-HA antibody (hemagglutinin, Biolegend, formerly Covance), mouse anti-β-actin antibody (Sigma Aldrich or anti −β-actin HRP clone 2F1-1 Biolegend cat#643807), mouse anti-LC3B antibody (Cell Signaling), mouse anti-IVF 12s antibody (Developmental Studies Hybridoma Bank), rat anti-ATP6V1B1 antibody (BiCell #20901), rabbit anti-mTOR antibody and phospho mTOR antibody (Cell Signaling), Rabbit anti-4E-BP1 antibody and phospho 4E-BP1 (Ser65) antibody (Cell Signaling), mouse anti-p62 antibody (Abcam), mouse anti-p53 antibody (Cell Signalling), rabbit anti-Cleaved caspase 3 antibody (Cell Signalling), mouse monoclonal anti Na⁺/K⁺-ATPase H-3 antibody (Santa Cruz Biotechnology, Dallas, TX), goat anti-mouse antibody horseradish peroxidase conjugated (HRP) (Cell Signaling Technology), Cy3-conjugated donkey anti-mouse antibody, anti-rabbit and anti-goat antibodies (Jackson Immunoresearch), X-tremeGENE HP DNA transfection reagent (Roche), Seahorse XFe consumables (Agilent).

Cell Lines, Transfections and Viral Infection

Mouse inner medullary collecting duct (mIMCD) cells (ATCC# CRL2123) were used for preparing kAE1 wild type and mutant cell lines. The pLVX TRE3G kAE1 construct was generated from the shuttling of human kAE1 cDNA with an external hemagglutinin (HA) epitope in position 557 (on eAE1) into pLVX-TRE3G plasmid (Clontech)⁶¹. This construct encodes a protein described as kAE1 throughout this paper. The kAE1 S525F and R589H mutants were generated with Q5 site-directed mutagenesis. All plasmids were introduced into the mIMCDs using a viral single shot packaging kit (Clontech).

Mouse intercalated cell isolation and tissue homogenate preparation

Kidney tissue homogenates were prepared as previously described⁶². Briefly, after decapsulation, freshly dissected kidneys were homogenized in cold lysis buffer (0.3 M Sucrose, 25 mM Imidazole, 1mM EDTA, 8.5 µM Leupeptin, 1 mM PMSF), and vortexed over one hour every 15 min. Thie homogenates were then centrifuged at 14000 rpm for 15 minutes at 4 °C prior to measurement of protein concentration by Bicinchoninic Acid Protein Assay. Primary intercalated cells were prepared from homozygous kAE1 R607H transgenic mice. After cardiac perfusion with PBS, heparin, and collagenase B (Sigma Aldrich), kidneys were homogenized by MACS dissociation and intercalated cells enriched using CD 117 magnetic sorting (Miltenyi Biotec) as previously described⁶³. During the selection, intercalated cells were kept in MACS buffer (PBS, 2mM EDTA and and 0.5 % FBS). Cells were lysed with RIPA lysis buffer (2 mM EDTA, 2 % Deoxycholate, 0.3 M NaCl, 20 mM Tris/HCl pH 7.5, 2 % Triton X-100, 0.2 % SDS, pH 7.4), supplemented with complete EDTA-free protease inhibitors, and PhoSTOP phosphatase inhibitor (Roche), PMSF, pepstatin, leupeptin and aprotinin. An aliquot was saved for bicinchoninic acid assay to determine protein concentration and remaining lysates were kept in Laemmli buffer at −20°C for immunoblotting.

