Reduction of lithium induced interstitial fibrosis on co-administration with amiloride

<jats:title>Abstract</jats:title><jats:p>Long-term administration of lithium is associated with chronic interstitial fibrosis that is partially reduced with exposure to amiloride. We examined potential pathways of how amiloride may reduce interstitial fibrosis. Amiloride was administered to a rat model of lithium induced interstitial fibrosis over a long term (6 months), as well as for short terms of 14 and 28 days. Kidney cortical tissue was subjected to RNA sequencing and microRNA expression analysis. Gene expression changes of interest were confirmed using immunohistochemistry on kidney tissue. Pathways identified by RNA sequencing of kidney tissue were related to ‘promoting inflammation’ for lithium and ‘reducing inflammation’ for amiloride. Validation of candidate genes found amiloride reduced inflammatory components induced by lithium including NF-κB/p65<jats:sup>Ser536</jats:sup> and activated pAKT<jats:sup>Ser473</jats:sup>, and increased p53 mediated regulatory function through increased p21 in damaged tubular epithelial cells. Amiloride also reduced the amount of <jats:italic>Notch1</jats:italic> positive PDGFrβ pericytes and infiltrating CD3 cells in the interstitium. Thus, amiloride attenuates a multitude of pro-inflammatory components induced by lithium. This suggests amiloride could be repurposed as a possible anti-inflammatory, anti-fibrotic agent to prevent or reduce the development of chronic interstitial fibrosis.</jats:p>

section_content: Reduced fibrosis and inflammatory signatures with amiloride exposure are evident in the short-term. The results of our long-term amiloride exposure suggested amiloride had anti-inflammatory and anti-fibrotic properties. We next determined if these changes occurred with early exposure to amiloride administered concurrently with lithium from day zero, using shorter treatment periods of 14 and 28 days. As expected, the lithium animals all demonstrated NDI which was reduced in the amiloride group (Supplementary Table 1 ). 

Following 14 days of lithium treatment alone or combined with amiloride, the characteristic morphological changes of distal tubular cystic dilations (Fig. 2a ) were evident. There was some interstitial fibrosis evident, but this did not reach significance compared to controls (Fig. 2c ). After 28 days of treatment, the same morphological changes were visible to a greater extent in both the lithium and lithium-amiloride groups (Fig. 2b ). Interstitial collagen deposition was increased in the lithium alone group to compared to control and lithium-amiloride groups at 28 days (Fig. 2d ). 

RNA Seq performed on kidney cortical tissue from all 14-and 28-day treatment groups showed most of the 14-day samples (except one) clustered within their respective treatment in the Principal Component Analysis (Supplementary Fig. S1b ). The heatmap of the most variable top 200 gene counts suggested the gene expression changes were treatment related (Fig. 2e ). In the 28-day data only the control group clustered as a separate group, with the lithium and lithium-amiloride groups clustering together (Supplementary Fig. S1c ). This was suggestive that actions of amiloride on modulating lithium induced gene expression may vary over this time course. (Fig. 2f ). 

Analysis of significantly differential gene expression is given in Supplementary Dataset 1. 

In line with studies over a shorter time frame 9, ontologies of differentially expressed genes increased with lithium administration at 14 days (n = 1264) were enriched for 'inflammatory response', 'lymphocyte activation', 'innate immune response' and 'cytokine-mediated signalling pathway' (Supplementary Dataset 2b). Specific genes differentially expressed in the lithium group correlating with the inflammatory response included Nfkb2 (0. 76log2FC), subunit of NF-κB (Supplementary Dataset 1 and Supplementary Fig. S3a ). These also included genes that encode T cell receptor CD3 (cd3d, 1. 92, cd3e, 1. 64 and cd3g, 1. 54log2FC), CD8 cytotoxic T cell (cd8b, 1. 13 and cd8a, 0. 69log2FC), along with activator Zap70 (1. 40log2FC) and fos (3. 48log2FC) important for activation and differentiation of leukocytes 12. Other significantly expressed genes were targets of tumor suppressor protein 53 (p53) including cdkn1a/p21 (4. 14log2FC), and cyclin D1 (0. 91log2FC) and tert (0. 71log2FC), which are associated with epithelial cell proliferation [13] [14] [15]. Of interest, GSK3β gene expression was not differentially altered, despite the key role this kinase plays in mediating the observed changes induced by lithium. 

