Inactivation of Invs/Nphp2 in renal epithelial cells drives infantile nephronophthisis like phenotypes in mouse

Article activity feed

1. 

   Version published to 10.7554/elife.82395 on eLife

   Mar 15, 2023
2. 

   eLife

   Feb 20, 2023

   Author Response

   eLife assessment

   Germline inactivation of NPHP2, which encodes a protein that localizes to the transition zone at the base of the primary cilium, results in infantile kidney cysts and fibrosis. In this study, the authors provide solid evidence that increased cell proliferation and fibrosis precede cyst formation in Nphp-2 mouse models, that mutant renal epithelial cells are responsible for the phenotype, and that genetic inhibition of ciliogenesis in this model reduces disease severity. They also show that valproic acid, a drug that affects a number of cellular targets and is used to treat other human conditions, slows disease progression. One limitation of the study is that it provides limited insights into the mechanisms responsible for any of its interesting observations.

   To our knowledge, our study is the first …

   Author Response

   eLife assessment

   Germline inactivation of NPHP2, which encodes a protein that localizes to the transition zone at the base of the primary cilium, results in infantile kidney cysts and fibrosis. In this study, the authors provide solid evidence that increased cell proliferation and fibrosis precede cyst formation in Nphp-2 mouse models, that mutant renal epithelial cells are responsible for the phenotype, and that genetic inhibition of ciliogenesis in this model reduces disease severity. They also show that valproic acid, a drug that affects a number of cellular targets and is used to treat other human conditions, slows disease progression. One limitation of the study is that it provides limited insights into the mechanisms responsible for any of its interesting observations.

   To our knowledge, our study is the first to pinpoint defective epithelial cells as the main driver for both epithelial cysts and interstitial fibrosis in a NPHP model. The discovery that abnormal signaling from epithelial cells triggered a profibrotic response in the absence of cyst formation is also novel. Our Ift88 Nphp2 double mutant results, combined with tissue-specific function of NPHP2, suggest that NPHP2 functions as a negative regulator of a profibrotic and pro-cystic pathway that interacts with cilia-mediated signaling in epithelial cells and that abnormal signaling from epithelial cells triggers interstitial fibrosis. Moreover, we identified the HDAC inhibitor VPA as a potential candidate drug for treating NPHP. Although the precise molecular function of NPHP2 remains undefined, our results suggest that epithelial specific function and epithelial-stromal crosstalk underlie NPHP like phenotypes in Nphp2 mutant kidneys. Furthermore, although whether NPHP2 interacts with polycystin-mediated signaling remains an outstanding question, our results ruled out the involvement of NPHP2 in ciliary localization of PC2.

   Reviewer #1 (Public Review):

   Nephronophthisis (Nphp) is a multigenic, recessive disorder of the kidney presenting in childhood that is characterized by cysts predominantly at the cortico-medullary junction and progressive fibrosis. An infantile form of the disease presents earlier with more diffuse cystic change. The condition is considered a ciliopathy because most of the genes linked to the condition encode proteins involved in ciliary biogenesis or function. Germline mutations in NPHP2 are associated with a particularly severe, infantile form of the disease. Given that interstitial fibrosis is a more prominent feature of Nphp compared to many other forms of polycystic kidney disease, the authors sought to determine the mutant cell types responsible for the phenotype.

   In the current study, the authors generated and characterized mouse lines with Nphp2 selectively inactivated in either renal epithelial cell or stromal cell lineages and found that inactivation in renal epithelial cells was both necessary and sufficient to cause disease. They further showed that markers of interstitial fibrosis and proliferation increase in mutants prior to the onset of histologically evident cystic disease, suggesting that aberrant epithelial-stromal cell signaling is an early and primary feature of the condition (Figures 1-4). The study design was straightforward and appropriate to address the question, and the results support their conclusions.

   They next tested whether the cilia-dependent cyst-activating pathway (CDCA) that is "unmasked" by loss of other PKD-related genes is similarly active in Nphp2 mutants by generating Nphp2/Ift88 double mutants. Their studies found that the severity of cystic disease and markers of proliferation and fibrosis was attenuated in double-mutants (Fig 5, 6). These studies were also appropriate for testing the hypothesis and the results were similarly consistent with their interpretation.