Bicarbonate transport assay

This assay has been previously described⁶⁴. Briefly, confluent kAE1 WT-HA or mutant mIMCDs cells grown on coverslips were incubated with 1 μg/ml doxycycline (Sigma-Aldrich) for 18 - 24 hours at 37°C to induce kAE1 expression. They were then incubated with 2’,7’-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester (BCECF – AM, Thermo Scientific), a fluorophore which excites at 440 and 490 nm and emits 510 nm wavelength for 30 minutes at 37°C. Using a fluorometer from Photon Technologies International (PTI) (London, Ontario, Canada), coverslips were perfused with NaCl-based Ringer’s buffer (5 mM glucose, 5 mM potassium gluconate, 1 mM calcium gluconate, 1 mM magnesium sulfate, 10 mM HEPES,

2.5 mM sodium dihydrogen phosphate, 25 mM sodium bicarbonate,140 mM sodium chloride) for 5 to 10 minutes. Once stable, initial fluorescence (corresponding to steady -state pHi)) was recorded for the first 30 seconds before switching to chloride-free containing sodium gluconate based Ringer’s buffer of same osmolality. BCECF fluorescence was calibrated by perfusing cells with different pH buffers (6.5, 7, 7.5) in the presence of 10 μM nigericin. The Ringer’s buffers were continuously bubbled with an air: CO2 mixture (19:1), providing 5 % CO2. Transport rates of the cells were determined by linear regression of the initial fluorescence variations (over the first 60 seconds), normalized to pH calibration measurements. All measurements were done using PTI FelixGX software.

Cell Treatments and immunoblotting

For autophagy experiments kAE1 WT or mutant cells were seeded to 70 % confluency on 10 cm culture plates and treated with 1 μg/ml doxycycline (Sigma-Aldrich) for 48 hours. Cells were then either treated with 400 nM bafilomycin A1 for four hours to inhibit autophagy or with Hanks balanced salt solution (HBSS, Gibco) to starve cells and induce autophagy or given no treatment. To chemically modify pHi of cells, 90 – 100 % confluent cells were treated with 1µg/ml of doxycycline (Sigma-Aldrich) overnight. Cells were then incubated in Ringers’ buffer with pH 6.6, supplemented with 0.03 μM nigericin with a final potassium concentration of 168mM for two hours at 37°C. Steady-state cells were incubated in normal pH media without nigericin prior to lysis. Under treated conditions, pHi was similar to pHe (Supplementary Figure 1). Cells were lysed with RIPA lysis buffer (1% deoxycholate, 1mM EDTA, 0.15 M NaCl, 0.1% SDS, 10 mM TRIS/HCL (pH 7.5), 1% Triton X-100) with phosphatase inhibitors (cat. no. 04906837001; Roche PhosSTOP) and protease inhibitors (cat. no. 04693159001; Roche Complete Tablets, Mini EDTA-free) and stored at −20 °C with or without 2x Laemmli buffer. The aliquot without Laemmli buffer was used for a bicinchoninic acid (BCA) assay to determine protein concentration. Following the BCA, 10 - 30 μg of total protein was loaded on SDS-PAGE gels. Proteins were transferred to PVDF membranes and antibodies listed above were used for detecting the proteins of interest. Primary antibodies were diluted in 1 % milk and incubated on membranes overnight at 4 °C followed by secondary antibodies linked with horseradish peroxidase (HRP) for one hour at room temperature. Protein detection was done with the Enhanced Chemiluminescence reagent (ECL Prime, Invitrogen), and a BioRad Imager. The ImageLab software (BioRad) was utilized for the quantification of relative band intensities.

Cell Surface Biotinylation assay

mIMCD3 cells stably expressing kAE1 WT, S525F or R589H were seeded to 70-80% confluency. The cells were incubated with sulfo-N-hydroxysuccinimide-SS-biotin (Thermo cat.# 21331) (1.5 mg/ml in ice-cold PBS) for an hour at 4°C, quenched with 100mM glycine and lysed with RIPA lysis buffer, supplemented with complete EDTA-free protease inhibitors, and PhoSTOP phosphatase inhibitor (Roche), PMSF, pepstatin, leupeptin and aprotinin. Total protein levels were measured by BCA, and an aliquot was saved as “Total” fraction. 450 μg of each lysate was subsequently incubated with 100 μl streptavidin slurry beads for 1 h on a rocker at 4°C. Following centrifugation, the supernatant was collected, and an aliquot kept as the “unbiotinylated” fraction. After six washes, the beads were resuspended in 50 μl of 2X Laemmli buffer and incubated at room temperature for 30 min. The eluted biotinylated proteins were subsequently collected by centrifugation (“Biotinylated” fraction). The biotinylated fraction (45 μl) was loaded on SDS-PAGE gel for immunoblot analysis along with 3 μg of the Total fraction and a matched volume of unbound fraction per well. In addition to anti-HA antibody, the blots were probed for actin to ensure cell membrane integrity was intact during the biotinylation procedure, and for Na⁺/K⁺-ATPase as cell surface control.