We focused on the genes modulating inflammation, cellular proliferation and fibrosis. Overrepresentation analysis of common genes increased with lithium treatment and decreased in response to lithium-amiloride at 14 days (n = 218), showed lithium induced positive enrichment of 'inflammatory response', 'cytokine production', 'cell adhesion', 'innate immune response' and 'wound healing' gene sets as previously reported 9 (Supplementary Fig. S4b ). Consistent with data at six-months, ' Aldosterone-regulated sodium reabsorption' was overrepresented amongst common genes decreased by lithium and increased with lithium-amiloride (n = 43) (Supplementary Fig. S4C ). 

At 28 days, lithium induced increased genes were overrepresented in 'Cell adhesion molecules (CAMs)' and 'inflammatory response' signalling pathway. Lithium-amiloride had ' Aldosterone-regulated sodium reabsorption' pathway overrepresented amongst increased genes (Supplementary Dataset 2b). Here there were fewer differential and common genes (n = 19) consistent with the groups clustering together (Fig. 2f ). These common genes were enriched in response to nutrient levels and wound healing amongst others (Supplementary Fig. S4e ). Many of the significantly differentially expressed genes from the Li/Control and LiAM/Li comparisons at six months were altered in the 14-day data. The pro-fibrotic and inflammatory genes decreased in the LiAM/Li comparison at both 6 months and 14 days included serpine1, osmr, dll1, socs3, myc, angptl4, lamb3, havcr1 and metalloproteinase genes. Short-term treatment demonstrated that amiloride produced a reduction in the anti-inflammatory gene signatures before a significant difference in interstitial fibrosis was evident.

section_content: The RNA Seq analyses suggested amiloride reduces multiple facets of lithium induced inflammation and T-cell mediated immune response. NF-κB a key inflammatory pathway can be positively and negatively modulated by Wnt/βcatenin signalling 16 and negatively regulated by p53 signalling 17. The Wnt/βcatenin pathway is regulated by GSK3β, a known target for lithium 18, 19. Therefore, we first investigated β-catenin expression, which had increased nuclear translocation in the tubular epithelial cells in the lithium samples, but was significantly reduced in the lithium amiloride group (Fig. 3 ). 

We next investigated if the inflammatory marker nuclear p65 ser536 /RelA, a subunit of the NF-κB complex was differentially expressed between treatment groups. Increased nuclear p65 ser536 staining was found in the dilated tubules, and a higher percentage of p65 ser536 positive cells was found at 14 and 28 days, and six months in the lithium and lithium-amiloride groups compared to controls (Fig. 4a, b ). The lithium-amiloride group at 14 days and six months had reduced p65 ser536 staining compared to the lithium group but this was still increased compared to controls. CD3 staining for T cells showed these cells were largely present in the interstitium (Fig. 4c ). The lithium groups had the highest number of T cells with the largest amount evident in the six-month cohort (Fig. 4d ). Lithium-amiloride significantly reduced the number of T cells at all time points, most evident at six months compared to lithium alone. The findings of reduced NF-κB signalling and T-cell infiltration is consistent with amiloride having profound anti-inflammatory effects in this model.
section_name: Reduced inflammation and T-cell infiltration with amiloride exposure.