   In the last set of studies, they tested whether valproic acid (VPA), a drug that has multiple modes of action including acting as a broad inhibitor of HDACs and previously used by the investigators in other forms of polycystic kidney disease, would have similar effects in Nphp2 mutants. The authors tested daily injection from days P10 through P28 in both control and Nphp2 mutant mice with VPA or an appropriate vehicle control and found that VPA was beneficial (Fig 7). The study design was acceptable and the results generally support their conclusions. The one perplexing result is shown in Fig 7B. The Nphp2 mutants, regardless of treatment status, have body weights (BW) that are significantly lower than the controls, with treated mutants even trending lower than their untreated mutant counterparts. This is unexplained and should be addressed. In the mutants with more widespread epithelial cell knock-out of Nphp2 (Ksp-Cre, Fig 1), total body weight decreased as mice became more severely cystic with renal impairment. In the milder form of disease produced with the Pkhd1- Cre (Fig 7), total body weight is inexplicably approx. 2g lower on average despite having much more modestly elevated KBWs and BUNs. Moreover, one might have expected that mutants treated with VPA would have had BWs intermediate between untreated mutants and controls since the severity of the disease was moderately attenuated. These differences raise the question as to whether body weight differences are due to factors independent of disease status, the most likely of which would be that the controls were not littermates. This prompted a careful review of the text for descriptions of the control mice. Throughout the study, the investigators describe selecting animals from the same "cohort", but this term is imprecise.

   There is little information provided about background strains, whether any of the lines were congenic, or whether any of the studies were done using littermate controls. This must be addressed. It would help if the investigators identified the litter status in their plots. This would clearly show relationships between animals and the number of litters that had animals with these properties. If littermates were not used for each study, the authors must explain both why they didn't do so and how they then selected which animals to use. This information is especially important for interpreting the results of their genetic interaction (fig 5) and drug treatment studies (fig 7).

   We thank the reviewer for the multiple positive comments.

   To address the issue of body weight, we examined the time course of body weight change more carefully and added Figure 7-figure supplement 1 to present the results. Although Nphp2flox/flox;Pkhd1-Cre mice displayed reduced body weight at P28 in comparison to controls, this reduction was more moderate than that of Nphp2flox/flox;Ksp-Cre mice (Figure 7-figure supplement 1A). Notably, the trend of body weight difference started at around P21 in both Nphp2flox/flox;Pkhd1-Cre and Nphp2flox/flox;Ksp-Cre mice, coinciding with weaning (Figure 7-figure supplement 1B). It is possible that mutants with compromised kidney function were less capable to thrive and gain weight at around this transition time. In terms of VPA treatment, body weight trended down in both wild type and mutant mice subjected to the treatment, although the difference did not reach statistical significance (Fig. 7B). We cannot rule out the possibility that side effect of VPA contributed to weight loss in treated mice. In addition, VPA may affect body weight increase through HDAC: the HDAC inhibitor Trichostatin A was shown to inhibit adipogenesis (PMID: 34232916) and 4-hexylresorcinol, another HDAC inhibitor, reduced body weight in treated rats (PMID: 34445640). To include the additional data and references, we added the following in the Results section:

   "We analyzed body weight change of Nphp2flox/flox;Pkhd1-Cre mice in more detail and compared it to Nphp2flox/flox;Ksp-Cre mice. At P28, the reduction of body weight in Nphp2flox/flox;Pkhd1-Cre mice in comparison to control mice was more moderate than that in Nphp2flox/flox;Ksp-Cre mice (Figure 7-figure supplement 1)."

   " However, the reduced body weight phenotype in mutant mice was not suppressed by VPA treatment (Fig. 7B). We cannot rule out the possibility that the side effects of VPA contributed to weight loss in treated mice. In addition, VPA may reduce body weight through inhibiting HDAC during the growth period: the HDACI Trichostatin A was shown to inhibit adipogenesis (51)."

   Regarding genetic background, all mice analyzed in figures 5 and 7 are in the same genetic background (C57/BL6J). We added more detailed description of genetic background in the Materials and Methods section. Littermate status is now also indicated in figure legends.