Magic Red Assay

80 % confluent kAE1 WT or mutant mIMCD3 cells were treated with 1 μg/ml doxycycline (Sigma-Aldrich) for 48 hours to induce kAE1 expression. Different treatments including: a 4-hour incubation with 400 nM bafA1 to inhibit autophagy, a 2-hour starvation in HBSS to induce autophagy or no treatment (steady state) were applied. Cells were then incubated with 1 % Magic Red reagent (ImmunoChemistry Technologies) in DMEM-F12 medium at 37°C in the dark for 30-60 minutes. Cells were then fixed with 4 % PFA, quenched with 100 mM glycine, permeabilized with 0.2 % Triton X-100, blocked with 1 % BSA and incubated with mouse anti-HA primary antibody and donkey anti-mouse Alexa fluor 488 conjugated secondary antibody for 30 mins each. Cells were then incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 5 mins before mounting using DAKO Mounting Medium (Agilent Technologies). A WaveFX confocal microscope was used to image the slides, and the images were analyzed blindly using the Fiji software.

Autophagy flux Assay

kAE1 WT-HA or mutant mIMCD3 cells seeded to 70 % confluency in a 6-well plate on coverslips were transiently transfected with the eGFP-RFP-LC3 cDNA construct (kind gift from Dr. Goping, Department of Biochemistry, University of Alberta) using the X-tremeGENE HP DNA transfection reagent (Roche). Four hours after transfection, the cells were incubated with 1 μg/ml doxycycline for 48 hours at 37°C to induce kAE1 expression. Following this incubation, cells were incubated with blocking medium, mouse anti-HA primary antibody (1:200) and donkey anti-mouse Alexa fluor 649 (Jackson ImmunoResearch). Hoescht stain (ImmunoChemistry Technologies) was used to stain cellular nuclei. A WaveFX confocal microscope together with the Velocity and Fiji software were used to capture and analyze images.

Assessment of mitochondrial content

kAE1 WT-HA or mutant mIMCD3 cells seeded to 50 % confluency in a 6-well plate on coverslips were incubated with 1 μg/ml doxycycline for 48 hours at 37°C and overnight in complete medium with no antibiotics. Cells were then fixed with 4 % PFA, quenched with 100 mM glycine, permeabilized with 0.2 % Triton X-100, blocked with 1 % BSA and incubated with mouse anti-HA and rabbit anti-TOM 20 primary antibodies and then with donkey anti-mouse Alexa fluor 488 and anti-rabbit Cy3 conjugated secondary antibodies. Cells were then incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 5 mins before mounting using DAKO Mounting Medium (Agilent Technologies). A WaveFX confocal microscope was used to image the slides, and the images were analyzed blindly using the Fiji software.

Metabolic flux analysis

kAE1 WT-HA or mutant mIMCD3 cells were treated with 1 μg/ml doxycycline for 48 hours followed by an overnight incubation in complete media with no antibiotics and cells seeded at 2 x 10⁴ cells per well in Seahorse XFe 96 well plates overnight to form a uniform monolayer. On the day of assay, culture medium was replaced with XF DMEM Medium pH 7.4 (103575-100, Agilent Technologies) with glucose (10 mM), pyruvate (1 mM) and L-Glutamine (2 mM) and incubated in a non-CO₂, 37°C incubator for one hour prior to their placement into the XFe96 Analyzer. Using the ATP production rate assay kit (#103592-100, Agilent Technologies) and XF cell Mito Stress Test kit (#103015–100, Agilent Technologies), metabolic indices were obtained from the Seahorse XFe96 Analyzer following manufacturer’s procedures previously described²³. The total ATP rate is the sum of ATP production rate from both glycolysis and oxidative phosphorylation. Glycolysis releases protons in a 1:1 ratio with ATP hence the glycolytic ATP rate is calculated from the glycolytic proton efflux rate (glycoPER). GlycoPER is determined by subtracting respiration linked proton efflux from total proton efflux by inhibiting complex I and III. The empty vector transfected cells provided a control for a potential effect of doxycycline on measurements. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured at various time points at basal state followed by injections of oligomycin (1.5 μM) and Rotenone +Antimycin A (0.5 μM).

