Allelic and Gene Dosage Effects Involving Uromodulin Aggregates Drive Autosomal Dominant Tubulointerstitial Kidney Disease

  1. Guglielmo Schiano
  2. Jennifer Lake
  3. Marta Mariniello
  4. Céline Schaeffer
  5. Marianne Harvent
  6. Luca Rampoldi
  7. Eric Olinger
  8. Olivier Devuyst

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Abstract

Missense mutations in the UMOD gene encoding uromodulin cause autosomal dominant tubulointerstitial kidney disease (ADTKD), one of the most common monogenic kidney diseases. A pressing need for ADTKD is to bridge the gap between postulated gain-of-function mutations and organ damage - a prerequisite for therapeutic development. Based on two missense UMOD mutations associated with divergent progression of ADTKD, we generated Umod C171Y and Umod R186S knock-in mice that showed strong allelic and gene dosage effects, with distinct dynamic pathways impacting on uromodulin trafficking, formation of intracellular aggregates, activation of ER stress, unfolded protein and immune responses, kidney damage and progression to kidney failure. Deletion of the wild-type Umod allele in heterozygous Umod R186S mice increased the formation of uromodulin aggregates and ER stress, indicating a protective role of wild-type uromodulin. Studies in kidney tubular cells confirmed biochemical differences between distinct uromodulin aggregates, with activation of specific quality control and clearance mechanisms. Enhancement of autophagy by starvation and mTORC1 inhibition decreased the uromodulin aggregates, suggesting a therapeutic strategy. These studies substantiate a model for allelic effects and the role of toxic aggregates in the progression of ADTKD- UMOD , with relevance for toxic gain-of-function mechanisms and for strategies to improve clearance of mutant uromodulin.

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    Referee #3

    Evidence, reproducibility and clarity

    Schiano and colleagues present data on two mouse knock-in models with a missense mutation in uromodulin (C171Y and R186S). A strength of the paper is that the mutations are found in patients with autosomal dominant tubulointerstitial kidney disease (ADTKD) but lead to divergent disease progression. The mouse models are characterized in detail examining changes in uromodulin processing, plasma and urine biochemistry and transcript levels by RNA-sequencing. These findings combined with studies in collecting duct lines provide evidence that the extent of uromodulin aggregate formation is related to the severity of the disease and mechanisms are provided to explain these findings including clearance pathway which might be targeted in the future. Overall, there is a large quantity of good data in the manuscript which moves our understanding of uromodulin mutations forward. However, there are some issues that need to be addressed as outlined below.