   In Figure 5, multiple genotypes (i.g. Nphp2flox/flox;Ksp-Cre, Nphp2flox/flox;Ift88flox/flox;Ksp-Cre and Ift88flox/flox;Ksp-Cre) were analyzed. Because of the limited number of animals per litter and low yield of desired genotypes, non-littermates had to be included in some cases. Littermate status is now highlighted by colors in the data tables of Figure 5 source data.

   In Figure 7, because of the limited number of animals per litter and the need to subject each genotype to VPA and vehicle treatment, non-littermates had to be included in some cases. Littermate status is now indicated by highlight colors in the data tables of Figure 7 source data.

   Several other considerations. The authors state that the effects of VPA are mediated through the drug's inhibition of HDACs and suggest that future studies could be directed at refining the specific HDAC. While this is certainly possible, the authors should acknowledge that VPAs have been reported to have numerous pharmacologic effects and targets and which of these is mediating the effects in their model is unknown (text). They would need mechanistic studies to show this, though it doesn't discount their possible efficacy as a therapy for PKD.

   We agree that it is an important point to clarify and added in Discussion: "It is also worth noting that VPA could affect targets other than HDACs and testing newly approved HDACIs will provide useful insight."

   The authors also state in their abstract that their double knock-out studies "support a significant role of cilia in Nphp2 function in vivo." It is not clear to me how their studies show this nor how they can exclude that ciliary activity is operating in an Nphp2-independent, parallel fashion that modulates some common downstream pathways.

   We agree with the reviewer that our results do not exclude the possibility that NPHP2 and ciliary activity feed into a common downstream pathway, i.e., a cilia-dependent cyst-activating pathway could operate outside of cilia. We changed the sentence in abstract to "supporting a significant interaction of cilia and Nphp2 function in vivo." In addition, we added "Although cilia-dependent, the downstream pathway could potentially operate outside of cilia and receive parallel signals from both ciliary activity and Nphp2." to Discussion to clarify and reflect the results and model more precisely.

   Reviewer #2 (Public Review):

   The manuscript by Li et al demonstrates the role of Nphp2/Invs in renal epithelia in preventing NPHP-like phenotypes, such as epithelial/stromal proliferation and stromal fibrosis, in mice. Previously, mutants of the Nphp2 allele in mice, generated by insertional mutagenesis, showed severe cystic kidney disease and fibrosis in neonates.

   The authors nicely show that the NPHP-like phenotypes in mutant kidneys arise from abnormal signaling specifically within and from renal epithelial cells. Furthermore, the fibrotic response and abnormal increase of cell proliferation precede cyst formation and could be initiated independently of cyst formation. The authors also show that the removal of cilia reduces the severity of Nphp2 phenotypes. The authors suggest that similar to polycystins, NPHP2 inhibits a cilia-dependent cyst and fibrosis-activating pathway. Finally, the histone deacetylase (HDAC) inhibitor valproic acid (VPA) reduces these phenotypes and preserves kidney function in Nphp2 mutant mice, supporting HDAC inhibitors as potential candidate drugs for treating NPHP.

   Overall, understanding the mechanisms driving NPHP phenotypes is important and drugging relevant pathways in treating this disease is an important unmet need in patients. The authors have provided insights into both these aspects in this study. The manuscript is nicely written, and the assays shown are rigorous and insightful.

   We thank the reviewer for the positive comments.

   Reviewer #3 (Public Review):

   In this manuscript, Li et. al, investigate whether epithelial or stromal Nphp2 loss, a gene causative of nephronophthisis (NPHP), drives polycystic kidney disease (PKD) and kidney fibrosis in a novel floxed model of Nphp2. The authors found that only epithelial and not stromal Nphp2 loss results in NPHP-like phenotypes in their mouse model. In addition, the authors show that concurrent cilia, via Ift88 loss, and Nphp2 loss within the kidney epithelium as well as HDAC inhibition results in less severe PKD/kidney fibrosis, as has been shown in mouse models of other non-syndromic forms of PKD, such as autosomal dominant PKD caused by mutations to Pkd1 or Pkd2.

   The authors aimed to understand (1) whether the published NPHP phenotype (kidney cysts and fibrosis), known from the global Nphp2 knockout mouse, is driven by the function of NPHP2 in the kidney epithelium or stromal cells; (2) if kidney fibrosis in NPHP is linked to kidney damage caused by cysts, or independent and preceding of the PKD phenotype; (3) whether cilia are required, causative, or prohibitive of NPHP cystogenesis; and (4) if a broad spectrum HDAC inhibitor is a potential therapeutic approach for NPHP.