section_content: Fibrosis is a consequence of persistent tubular epithelial injury leading to maladaptive repair and inflammation 20. RNA seq analysis highlighted cdkn1a/p21, a key target of p53 at all time points (1. 25log2FC at six months, 3. 19 log2FC at 28 days and 4. 14log2FC at 14 days). Tumor suppressor p53 has a well-established role in responding to stress and preventing inflammation which may be modified with lithium and amiloride treatment 21, 22. To test if p53 was altered with lithium and/or amiloride exposure, p53 and its downstream target (p21), a measure of activated p53, were investigated using immunohistochemistry. p53 and p21 were almost exclusively found in the nuclei of cells in dilated distal tubules (Fig. 5a ). In the lithium and lithium-amiloride treatment groups, p53 staining was increased at all time points compared to controls (Fig. 5b ). The percentage of p53 staining in lithium induced dilated tubular cells was highest at six months and of interest, this was evident most in the lithium-amiloride group. Associated with this was an increased expression of miRNA34a which is known to be transactivated by p53 23 (Supplementary Fig. S5 ). At 14 days, p53 staining was again highest in the lithium-amiloride group; however, no difference between the lithium-amiloride and lithium groups was found at 28 days (Fig. 5b ). The p21 results largely followed the p53 results at six months and 28 days; however, p21 staining was highest in lithium treated animals at 14 days (Fig. 5c ). These results suggest p53 responds to lithium induced cellular damage. The enhanced p53 response with amiloride exposure is consistent with p53 contributing to amiloride's anti-inflammatory properties with reduced p65/NF-κB expression. 

We next explored if amiloride's effect on p53 would attenuate pro-inflammatory regulators that can function in the absence of p53, such as activated pAKT ser473 that can promote p53 degradation [24] [25] [26] [27]. An analysis of pAKT ser473 expression in all treatment groups found pAKT ser473 was localised in the dilated tubular epithelial cells and was increased in lithium and lithium-amiloride groups compared to controls. At 14 days and 6 months pAKT ser473 expression was highest in lithium compared to the corresponding lithium-amiloride group (Fig. 5d ). Again, at 28 days pAKT ser473 showed no difference between the lithium and lithium-amiloride groups (Fig. 5d ). These results suggest amiloride reduces pAKT ser473 function supporting an anti-inflammatory role for amiloride. The lack of difference observed in the number of p65 and pAKT positive cells between lithium and the lithiumamiloride groups at 28 days in particular was interesting. While we do not fully understand this phenomenon, we propose a transition from acute injury between 14 and 28 days to chronic injury seen at 6 months. Specifically, it could be an acute inflammatory response to lithium at 14 days reduced by amiloride and a potential wound healing stage at 28 days where the effect of lithium and lithium-amiloride on inflammation equilibrate. Lower doses of lithium have been shown promote tubular epithelial cell recovery by directly inhibiting GSK3β, or indirectly via phosphorylating Akt. The short term beneficial effect of lithium at 14 days could be due to processes that are intrinsically linked with cell proliferation by upregulation of cell cycle kinases thereby preventing apoptosis with reduced fibrosis 28. 

Amiloride co-administration reduces pericytes and Notch1 accumulation in the interstitium. PDGFrβ positive pericytes transforming into myofibroblasts is a key component in the development of interstitial fibrosis 29, 30. Based on our network plot analyses at six months (Supplementary Fig. S2 ), we identified that PDGFrβ expression was reduced with the lithium-amiloride group. This was suggestive that activated pericytes may be recruited and mediate in part the lithium induced interstitial fibrosis and amiloride may downregulate this activation. To explore this further, we used PDGFrβ immunohistochemistry to examine the role of activated pericytes in this model. Lithium treatment alone demonstrated increased PDGFrβ positive cells in the interstitium around dilated tubules at 28 days and 6 months (Supplementary Fig. S6c and Fig. 6a ) with fewer PDGFrβ positive cells in the lithium-amiloride group. Additional marker characterization of PDGFrβ positive cells revealed many were p65 Ser536 positive and some were also positive for the myofibroblast marker αSMA (Supplementary Fig. S7 ). These data suggest amiloride exposure reduced the amount of activated pericytes in the kidney interstitium surrounding the dilated tubules. 

Transition of PDGFrβ positive pericytes into myofibroblasts has been associated with Notch1 27. Although Notch1 was not significantly increased, the Notch1 ligand Jag1 expression was increased with lithium alone (0. 63log2FC) and decreased with lithium-amiloride (-0. 49 log2FC) at 6 months. Increased Jag1 expression has been found to be dependent upon activation of NF-κB/RelA 31. 