    Major Comments

    1. In the Introduction, the authors state that the current mouse models have only provided limited information warranting this new study. More information is required here to provide a stronger rationale. What are the specific weaknesses of the prior approaches and what precise questions remain unanswered and how is this hindering therapeutic development. Subsequently, how does this study fill these gaps in our knowledge? This narrative of highlighting the new aspects of this study should also run through the Abstract of the paper more prominently.
    2. The authors have selected two missense mutations from the Belgo-Swiss ADTKD Registry to subsequently model in mice. Are these mutations also present at a high prevalence in other genetic studies of ADTKD? The authors indicate that the patients with a Arg185Ser mutation have a faster progression than Cys170Tyr. One caveat here is that in Supplementary Table 1-2, the patients with Arg185Ser are predominately male and those with Cys170Tyr predominately female. Therefore, is gender playing a role here with males more susceptible to renal disease. Taking this concept forward, if the generated mice are separated by gender are comparable results seen in pathology and renal function parameters than if the animals are grouped together as presented in the paper.
    3. In Figure 1D, an examination of kidney biopsies is undertaken. Can the authors provide any quantification across multiple samples/sections/cells to strengthen this data? The authors measure CD3+ cells in their mouse models - any evidence of these cells in the human biopsies.
    4. In Figure 2C, the quantification presented does not seem to fully reflect the pattern of the blot shown, for example, increase in total signal seen in homozygous mice versus heterozygous C171Y mice. As one of the focuses of the paper is the formation of uromodulin aggregates, perhaps there is a rationale for the core and HMW proteins to be quantified separately, rather than the ratio between them.
    5. The authors use electron microscopy (Figure 2F) to conclude that expansion and hyperplasia of the ER occurs in their mutant mice. A representative snapshot is shown, but can quantification be provided to strengthen this data.
    6. A detailed assessment of plasma and urine biochemistry has been made. As highlighted above, separating this data by sex could be helpful. It is stated that the C171Y mice have a progressive increase in BUN at 4 months, but this statement requires clarification. Are the authors referring to a progressive change over time or with respect to gene dosage? An additional measurement of creatinine clearance might also be useful here. Are there any changes in glomerular function? Significant changes are also found in the urine of C171 heterozygous mice (in sodium and creatinine) but not in the homozygous animals. Any explanation for these findings which are not mentioned in the text? Some of the data is not reported corrected, for example it is stated that uric acid excretion is reduced at 1 month, but this has not been measured then. The conclusion that there are strong gene-dosage effects in both models seems strong. The reviewer agrees this holds for BUN but is not so clear cut for other parameters such as diuresis and osmolarity in C171Y mice. This should be refined.
    7. An interesting analysis is presented on the effect of partial and total denaturation treatments of uromodulin. The reproducibility of these experiments is unclear. Please clarify. Do the authors have any information on how the protein structure of uromodulin might change due to these mutations, for example by structural modelling?
    8. Next, the authors delete a wild-type allele in the R186S mice and examine the severity of disease. In Figure 4D and E it would be more informative to also present the specific changes in HMW and core proteins separately. Is there really a pronounced reduction in premature uromodulin in Figure 4E? Why have the authors focused on CD3+ cells as a marker of inflammation, how about other cell types such as macrophages? The rationale needs to be provided here. Are there changes in fibrosis by histology? Importantly, there appears to be no changes in clinical parameters when the wild-type allele is deleted, so is the main conclusion of this part that the deletion of the wild-type allele has no effect on disease severity, despite some of the gene changes observed.
    9. In Figure 5, the relationship between the amount of uromodulin aggregates and the UPR pathway, fibrosis and inflammation is examined. As highlighted above, the methodology to determine the number of uromodulin aggregates needs to be considered. It is unclear in Figure 5C how this parameter has been generated. Can the authors present the data in this panel as individual mice of all six groups rather than the grouped analysis currently done. This would distinguish if the individual mice with greatest uromodulin aggregates also had the most fibrosis and inflammation and strengthen the presentation of this data.
    10. In your RNA-sequencing data, please clarify if the mice were of the same sex. Interesting changes are found, but the final conclusion is that the transcription signals recapitulate severe ADTMD. This seems an overinterpretation and to strengthen this section the authors could go back to their biopsy samples and examine some of the expression patterns of the novel genes they have identified. Similarly, can any of the novel transcripts identified in the RNA-seq be examined (and/or) altered in the cell lines they have generated with the same mutations in uromodulin.
    11. Using their cells the authors show the autophagy may be involved in the clearance of uromodulin in R185S mutants. However, this pathway is not explored in vivo, an assessment of autophagy in these mice would strengthen this connection.

    Minor

    1. The authors should present full Western blots in their Supplementary data
    2. Figure 2C (and others). Please clarify and label clearly the blots from 1 month and 4-month-old mice.

    Significance

    Schiano and colleagues present data on two mouse knock-in models with a missense mutation in uromodulin (C171Y and R186S). A strength of the paper is that the mutations are found in patients with autosomal dominant tubulointerstitial kidney disease (ADTKD) but lead to divergent disease progression. The mouse models are characterized in detail examining changes in uromodulin processing, plasma and urine biochemistry and transcript levels by RNA-sequencing. These findings combined with studies in collecting duct lines provide evidence that the extent of uromodulin aggregate formation is related to the severity of the disease and mechanisms are provided to explain these findings including clearance pathway which might be targeted in the future. Overall, there is a large quantity of good data in the manuscript which moves our understanding of uromodulin mutations forward. However, there are some issues that need to be addressed; in particular the authors should (i) precisely outline the novelty of their study compared with the prior literature; (ii) clarify the reproducibility of their experiments; (iii) refine areas of overinterpretation in the manuscript; (iv) consider the potential role of gender in their findings and (v) complete the circle in some of their findings, for example examining the novel genes identified in their RNA-sequencing in their human biopsy samples and examining autophagy in their mouse models. These changes will considerably strengthen their article.