   With the provided results, the authors established that epithelial Nphp2 loss is likely a predominant driver of PKD in their model; however, they cannot exclude that stromal NPHP2 does not play a role in cysts growth post-initiation because the authors failed to directly compare their cell type-specific models to a global cre knockout (e.g. Cagg-cre).

   We agree with the reviewer that we cannot rule out the possibility that stromal NPHP2 plays a role post cyst initiation and added "However, our result does not rule out functional significance of interstitial cells once a pro-cystic and fibrotic response is triggered in mutant epithelial cells." to the Discussion section.

   A direct comparison between epithelial specific and global knockout models is an attractive idea, but technically challenging. For an interpretable comparison, it is essential that the stage and knockout efficiency in epithelial cells are equivalent between the two models. However, Ksp-Cre is expressed in the distal nephron specifically, sparing epithelial cells in other segments, while epithelial cells in all segments would be affected by Cagg-Cre. In addition, global knockout of Nphp2 leads to peri-natal lethality. Inducible Cagg-Cre could potentially be used to bypass earlier functional requirements. But matching stage and knockout efficiency in renal epithelial cells between Ksp-Cre and inducible Cagg-Cre mediated knockout remains challenging. These factors make a direct comparison problematic. Finally, our results revealed the role of defective epithelial cells in triggering the phenotypes but did not rule out a role of interstitial cells once abnormal signaling is initiated in epithelial cells. To clarify this point, we added " However, our result does not rule out functional significance of interstitial cells once a pro-cystic and fibrotic response is triggered in mutant epithelial cells." to the Discussion section.

   In addition, it is possible that cyst initiation/growth upon stromal Nphp2 loss occurs substantially slower compared to epithelial Nphp2 loss. However, it seems the authors did not look at kidney phenotypes beyond 28 days of age. Publications from the ADPKD field suggest, that stromal Pkd1 loss initiates cystogenesis much slower than epithelial Pkd1 loss.

   We have expanded our analysis to 8-week-old mice. We now show that Nphp2flox/flox;Foxd1-Cre mice show normal kidney weight, kidney/body weight ratio, kidney function and histology at P56, supporting our original conclusion that deletion of Nphp2 in interstitial cells fails to trigger obvious renal phenotypes, up to young adult stage. These results were presented in Figure 4- figure supplement 1 and the Results section.

   Further, while the authors suggest that kidney fibrosis precedes cyst development, the results supporting this conclusion are limited to one time point, analyzing IF staining of a single marker that can be compared between non-cystic and cystic time points. These analyses need to be extended to make any firm conclusions.

   At the precystic kidney stage (P7), we analyzed SMA and vimentin levels via immunostaining. Their mRNA levels were additionally quantified via RT-qPCR. We have now analyzed vimentin levels at multiple timepoints (P9, 14 and 21) and results were added to Figure 2. Combined, these data support the initiation of a fibrotic response prior to cyst formation.

   The most interesting finding of the manuscript, and likely most impactful to the field, is, that loss of cilia within the setting of epithelial Nphp2 loss reduces PKD severity. This finding parallels published findings for Pkd1 and Pkd2 which are suggested to function in a cilia- dependent cyst-activation mechanism. Unfortunately, the here shown studies, do not add to the mechanistic insight beyond showing the descriptive finding. Most importantly, it remains unclear whether NPHP2 functions in the same pathway as polycystin-1 or -2 (the Pkd1, Pkd2 gene products) or in a separate independent pathway.

   Our Ift88 Nphp2 double mutant results, combined with tissue-specific function of NPHP2, which to our knowledge is completely novel in a NPHP model, suggest that NPHP2 functions as a negative regulator of a profibrotic and pro-cystic pathway that interacts with cilia-mediated signaling in epithelial cells and that abnormal signaling from epithelial cells triggers interstitial fibrosis. We agree with the reviewer that whether NPHP2 functions in the same pathway as polycystins is an interestingly question. However, we feel it is out of the scope of this manuscript and would pursue this research direction in our future studies.