Here lithium alone was associated with increased expression of p65 ser536 (NF-κB/RelA) (Fig. 4a, b ) with increased Jag1 evident from the network plot (Supplementary Fig. S2a ). Therefore, we investigated if Notch1 expression was increased. The spatial distribution of Notch1 was determined using RNAscope which was found predominantly in the interstitium with minimum expression in the tubular cells (Fig. 6b ). Regions associated with increased Notch1 expression overlapped with PDGFrβ positive cells, suggesting that these were Notch 1 positive pericytes. Furthermore, the in-situ images demonstrated reduced Notch 1 positive pericytes with amiloride exposure, further supporting an anti-inflammatory function for amiloride use.
section_name: Amiloride affects inflammatory regulators upstream of NF-κB signalling.

section_content: In this study, we identified genes and pathways involved in lithium induced progressive interstitial fibrosis and how amiloride interacts with these pathways to down-regulate the progression of interstitial fibrosis (Fig. 7 ). The top candidates identified by RNA sequencing, validated using immunohistochemistry and immunofluorescence, included activated PI3k/Akt, p53 and its downstream target p21, NF-κB/p65 ser536, and CD3 T cells. Other targets included Notch1 positive PDGFrβ pericytes involved in pericyte to myofibroblast transformation, recently identified as a regulator of kidney fibrosis 30. On the basis of these results, we highlight molecular targets that are modified by the actions of amiloride in combination with lithium. 

Lithium induced interstitial fibrosis is initiated following G2M arrest that is associated with a maladaptive response with persistent inflammation 8, 20. Lithium directly inhibits GSK3β 32 and indirectly via activation of AKT through PI3k/Akt signalling 33. Physiologically, GSK3β is constitutively expressed and required for cell cycle turnover 34, 35, but inhibition of GSK3β leads to stabilization of β-catenin, as seen with increased β-catenin expression following lithium treatment in the tubular epithelial cells lining the dilated distal tubules 36, 37. Activated Wnt causes cell arrest in the G2M phase 38, which is associated with increased interstitial fibrosis 39 mediated via increased activation of pericytes along with pericyte to myofibroblast transformation 40. Of interest, we did not observe any changes in GSK3β gene expression. A possible explanation is that as GSK3β is ubiquitously expressed, total GSK3β may not change, rather the intracellular ratio of active GSK3β versus inactive pGSK3β is critical for the observed changes seen. As the role of GSK3β in lithium induced changes are well documented in other studies 36, 37 we focused on the regulatory pathways upstream of GSK3β as well as other linked pathways associated with lithium induced fibrosis. p53 and NF-κB signalling pathways are both activated under cellular stress and have opposing outcomes with p53 promoting anti-inflammatory responses and NF-κB promoting pro-inflammatory responses 21, 22. p53 expression was increased in dilated tubular epithelial cells suggesting increased stress following lithium exposure. A robust p53 response may be compromised with lithium, given that GSK3β activity is required for p53 function and lithium inhibition of GSK3β would indirectly down-regulate p53 function 41. In addition, pAKT ser473 which was increased in the dilated tubular epithelial cells with lithium, would also down-regulate p53 function by directly interacting with MDM2 leading to ubiquitination of p53 24. From the RNA seq data, we found MDM4 at 6 months and MDM2 gene expression at 14 days significantly increased with lithium consistent with lithium reducing p53. Other negative regulators of p53, were increased including Tert 42, 43. Given activated pAKT ser473 increases Tert 44, upregulation of Tert offers another explanation for the reduced p53 function following lithium induced damage. 

This study found that co-administration of amiloride significantly reduced inflammation and fibrosis. Amiloride reduced pAKT ser473 expression and increased p53 which could explain the decrease in NF-κB/p65 expression, and decreased recruitment of Notch1 positive PDGFrβ and CD3 T cells in the interstitium. Collectively these changes were associated with reduced inflammation and a decrease in profibrotic cytokines TGFβ1 and CTGF with a marked reduction in interstitial fibrosis 3. Lithium-amiloride could also potentially reduce pGSK3β ser9 (inactive) expression, allowing active GSK3β mediated down-regulation of Notch1 expression further contributing to the downregulation of activated pericytes as previously demonstrated 45, 46. In this study, we were not able to demonstrate successful GSK3β and pGSK3β ser9 co-staining with Notch1 to further support this conclusion. 