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    Referee #2

    Evidence, reproducibility and clarity

    UAKD, a subtype of ADTKD, is extensively studied, although it is an rare inherit kidney disease. Using a knock-in strategy, the authors raised a novel concept that the differences in allelic and gene dosage of Umod mutation triggered distinct protein catabolic pathways, yielding distinct phenotypes and prognosis. The functional mechanisms include that UmodR186S mutation caused insoluble uromodulin aggregates resulting in activation of autophagy, and UmodC171Y mutation led to uromodulin misfolding and touched off ubiquitin-dependent ERAD pathway. Accordingly, the authors tested whether enhancing autophagy attenuates the accumulation of UmodR186S protein in cell cultures. Based on these observations, the authors suggested a strategy to improve clearance of mutant uromodulin. This study was carried out by a team with strong reputation in this area. However, the story appears to be incomplete and in vivo testing of their therapeutic strategy is needed to improve this research.

    Specific comments

    1. Figure 1D: Images at low magnification do not show DAPI, therefore there is no information on the total number of cells in the selected field. Nephron loss (represented by glomeruli) did not appear to differ between UMOD p C170Y and UMOD p R185S, which is inconsistent with the overall conclusions. In addition, PAS staining should be added in Figure 1D.
    2. Figure 2E: in image of C171Y/+, this is no corresponding tubules which is represented by the insert. Figure 2F lower panel, the bars in EM fields are same, indicating a hypertrophy of nuclei in R186S? Figure 2G: how about serum creatinine in these mice? In addition, signs of catabolism (e.g., loss of body weight) are associated with these KI mice?
    3. Figure 3C: what is rationale of using two high speed centrifuges. Please state briefly in method.
    4. Figure 4: histologic assessment of progression is missing here, please add images of PAS, Masson staining at low magnification
    5. Figure 5: Can the authors provide low magnification images (40X) for each condition? A histological evaluation of kidney damage is critical to support the conclusion.
    6. Figure 6: Why are no ubiquitin-related catabolic processes or pathways enriched in C171Y? The authors should perform GSEA analysis to determine whether defined gene sets have significant differences between C171Y and R186S.
    7. Following the experiments in Figures 7 and 8, the authors should assess whether administration of autophagy agonists could improve kidney injury and function in R186S mice.

    Significance

    Although ADTKD is an rare inherit kidney disease, the authors provide new insight into its pathogenesis. As nephrologist, I agreed with the observations and conclusions provided by the study. However, sufficient histological assessment and in vivo validation of the proposed therapeutic strategy would significantly improve this study.

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    Referee #1

    Evidence, reproducibility and clarity

    Uromodulin (Tamm-Horsfall protein) is the most abundant protein excreted in human urine.
    It plays role in protection against urinary tract infections and renal stones. Mutations in UMOD gene encoding uromodulin cause Autosomal Dominant Tubulointerstitial Disease (ADTKD) that slowly progresses to chronic kidney disease.

    In this manuscript, Schiano et al. isolate 12 missense UMOD mutations, which they classify into two groups by age occurrence. They then proceed to study two of these mutations: one from the earlier-onset - Arg185Ser - and the second from the later-onset - Cys170Tyr.

    The authors generate UmodC171Y and UmodR186S knock-in mice with distinct dynamic pathways impacting on ADTKD progression. These mutations are equivalent with UMOD mutations (C170Y and R185S) in patients. UmodC171Y and UmodR186S knock-in mice show impaired uromodulin biogenesis, with strong allelic and gene-dosage effects. The trafficking problem of ADTKD-UMOD mutants, involving ER retention, ER stress, and activation of the UPR is recapitulated in mIMCD-3 cells, where the R185S mutant reveals more aggregates that are triggering PERK and IRE1 pathways and ER stress responses.

    The manuscript is well written, experiments are in general well described and performed, results offer important insights on cellular events eventually leading to organ damage in ADTKD resulting from missense mutation in the UMOD gene.
    The part of the work investigating the degradation mode of two different UMOD mutants, one relying on proteasomal and one relying on lysosomal clearance, is the most interesting for a general audience. Unfortunately, this last part of the work is too preliminary to be accepted as it is.

    Comments/Suggestions:

    • Selection of the UMOD variants, page 5: "R185S and C170Y are the most prevalent mutants in the clusters" please document/add reference.