   With respect to the HDAC preclinical studies, the authors show supporting data that a broad- spectrum HDAC inhibitor may be suitable for slowing cyst growth in their model of NPHP. Overall, these studies are not novel to the field, as HDAC inhibition has been shown to slow PKD progression in various models of PKD al while not in NPHP specifically. Further, the studies seem like an add-on, which does not directly link to the prior cell type-specific studies of NPHP2, and no mechanisms linking the two concepts are provided.

   Although we and others showed that HDACIs slow cyst progression in other PKD models, this study is the first to show its impact on a NPHP model. Given the current lack of treatment for NPHP, we feel it important to communicate the results to the research community even though the molecular mechanism remains to be defined.

   Read the original source
3. 

   eLife

   Feb 20, 2023

   eLife assessment

   Germline inactivation of NPHP2, which encodes a protein that localizes to the transition zone at the base of the primary cilium, results in infantile kidney cysts and fibrosis. In this study, the authors provide solid evidence that increased cell proliferation and fibrosis precede cyst formation in Nphp-2 mouse models, that mutant renal epithelial cells are responsible for the phenotype, and that genetic inhibition of ciliogenesis in this model reduces disease severity. They also show that valproic acid, a drug that affects a number of cellular targets and is used to treat other human conditions, slows disease progression. One limitation of the study is that it provides limited insights into the mechanisms responsible for any of its interesting observations.

   Read the original source
4. 

   eLife

   Feb 20, 2023

   Reviewer #1 (Public Review):

   Nephronophthisis (Nphp) is a multigenic, recessive disorder of the kidney presenting in childhood that is characterized by cysts predominantly at the cortico-medullary junction and progressive fibrosis. An infantile form of the disease presents earlier with more diffuse cystic change. The condition is considered a ciliopathy because most of the genes linked to the condition encode proteins involved in ciliary biogenesis or function. Germline mutations in NPHP2 are associated with a particularly severe, infantile form of the disease. Given that interstitial fibrosis is a more prominent feature of Nphp compared to many other forms of polycystic kidney disease, the authors sought to determine the mutant cell types responsible for the phenotype.

   In the current study, the authors generated and characterized mouse …

   Reviewer #1 (Public Review):

   Nephronophthisis (Nphp) is a multigenic, recessive disorder of the kidney presenting in childhood that is characterized by cysts predominantly at the cortico-medullary junction and progressive fibrosis. An infantile form of the disease presents earlier with more diffuse cystic change. The condition is considered a ciliopathy because most of the genes linked to the condition encode proteins involved in ciliary biogenesis or function. Germline mutations in NPHP2 are associated with a particularly severe, infantile form of the disease. Given that interstitial fibrosis is a more prominent feature of Nphp compared to many other forms of polycystic kidney disease, the authors sought to determine the mutant cell types responsible for the phenotype.

   In the current study, the authors generated and characterized mouse lines with Nphp2 selectively inactivated in either renal epithelial cell or stromal cell lineages and found that inactivation in renal epithelial cells was both necessary and sufficient to cause disease. They further showed that markers of interstitial fibrosis and proliferation increase in mutants prior to the onset of histologically evident cystic disease, suggesting that aberrant epithelial-stromal cell signaling is an early and primary feature of the condition (Figures 1-4). The study design was straightforward and appropriate to address the question, and the results support their conclusions.

   They next tested whether the cilia-dependent cyst-activating pathway (CDCA) that is "unmasked" by loss of other PKD-related genes is similarly active in Nphp2 mutants by generating Nphp2/Ift88 double mutants. Their studies found that the severity of cystic disease and markers of proliferation and fibrosis was attenuated in double-mutants (Fig 5, 6). These studies were also appropriate for testing the hypothesis and the results were similarly consistent with their interpretation.