Further support for an enhanced role for p53 with amiloride, comes from the lithium-amiloride treatment group having increased microRNA34a expression. This microRNA is transactivated by p53 23 and together are able to suppress Wnt/βcatenin signalling that has been implicated in kidney fibrosis 47. Other studies have suggested that miRNA34a is downregulated by Notch1 48. This also supports the observations seen in this study. Overall, the results suggest that reduced pAKT ser473 signalling is an essential component in amiloride-induced reduction in interstitial fibrosis. Increased p53 expression could be potentiated via a feedback loop mediated through PTEN further inhibiting pAKT 49. 

Increased expression of PDGFrβ positive pericyte accumulation is an essential component of interstitial proliferation in response to injury and the development of interstitial fibrosis [50] [51] [52]. Notch signalling plays an important role in the development and transition of pericytes into myofibroblasts 27. Studies have shown active pAKT ser473 is essential for activation of Notch signalling 53. Co-labelling sections with αSMA clearly showed sparse but distinct co-localisation of PDGFrβ postive pericytes to αSMA labeled myofibroblasts in the interstitium. Taken together the co-localisation of Notch1 and PDGFrβ (Fig. 6 ) and pAKT ser473 and PDGFrβ expression in PDGFrβ production is reduced resulting in reduced Notch1 positive PDGFrβ pericytes in the interstitium and a decrease in profibrotic cytokines TGFβ1 and CTGF (dashed black line). The reduced inflammation leads to reduced fibrosis 3. From the literature: Lithium directly inactivates constitutively active glycogen synthase kinase 3β (GSK3β) by phosphorylation (pGSK3β) 32. Inactive pGSK3β activates and increases Wnt/βcatenin signalling that causes cell arrest contributing to pericyte recruitment 38, 78 and fibrosis 39. Active GSK3β is required for transactivation of p53 41 (dashed yellow line) and inactive pGSK3β enhances Notch1 activity. Lithium-Amiloride could potentially be reducing the inactive pGSK3β that could lead to reduced expression of Notch1 (dashed black lines) 45, 46. *Data derived through cell culture (not shown, unpublished study). interstitial cells (Supplementary Fig. S8 ) upon lithium exposure supports the concept of Notch-mediated PDGFrβ positive pericytes being recruited to sites of damaged tubules and transitioning into myofibroblasts mediated via active pAKT ser473 in response to chronic lithium exposure. 

Co-administration of amiloride with lithium from day 0 did not prevent the changes in cell proliferation and G2M arrest leading to the dilated cystic tubules that were evident at 14 and 28 days. Lithium at therapeutic doses is thought to suppress GSK3β activity by approximately 25% 35, 54. So clearly in this model there is not complete inhibition of GSK3β activity. Amiloride would reduce cellular uptake of lithium via inhibition of sodium channels and therefore potentially reduce intracellular lithium concentrations which may allow a reduction in GSK3β inhibition. This was observed with increase in p53, p21, reduced Notch1 and reduced NF-κB/p65 mediated inflammation that was evident from as early as day 14 with amiloride co-treatment. However, clearly amiloride did not counteract all of the pathways associated with lithium induced cellular injury, especially those related to tubular epithelial cell proliferation and subsequent G2/M arrest [6] [7] [8]. 

Activated p53 can lead to induction of downstream target genes including cdkn1a/p21 and Bax, and depending on the stimulus, causes either cell arrest 55 or apoptosis 56 respectively. Following short term (4-13 days) lithium treatment, de Groot et al. 8 demonstrated that proliferating principal cells were arrested at the G2/M phase which may contribute towards fibrosis 8. In the current study, lithium alone and lithium-amiloride was associated with increased p21 expression compared to controls. In other models of kidney fibrosis, an increase in p21 expression has been linked to G2/M cell arrest 57. This would suggest that lithium is having a direct effect on p21 expression which is not modified by amiloride. The proliferating tubular epithelial cells arrested at the G2/M phase, following lithium induced increased expression of p21, leads to a pro-inflammatory state with activation of NF-κB/ p65 mediated inflammation and increased expression of pro-fibrotic cytokines 39. However, the robust increase in p53 expression as well as the observed increased p21 expression in the dilated tubular epithelial cells in the lithium and amiloride, may in part explain 8 the reduced fibrosis when compared to the lithium alone group. 