    • Fig. 1D: please show the position of the insets in the UMOD and BiP panels. Please separate the IF panels from the Picrosirius red panels (these are not the same samples that are shown),
      Formally, the BiP panels in Fig. 1D reveal that there is more BiP in cells expressing R185S. That this correlates with UPR induction (as confirmed in Fig. x) should be written at the end of page 5 to make this issue clear for non-experts.
      In Fig. 1D, the signal of BiP is not visible in WT and C170Y tissue/cells, which is odd because BiP is abundant protein. Moreover, the differences in BiP levels quantified in WB (semi-quantitative analyses) are not that dramatic in the mouse model (SFig. 3). Which panel in SFig. 3 (mouse) should be representative of the IF shown in Fig. 1D (patients)?
      Fig. 1D: Magnification of these images is not sufficient to conclude that R185S accumulates in the ER, and that WT and C170Y are at the apical cell's membrane as written (page 5). Authors should refer to Suppl Fig 1C, where individual cells are visible.
      Authors should briefly explain at the end of page 5 how the P. red staining in Fig. 1D informs on fibrosis.

    • In the analyses of misfolded UMOD mutants (e.g., Fig. 2, 3, 4, ...) one would expect a test showing that BiP associates with R185S>C170Y>WT.

    • Fig. 2F: in R186S there is a dramatic enlargement (at least 2x) of nuclei. Can the authors comment on that?

    • Fig. 7E: Shouldn't one expects apical signal for C170Y?

    • Fig. 7F: Why there is apical signal for R185S (and not for C170Y)?

    • The part covering the degradation of the two UMOD variants would be of great interest for a wide audience of cell biologists. However, these data are too preliminary and, in this form, inconclusive.
      Few examples: MG132 is a non-specific inhibitor of the proteasome, which may enhance endogenous and trans-gene expression (check in Pubmed "mg132 promoter" for relevant literature). Thus, an increase in the intracellular level of C170Y on MG132 treatment does not necessarily indicate inhibition of the protein's proteasomal turnover. It could also, at least in part, be caused by an increased synthesis of UMOD. The authors should show that MG132 does not increase synthesis of mutant UMOD (or use the more selective proteasome inhibitor PS-341 in their experiments); similarly, the data on R185S do not prove that this protein is client of autophagy. They rather show that autophagy removes the protein when cells are under nutrient restriction (note that starvation activates bulk autophagy, the non-selective lysosomal clearance of cellular components). To show that misfolded R185S is removed from cells by misfolded protein-induced ER-phagy (i.e., ER-to-lysosome-associated degradation), the authors should monitor in WB the accumulation of R185S in the presence of BafA1 and/or in IF the accumulation of R185S within lysosomes in the presence of BafA1.

    Minor comments

    • Figure 1B: dotted lines should be defined in the legend.
    • Figure 1C: "phenotypes are denoted as indicated". The color-code used for the phenotype is unclear to me. For example, what is the phenotype of the V.2 (grey square)?
    • The meaning of "Unlike in UMOD R185S cells, higher SQSTM1 puncta colocalizing with uromodulin were initially present in C170Y mutant cells and further accumulated in MG132-treated cells (Supplementary Figures 10A, B). These data suggest that mutant cells respond differently to UPS inhibition, with C170Y mutant uromodulin being mainly targeted to this pathway." (page 14) and the interpretation of the results shown in 10A and 10B is unclear to me.
    • Page 7: "The UmodC171Y mice showed a progressive increase in BUN at 4 months" please define BUN.
    • Please, provide a complete list of primary antibodies used for immunoblotting, immunohistochemistry, and immunofluorescence staining.

    Significance

    The manuscript is well written, experiments are in general well described and performed, results offer important insights on cellular events eventually leading to organ damage in ADTKD resulting from missense mutation in the UMOD gene.
    The part of the work investigating the degradation mode of two different UMOD mutants, one relying on proteasomal and one relying on lysosomal clearance, is the most interesting for a general audience. Unfortunately, this last part of the work is too preliminary to be accepted as it is.

    My expertise: protein quality control, ER-phagy

  9. Version published to 10.1101/2022.09.13.507770v2 on bioRxiv
  10. Version published to 10.1101/2022.09.13.507770v1 on bioRxiv