   In the last set of studies, they tested whether valproic acid (VPA), a drug that has multiple modes of action including acting as a broad inhibitor of HDACs and previously used by the investigators in other forms of polycystic kidney disease, would have similar effects in Nphp2 mutants. The authors tested daily injection from days P10 through P28 in both control and Nphp2 mutant mice with VPA or an appropriate vehicle control and found that VPA was beneficial (Fig 7). The study design was acceptable and the results generally support their conclusions. The one perplexing result is shown in Fig 7B. The Nphp2 mutants, regardless of treatment status, have body weights (BW) that are significantly lower than the controls, with treated mutants even trending lower than their untreated mutant counterparts. This is unexplained and should be addressed. In the mutants with more widespread epithelial cell knock-out of Nphp2 (Ksp-Cre, Fig 1), total body weight decreased as mice became more severely cystic with renal impairment. In the milder form of disease produced with the Pkhd1-Cre (Fig 7), total body weight is inexplicably approx. 2g lower on average despite having much more modestly elevated KBWs and BUNs. Moreover, one might have expected that mutants treated with VPA would have had BWs intermediate between untreated mutants and controls since the severity of the disease was moderately attenuated. These differences raise the question as to whether body weight differences are due to factors independent of disease status, the most likely of which would be that the controls were not littermates. This prompted a careful review of the text for descriptions of the control mice. Throughout the study, the investigators describe selecting animals from the same "cohort", but this term is imprecise. There is little information provided about background strains, whether any of the lines were congenic, or whether any of the studies were done using littermate controls. This must be addressed. It would help if the investigators identified the litter status in their plots. This would clearly show relationships between animals and the number of litters that had animals with these properties. If littermates were not used for each study, the authors must explain both why they didn't do so and how they then selected which animals to use. This information is especially important for interpreting the results of their genetic interaction and drug treatment studies.

   Several other considerations. The authors state that the effects of VPA are mediated through the drug's inhibition of HDACs and suggest that future studies could be directed at refining the specific HDAC. While this is certainly possible, the authors should acknowledge that VPAs have been reported to have numerous pharmacologic effects and targets and which of these is mediating the effects in their model is unknown. They would need mechanistic studies to show this, though it doesn't discount their possible efficacy as a therapy for PKD. The authors also state in their abstract that their double knock-out studies "support a significant role of cilia in Nphp2 function in vivo." It is not clear to me how their studies show this nor how they can exclude that ciliary activity is operating in an Nphp2-independent, parallel fashion that modulates some common downstream pathways.

   Read the original source
5. 

   eLife

   Feb 20, 2023

   Reviewer #2 (Public Review):

   The manuscript by Li et al demonstrates the role of Nphp2/Invs in renal epithelia in preventing NPHP-like phenotypes, such as epithelial/stromal proliferation and stromal fibrosis, in mice. Previously, mutants of the Nphp2 allele in mice, generated by insertional mutagenesis, showed severe cystic kidney disease and fibrosis in neonates.

   The authors nicely show that the NPHP-like phenotypes in mutant kidneys arise from abnormal signaling specifically within and from renal epithelial cells. Furthermore, the fibrotic response and abnormal increase of cell proliferation precede cyst formation and could be initiated independently of cyst formation. The authors also show that the removal of cilia reduces the severity of Nphp2 phenotypes. The authors suggest that similar to polycystins, NPHP2 inhibits a …

   Reviewer #2 (Public Review):

   The manuscript by Li et al demonstrates the role of Nphp2/Invs in renal epithelia in preventing NPHP-like phenotypes, such as epithelial/stromal proliferation and stromal fibrosis, in mice. Previously, mutants of the Nphp2 allele in mice, generated by insertional mutagenesis, showed severe cystic kidney disease and fibrosis in neonates.

   The authors nicely show that the NPHP-like phenotypes in mutant kidneys arise from abnormal signaling specifically within and from renal epithelial cells. Furthermore, the fibrotic response and abnormal increase of cell proliferation precede cyst formation and could be initiated independently of cyst formation. The authors also show that the removal of cilia reduces the severity of Nphp2 phenotypes. The authors suggest that similar to polycystins, NPHP2 inhibits a cilia-dependent cyst and fibrosis-activating pathway. Finally, the histone deacetylase (HDAC) inhibitor valproic acid (VPA) reduces these phenotypes and preserves kidney function in Nphp2 mutant mice, supporting HDAC inhibitors as potential candidate drugs for treating NPHP.

   Overall, understanding the mechanisms driving NPHP phenotypes is important and drugging relevant pathways in treating this disease is an important unmet need in patients. The authors have provided insights into both these aspects in this study. The manuscript is nicely written, and the assays shown are rigorous and insightful.

