Distal Renal Tubular Acidosis Mutants of the Kidney Anion Exchanger Disrupt Cytosolic pH and Cause Abnormal Autophagy in Renal Epithelial Cells
Bibliographic record
Abstract
Background: Distal renal tubular acidosis (dRTA) is a disease caused by impaired renal acid secretion leading to metabolic acidosis and subsequent biochemical derangements which facilitate formation of kidney stones. This can result from a mutation in the kidney anion exchanger 1 (kAE1) protein, a chloride bicarbonate exchanger in the alpha intercalated cells (A-ICs) of the kidney collecting duct. Studies in human dRTA patients and mouse dRTA models have shown an unexplained depletion in A-ICs. dRTA mouse kidney sections also showed an accumulation of autophagy markers in the remaining A-ICs. Objective and Hypothesis: To better understand the link between dRTA kAE1 mutant expression and A-IC depletion, we hypothesised that dRTA mutant expression activates IC loss through abnormal autophagy and apoptosis. Methods: We used mouse inner medullary collecting duct (mIMCD3) cells expressing two dRTA kAE1 mutants (R589H or S525F) for in vitro studies and two dRTA kAE1 knockin mice L919X and R607H, the murine equivalents of human R901X and R589H mutations, respectively for in vivo studies. We assessed intracellular pH using the ratiometric pH-sensitive fluorescent probe BCECF-AM. Using live cell confocal microscopy, we measured autophagy flux with the eGFP-RFP-LC3 construct. Real time measurements of extracellular acidification and oxygen consumption rates were used to determine cellular glycolytic and oxidative ATP production rates. Results: In vitro, kAE1 R589H and S525F dRTA mutants had significantly more alkaline intracellular pH compared to wild type (WT). Both mutants had higher autophagy induction and accumulation of autolysosomes at steady state. Chemically acidifying cytosolic pH of mutants reversed abnormal autophagy in mutant cells. The kAE1 R589H mutant had significantly lower oxidative ATP production rate whereas S525F mutant had significantly lower glycolytic ATP production rate compared to WT. In vivo, dRTA R607H homozygous mutant mice showed reduced abundance of the vacuolar proton ATPase and of mature Cathepsin D compared to WT. Conclusion: These findings confirm that kAE1 mutant expression alters cytosolic pH and induces abnormal autophagy as a result of reduced overall ATP production and defective lysosomal function. Future work will analyse the processing/traffcking of lysosomal cathepsins and mitochondrial structure and function in mutant cells, and assessing collecting duct lysosome numbers in kidney sections by immunohistochemistry. Our work provides a lead explaining the loss of A-ICs observed in dRTA patients. Canadian Institutes of Health Research (CIHR), Graduate Student Engagement Scholarship — University of Alberta. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".