Tumor suppressor p53 regulates the cell cycle and the arrest after cell lineage decision is made via Notch signalling 58. Lineage decision, inductive signalling and later inhibition are the main functions of Notch signalling 59. Active Notch favors principal cell development, whereas inactive Notch favors intercalated cell development 60. Lefort et al., using a keratinocyte cancer and squamous cell carcinoma lines, showed that Notch1 is a target of p53 with an active role in tumor suppression 61. Lithium induced tubular injury is associated with principal cells de-differentiating to intercalated cells 6, 7, 62, 63. Increased Foxi1 transcription, a key marker of intercalated cells 60 with lithium treatment was significantly and differentially expressed at 28 days and 6 months (0. 94 and 1. 05 log 2FC, respectively) (Supplementary Dataset 1). Given lithium treatment can alter the ratio of principal to intercalated cells, it is not surprising that in this study Notch1 expression was predominantly seen in the interstitium and not in the tubular epithelial cells due to the reduction of principal cells. Notch1 signalling has also been implicated in enhancing fibrosis and its inhibition rescuing fibrosis 64. 

In conclusion, the previously demonstrated lithium induced overexpression of number of intracellular pathways including 'cell cycle signalling', 'NF-κB signalling', 'p53 signalling', 'Wnt signalling' and 'aldosterone upregulated genes' 9 was confirmed at later time points of 14 days, 28 days and at 6 months. Combined treatment with amiloride, significantly downregulated the expression of those gene pathways associated with inflammation resulting in a marked reduction in interstitial fibrosis. The interaction of these pathways are summarized in Fig. 7. However, the lithium induced cell proliferation and G2/M growth arrest which is associated with dilated distal tubules and altered tubular epithelial cells, was not modified by amiloride. This would suggest that amiloride does not modify 'cell cycle signalling' pathways directly. Further studies are underway to examine the anti-inflammatory actions of amiloride.
section_name: Discussion

section_content: Kidney fibrosis animal model. Rat kidney cortical tissue from our previously published long-term (6 months) animal model 2, 3 was used here alongside rat kidney cortical tissue from short term lithium treated animals (14 and 28 days). Animals were fed lithium for 6 months and co-administered amiloride for five months that maintained plasma lithium levels equivalent to that used therapeutically 2, 3, 65. The animal studies were performed in accordance with ARRIVE guidelines. 

Short term animal study for 14 and 28 days included male Wistar rats (~ 200 g) from the Hercus-Taieri Resource Unit, University of Otago that were separated into three groups; Control n = 6, lithium alone (Li, n = 6), and lithium with amiloride (LiAM, n = 6). Here animals were treated following the protocol from our previous study but were instead given amiloride from day zero along with lithium. Anesthetised animals were euthanized using decapitation (guillotine). Excised kidney tissue was either frozen and stored at -80 °C in RNAlater®, or fixed in 10% neutral buffered formalin (NBF) and processed into formalin fixed paraffin embedded (FFPE) blocks for downstream analysis. Ethical approval for the protocols described here was given by the University of Otago Animal Ethics Committee (81/14), under New Zealand National Animal Welfare guidelines. 

To induce chronic interstitial fibrosis, the protocol from Croft et al. was modified 3. The diets were administered as follows: control rats were given a normal chow diet (Speciality Foods, Perth, Australia), lithium alone group received 40 mmol lithium/kg dry food for the first 7 days, followed by 60 mmol lithium/kg dry food (lithium diet) for up to 14 or 28 days and the LiAM group were given standard chow with lithium and amiloride drinking water containing 0. 2 mmol/l amiloride (Sigma Aldrich, USA). This protocol results in plasma lithium levels comparable to therapeutic levels in human plasma (0. 8-1. 3 mmol/l) and minimizes the reduced weight gain that can be caused by lithium. To enable adequate mixing with the drug, all pellets were pulverized with a kitchen blender, sifted, ground into a fine powder in a small blender and sifted again. All rats administered lithium received a salt block to maintain sodium balance and to prevent lithium intoxication. Water and food intake was measured every day and body weight every other day. For the last two days of the experiment, rats were housed individually in metabolic cages on a 12:12 h light-dark cycle and allowed to settle for 21 h. 