   Read the original source
6. 

   eLife

   Feb 20, 2023

   Reviewer #3 (Public Review):

   In this manuscript, Li et. al, investigate whether epithelial or stromal Nphp2 loss, a gene causative of nephronophthisis (NPHP), drives polycystic kidney disease (PKD) and kidney fibrosis in a novel floxed model of Nphp2. The authors found that only epithelial and not stromal Nphp2 loss results in NPHP-like phenotypes in their mouse model. In addition, the authors show that concurrent cilia, via Ift88 loss, and Nphp2 loss within the kidney epithelium as well as HDAC inhibition results in less severe PKD/kidney fibrosis, as has been shown in mouse models of other non-syndromic forms of PKD, such as autosomal dominant PKD caused by mutations to Pkd1 or Pkd2.

   The authors aimed to understand (1) whether the published NPHP phenotype (kidney cysts and fibrosis), known from the global Nphp2 knockout mouse, is …

   Reviewer #3 (Public Review):

   In this manuscript, Li et. al, investigate whether epithelial or stromal Nphp2 loss, a gene causative of nephronophthisis (NPHP), drives polycystic kidney disease (PKD) and kidney fibrosis in a novel floxed model of Nphp2. The authors found that only epithelial and not stromal Nphp2 loss results in NPHP-like phenotypes in their mouse model. In addition, the authors show that concurrent cilia, via Ift88 loss, and Nphp2 loss within the kidney epithelium as well as HDAC inhibition results in less severe PKD/kidney fibrosis, as has been shown in mouse models of other non-syndromic forms of PKD, such as autosomal dominant PKD caused by mutations to Pkd1 or Pkd2.

   The authors aimed to understand (1) whether the published NPHP phenotype (kidney cysts and fibrosis), known from the global Nphp2 knockout mouse, is driven by the function of NPHP2 in the kidney epithelium or stromal cells; (2) if kidney fibrosis in NPHP is linked to kidney damage caused by cysts, or independent and preceding of the PKD phenotype; (3) whether cilia are required, causative, or prohibitive of NPHP cystogenesis; and (4) if a broad spectrum HDAC inhibitor is a potential therapeutic approach for NPHP.

   With the provided results, the authors established that epithelial Nphp2 loss is likely a predominant driver of PKD in their model; however, they cannot exclude that stromal NPHP2 does not play a role in cysts growth post-initiation because the authors failed to directly compare their cell type-specific models to a global cre knockout (e.g. Cagg-cre). In addition, it is possible that cyst initiation/growth upon stromal Nphp2 loss occurs substantially slower compared to epithelial Nphp2 loss. However, it seems the authors did not look at kidney phenotypes beyond 28 days of age. Publications from the ADPKD field suggest, that stromal Pkd1 loss initiates cystogenesis much slower than epithelial Pkd1 loss. Further, while the authors suggest that kidney fibrosis precedes cyst development, the results supporting this conclusion are limited to one time point, analyzing IF staining of a single marker that can be compared between non-cystic and cystic time points. These analyses need to be extended to make any firm conclusions.

   The most interesting finding of the manuscript, and likely most impactful to the field, is, that loss of cilia within the setting of epithelial Nphp2 loss reduces PKD severity. This finding parallels published findings for Pkd1 and Pkd2 which are suggested to function in a cilia-dependent cyst-activation mechanism. Unfortunately, the here shown studies, do not add to the mechanistic insight beyond showing the descriptive finding. Most importantly, it remains unclear whether NPHP2 functions in the same pathway as polycystin-1 or -2 (the Pkd1, Pkd2 gene products) or in a separate independent pathway.

   With respect to the HDAC preclinical studies, the authors show supporting data that a broad-spectrum HDAC inhibitor may be suitable for slowing cyst growth in their model of NPHP. Overall, these studies are not novel to the field, as HDAC inhibition has been shown to slow PKD progression in various models of PKD al while not in NPHP specifically. Further, the studies seem like an add-on, which does not directly link to the prior cell type-specific studies of NPHP2, and no mechanisms linking the two concepts are provided.

   Read the original source
7. 

   Version published to 10.1101/2022.02.10.479981v1 on bioRxiv

   Feb 10, 2022