Tissue Preparation and Immunostaining of kidney sections

Kidneys collected after perfusion were stored in 4 % PFA overnight at 4°C. The PFA solution was switched to 15 % sucrose for 2 hours and then transferred to 30 % sucrose solution overnight at 4°C. Thereafter, kidneys were fixed in O.C.T (Tissue-Tek^®) and snap frozen in liquid nitrogen. These tissues were stored at −80°C until cryo-sectioning. Ten (10) micron tissue sections were fixed on a charged glass slide (ThermoFisher) and used immediately or stored at −80°C until immunostaining. For immunostaining, the slices were first air-dried for 20 mins, washed with PBS for 5 minutes and fixed with 4 % PFA for 20 mins at 4°C. The sections were quenched with 100 mM glycine for 15 minutes, permeabilized and blocked with 5 or 10 % serum in 0.2 % Triton in PBS for one hour at room temperature. Slices were incubated in primary antibody diluted in 5 or 10 % serum overnight at 4°C followed by secondary antibody diluted in 5 or 10 % serum for one hour at room temperature. Slices were washed with 0.1 % Tween 20 in PBS and incubated with DAPI for 5 mins at room temperature, mounted with DAKO mounting medium and sealed. Slides were air-dried and then stored at −20^°C. Note that kidney sections analyzed in Figure 5H-K were obtained from mice fed a “salt-depletion with acid load” diet consisting of a low sodium and chloride diet for 8 days, complemented with 0.28 M NH₄Cl with 0.5 % sucrose in drinking water for 6 additional days as previously described². This diet triggered a metabolic acidosis, significantly lower plasma bicarbonate with a more alkaline urine in the homozygous KI mice compared to WT littermates.

Confocal imaging and image analysis

Immunofluorescent imaging was done with a WaveFX confocal microscope (Quorum Technologies, Guelph, Ontario, Canada) powered by a Volocity software (Quorum Technologies, Guelph, Ontario, Canada). Images were taken with 40 X oil immersion objective with z-stacks at 0.5- µm intervals. Quantitative image analysis was performed using the Volocity analysis software or by open-source cell image analysis softwares CellProfiler⁶⁵ and Fiji⁶⁶.

Image Analysis

Cell profiler was used to analyse TOM20 staining in mIMCD3 cells. After converting the three channel RGB images to grayscale using the split method, we manually outlined each cell in the channel corresponding to kAE1 staining and inputted back into the pipeline. The objects were then converted to a binary image and the TOM20 channel was used as the input channel to measure intensity of TOM20 staining in the manually outlined objects. The raw data including the sum of TOM 20 pixel intensity per cell were then analysed using GraphPad Prism software. A minimum of 30 cells were analysed.

Fiji software was used to analyse images from Magic Red staining and autophagy flux experiment. After each multichannel image was opened and merged in Fiji, the “multi point” tool was used to label all regions of interest. Freehand selection tool was used to manually draw the outline of all selected regions of interest, followed by the “Analyze” command to extract number and size of puncta in pixels. Pixel values were then converted to microns and data analysis was completed using GraphPad Prism. A minimum of 30 cells were analyzed.

Volocity software was used for analysis of kidney sections. From B1 H⁺-ATPase-positive single cells cropped from confocal images of medullary or cortical mouse kidney sections, the channel of interest was used to find objects using the “Find objects” command. Refinement within the selection was made based on object size. The minimum object size thresholds were 0.016 μm² for LAMP1 puncta and 0.02 μm² for TOM20 staining. This was then labelled as “population one”. From population one, touching objects were separated using a size guide of 0.02 μm² for LAMP 1 puncta only with the “Separate touching objects” function. For TOM20 staining, all RFP positive stain within individual cells were characterized under one mask without separating touching objects. The minimum object size was set to 0.02 μm² and everything less than that was considered background staining. The sum of TOM 20 fluorescence intensity per cell was collected and analysed using GraphPad Prism. For LAMP1 images, an exclusion criterion removing objects lesser than or equal to 0.02 μm² was used to remove small background objects. Once the regions of interest in the image were properly outlined, data including number of puncta, intensity of puncta, area/ volume of puncta, among other measurements were exported and data analysed with GraphPad Prism. A minimum of 60 cells were analysed.