Blood plasma and urine analysis. Blood and urine analyses were performed to characterize the effect of lithium and amiloride on kidney function. The analyses for the six-month study were performed and published previously 3. For the 14-and 28-day studies the osmolality of urine and plasma was measured using Wescor 5500 Vapro® Osmometer (Logan, UT, USA). Sodium and potassium ion concentrations were determined by flame photometry (SEAC FP20, Italy), and chloride ions in the plasma were determined electrometrically using a Cotlove chloride titrator (American Instrument Co., Silver Spring, MD, USA). Lithium-ion concentration in plasma was measured with a contrAA® 700 High-Resolution Continuum Source atomic absorption spectrometer (Analytik Jena AG, Germany). Creatinine in urine was measured according to Jaffe's method (Pointe Scientific Inc., Canton, MI, USA), and urea (blood urea nitrogen (BUN); Pointe Scientific Inc., Canton, MI, USA) was measured in plasma. Total urinary protein was determined with a bicinchoninic acid (BCA) assay (Pierce Chemical Co., ThermoFisher Scientific, Rockford, IL, USA). Blood and urine analysis are given in Supplementary Table 1.
section_name: Methods

section_content: Kidney sections of 4 µm stained with Masson's trichrome (MST) were used to estimate the percentage of collagen deposition indicative of interstitial fibrosis. Aperio Imagescope (Aperio, Vista, California, USA) software at × 200 magnification was used with the grid function. From ten random fields around the cortex, 4000 grids per field were made and MST staining in areas around the dilated tubules or tubules in the cortex of control group was counted. Thin layer of staining around the vessels was excluded. Positive number of squares per field were divided from 4000 grids per field to get the percent positive area of interstitial fibrosis. 

RNA extraction and quantification. RNA was isolated from the cortical region of the frozen kidney using TRIzol™ (Thermo Fisher Scientific), and the Total RNA Purification Kit (Norgen Biotek Corp, Ontario, Canada). RNA was quantified using NanoPhotometer N60/N50 spectrophotometer (IMPLEN, CA, USA) and integrity verified using RNA 600 Nano 6000 Assay kit with Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
section_name: Quantifying interstitial fibrosis.

section_content: Library generation for RNA sequencing was performed using Illumina TruSeq Stranded mRNA IDT for Illumina Unique Dual Indexing then run on the HiSeq 2500 V2 Rapid sequencing platform with Single end 100 base pair reads at the Otago Genomics Facility. Raw data was subject to Quality Control (QC) using FastQC version 0. 11. 9. Adaptor sequences and low quality sequences (Phred score < 30) were trimmed using Cutadapt version 2. 6 66. Reads were aligned against the rat reference genome (Ensembl Rnor6. 0) using HISAT2 version 2. 1. 0 67 and annotated with Rnor 6. 0. 85. gtf from Ensembl 68. Aligned reads were counted by exon and summarized by gene using featureCounts version v1. 5. 3 69. Counts were normalized for differential gene expression using Wald Test from DESeq2 package (version 1. 30. 1) 70 performed locally on R (version 4. 0. 2) and RStudio™ (version 1. 3. 1093) 71. P. values were adjusted for multiple testing using Benjamini Hochberg correction. Genes were considered statistically differentially expressed if the criteria of False Discovery Rate (FDR) < 0. 05 and log2 fold change of ± 0. 5 (FC ± 0. 5) were satisfied. Overrepresentation of ontologies and signalling pathways were derived using Metascape 72 and NetworkAnalyst 73. Genes of interest were selected based on function and validated using immunohistochemistry. MicroRNA data derived using nCounter Rat miRNA Assay Kit (cat#GXA-RMIR-12) normalized using NanoStriDE for pre-analysis 74 and NanostringNorm 75. 

Immunohistochemistry and immunofluorescence. Kidney sections (4 µm) from FFPE tissue blocks were subjected to immunohistochemistry and immunofluorescence (IHC) on a fully automated system (Leica Bond RXm, Leica Biosystems, Illinois, USA) according to the manufacturer's instructions. Antigen retrieval treatment and antibody dilution was optimized for each antibody (Supplementary Table 2 ). Slides were then scanned using Aperio Scanscope CS digital pathology system (Aperio, Vista, California, USA). Stained slides were analyzed for total and positive nuclear staining for p53, p21, p65 Ser536 and cytoplasmic staining for pAKT Ser473. This was performed by randomly selecting ten sections within the kidney cortex tissue at 200 × magnification. Positive and total number of cells were measured using cell counter tool on Fiji by ImageJ. Fluorescent staining was performed using the OPAL 7-Color Kit for Multiplex IHC (Akoya Biosciences, Massachusetts, USA). Multiplex staining using fluorophores OPAL 520, 570, 620, 690 and spectral DAPI was performed using automated staining. All fluorophores were used at a dilution of 1:100. Slides were mounted using ProLong™ Gold Antifade Mountant (Thermo Scientific) and scanned using Aperio VERSA digital pathology system (Leica Biosystems). Slide images were analyzed using Fiji by ImageJ software 76. Quantification of β catenin signal was performed using the Membrane v9 algorithm by Aperio Technologies (Vista, California). Intense positive signals from β catenin expression was scored as 3 + by the membrane v9 algorithm. Percentage of tubules with a score of 3 + positive tubules were used to graphing. 

RNAscope: in situ hybridization. RNAscope 2. 5 HD Assay-BROWN manual kit™ (Advanced Cell Diagnostics, Newark, CA, USA) was used to measure custom designed probe for Notch1 specific to Rattus Norvegicus (Ref: 847361), positive control Rn-UbC (Ubiquitin C) and negative control probe (the bacterial gene DapB) on FFPE serial tissue sections (4 µm). The manufacturer's protocol for the 2. 0 HD Detection Kit (BROWN) assay for kidney tissue was followed. Slides were scanned using Aperio VERSA digital pathology system (Leica Bio-systems). Semi-quantification was performed using trainable Weka segmentation for use with RNAscope in situ Hybridization on ImageJ 77. Statistical analysis. One-way ANOVA followed by Tukey's multiple comparisons test was performed using GraphPad Prism 9. 0. 0 (GraphPad Software, California USA, Version 9. 4. 1(458), https:// www. graph pad. com/ scien tific-softw are/ prism/). A P value less than 0. 05 (P < 0. 05) was considered significantly different.
section_name: RNA sequencing.

section_name: Acknowledgements

section_content: We would like to thank Dr Aaron Jeffs and the team at the Otago Genomics Facility, University of Otago, Dunedin, New Zealand for preparing libraries and performing RNA sequencing. We are grateful to Dr Jennifer J Bedford and Associate Professor John P Leader for supervising the animal studies and Leunie van der Tholen for assistance with the short-term animal studies. Additionally, we are grateful to Amanda Fisher and the Otago Micro and Nanoscale Imaging (OMNI) Histology Service Unit at the University of Otago.
section_name: Acknowledgements

section_name: Funding

section_content: Funding was provided by University of Otago and Otago Medical Research Foundation grant number Laurenson Award LA390.
section_name: Funding

section_name: Data availability

section_content: Raw files for RNA sequences (FASTQ format) and Nanostring data are deposited in Gene Expression Omnibus with accessions numbers: GSE178950 and GSE178695, respectively.
section_name: Data availability

section_name: Author contributions

section_content: R. W., T. S., P. M. and G. G. conceived the study, established the methodology, validated and visualized the data; P. M. -M. and G. G. performed the experiments; P. M., G. G., R. W. and T. S. analyzed data. P. M., T. S. and R. W. wrote the original manuscript, and all authors edited the manuscript; R. W. and T. S. secured funds, G. G., R. W. and T. S. administered and supervised the project.
section_name: Author contributions

section_content: The authors declare no competing interests.
section_name: Competing interests