Supplementary Figures

Supplementary Figure 1:

Supplementary Figure 2:

Supplementary Figure 3:

Supplementary Figure 4:

Supplementary Figure 5:

Data availability

Original blots shown in Figures 1, 2 and 3 are in the Supplementary Material file. All other relevant data supporting the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgements

We thank Kristina MacNaughton, Jared Bouchard, Kiera Smith and Hilmar Strickfaden for excellent technical assistance. Imaging experiments were performed at the University of Alberta Faculty of Medicine & Dentistry Cell Imaging Core, RRID:SCR_019200, which receives financial support from the Faculty of Medicine & Dentistry, the University Hospital Foundation, Striving for Pandemic Preparedness – The Alberta Research Consortium, and Canada Foundation for Innovation (CFI) awards to contributing investigators. Services were provided by the University of Alberta Faculty of Medicine & Dentistry Workshop, RRID:SCR_019181, which receives financial support from the Faculty of Medicine & Dentistry.

Additional information

Funding

This study was funded by the Canadian Institutes of Health Research (PJT#168871) and the Kidney Foundation of Canada (2020KHRG-666615) to E.C, by a grant from the Deutsche Forschungsgemeinschaft to M.J.S. (IRTG 1830), and operating funds from Natural Sciences and Engineering Research Council of Canada (RGPIN-2018-05783) and the Canadian Institutes of Health Research (PS#165816) to N.T. G.E. received a Graduate Student Engagement Scholarship, a Faculty of Medicine and Dentistry Delnor Scholarship and a Faculty of Medicine and Dentistry 75^th Anniversary award. M.R. received a Sir Frederick Banting and Dr. Charles Best Canada Graduate Scholarship-Master’s (CGS-M) from the Canadian Institutes of Health Research; Walter H. Johns Graduate Fellowship; a University of Alberta Faculty of Medicine and Dentistry/Alberta Health Services Graduate Student Recruitment Studentship (GSRS) and an Alberta Graduate Excellence Scholarship (AGES). A.K.M.S.U. received an NSERC CREATE graduate studentship. F.C. was supported by a Discovery Grant to E.C. from the Natural Sciences and Engineering Research Council (RGPIN-2017-06432), and was awarded a Graduate Recruitment scholarship from the University of Alberta. S.M.A.H. received a PhD scholarship from the DAAD (Deutscher Akademischer Austauschdienst).

Funding

Canadian Institutes of Health Research (CIHR) (PJT#168871)

• Emmanuelle Cordat

Natural Sciences and Engineering Research Council of Canada (NSERC) (RGPIN-2017-06432)

• Forough Chelangarimiyandoab

Kidney Foundation of Canada (KFOC) (2020KHRG-666615)

• Emmanuelle Cordat

Deutsche Forschungsgemeinschaft (DFG) (IRTG1830)

• Manfred J Schmitt

Deutscher Akademischer Austauschdienst (DAAD) (PhD Scholarship)

• Sarder MA Hasib

Canadian Institutes of Health Research (CIHR) (PS165816)

• Nicolas Touret

Natural Sciences and Engineering Research Council of Canada (NSERC) (RGPIN-2018-05783)

• Nicolas Touret

University of Alberta (UofA) (Multiple Scholarships)

• Forough Chelangarimiyandoab

• Grace Essuman

• Midhat Rizvi

• Priyanka Mungara

Canadian Institutes of Health Research (CIHR) (Canada Graduate Scholarship-Master's)

• Midhat Rizvi

Natural Sciences and Engineering Research Council of Canada (NSERC) (CREATE Graduate Studentship)

• Shahid AKM Ullah