Accumulation of short telomeres is a hallmark of aging. Mutations in telomerase or telomere-binding proteins lead to telomere shortening or dysfunction and are at the origin of human pathologies known as ‘telomere syndromes’, which are characterized by loss of the regenerative capacity of tissues and fibrotic pathologies. Here, we generated two mouse models of kidney fibrosis, either by combining telomerase deficiency to induce telomere shortening and a low dose of folic acid, or by conditionally deleting Trf1, a component of the shelterin telomere protective complex, from the kidneys. We find that short telomeres sensitize the kidneys to develop fibrosis in response to folic acid and exacerbate the epithelial-to-mesenchymal transition (EMT) program. Trf1 deletion in kidneys led to fibrosis and EMT activation. Our findings suggest that telomere shortening or dysfunction may contribute to pathological, age-associated renal fibrosis by influencing the EMT program.
Chronic kidney disease (CKD) is a disorder with high mortality, and its incidence is increasing owing to the demographic aging phenomenon1. Renal fibrosis is the primary determinant of end-stage renal disease characterized by fibroblast activation and excessive production and deposition of extracellular matrix (ECM), leading to destruction of renal parenchyma, an inflammatory and fibrotic response and decreased renal function2.
EMT is characterized by a change in cell identity of epithelia, which lose their apical–basal polarity and their cell-to-cell junctions maintained by epithelial cadherin (E-cadherin), into a mesenchymal phenotype characterized by loss of E-cadherin and expression of vimentin, neural cadherin (N-cadherin) and β3-integrin, among others. EMT has been associated with normal wound healing and regeneration3, but also with pathological processes such as fibrosis in various tissues, including kidney4. In vivo evidence for EMT in renal fibrosis is also reported in human biopsy studies5. The EMT program is mediated by the so-called EMT transcription factors (EMT-TFs), which include ZEB1 and ZEB2, SNAIL1 and SNAIL2, and TWIST1 and TWIST2. SNAIL1 and ZEB1 repress the expression of E-cadherin and induce the expression of genes associated with the mesenchymal state, such as vimentin. Overexpression of SNAIL1 is sufficient to induce kidney fibrosis4. In turn, EMT-TF programs are initiated by a number of signaling pathways, including transforming growth factor beta (TGFβ), WNT and Notch3. In particular, high levels of TGFβ are found in human fibrosis and promote EMT changes in kidney, liver and lung.
Telomeres protect chromosome ends from fusion and degradation6,7. In all vertebrates, telomeres are composed of tandem repeats of the TTAGGG sequence bound by the shelterin complex, encompassing TRF1, TRF2, POT1, TPP1, TIN2 and RAP1 proteins7. Telomere shortening is associated with cell division owing to the so-called ‘end-replication problem’, eventually leading to critically short and dysfunctional telomeres and activation of a DNA damage response, cell senescence and/or apoptosis6,8,9,10,11. Telomerase is a reverse transcriptase (telomerase reverse transcriptase (TERT)) that can synthetize telomere repeats de novo onto chromosome ends by copying an associated RNA molecule that serves as a template (telomerase RNA component (TERC)). Both TERT and TERC are essential for telomerase catalytic activity. Telomerase is active at the pluripotency stage during embryonic development, but it is silenced after birth leading to telomere shortening with aging10,12. Adult stem cells can activate telomerase during tissue regeneration but this does not compensate for overall telomere shortening with aging in stem cell compartments13. Telomere shortening with aging is proposed as one of the molecular causes of aging14, and has been shown to limit the regenerative capacity of tissues13. Indeed, mutations in telomerase or in some telomere-binding proteins are associated with human diseases known as ‘telomere syndromes’, which include some cases of aplastic anemia, dyskeratosis congenita and fibrosis of the lung, liver and kidney8,15,16,17,18.
In addition to the telomere syndromes where extremely short telomeres can contribute to kidney fibrosis, telomere shortening associated with aging also occurs in the kidney. In particular, age is a key predictor of CKD—11% of individuals older than 65 years have at least stage 3 CKD19.
In the past, we have developed mouse models that develop aplastic anemia20,21 and lung fibrosis associated with short telomeres22,23. These mouse models demonstrate the role of short telomeres in the origin of these diseases and point to potential therapeutic strategies. However, the role of short telomeres in kidney fibrosis remains less understood, in part due to the lack of appropriate mouse models.
Telomerase deficiency in mice leads to telomere shortening and anticipation of various degenerative pathologies including aplastic anemia, intestinal atrophy and infertility, among others24,25,26. However, it is not known whether telomerase-deficient mice with short telomeres develop renal fibrosis or they require additional insults to contribute to the disease.
As we found here that telomerase-deficient mice per se do not spontaneously develop kidney fibrosis, we set to generate a mouse model of kidney fibrosis associated with short telomeres by challenging telomerase-deficient mice with short telomeres24 to a low dose of folic acid (FA), a damaging agent to the kidney, which does not induce kidney fibrosis in wild-type mice but could synergize with short telomeres in the context of telomerase-deficient mice. The results show that short telomeres increase the susceptibility to the development of FA-induced kidney fibrosis. In addition, we also observed kidney fibrosis in an alternative mouse model based on the conditional deletion of the shelterin component Trf1, which induces telomere dysfunction, highlighting the importance of proper telomere function in protection from renal fibrosis. These mice are instrumental for the development of potential therapeutic strategies for kidney fibrosis.
A mouse model of kidney fibrosis is associated with short telomeres
To test the involvement of short telomeres in kidney fibrosis, we first analyzed the kidneys of wild-type mice, as well as mice lacking the telomerase catalytic subunit, Tert, that were bred for three generations (G3) to induce the presence of very short telomeres, that is, G3 Tert−/− mice24 (Extended Data Fig. 1a). Both wild-type (Tert+/+) and G3 Tert−/− mice aged 8–9 weeks displayed grossly normal kidney histology, with no evidence of glomerular or tubular defects and with no fibrosis or accumulation of collagen fibers as determined by Masson’s trichrome and Sirius red staining, respectively (Extended Data Fig. 1b,c). Similarly, we did not detect increased tubular injury or loss of brush borders in the kidney as determined by periodic acid-Schiff with diastase staining (PAS + D; Extended Data Fig. 1d). Also, in agreement with grossly normal kidneys in the G3 Tert−/− mice, we did not see differences in the presence of activated fibroblasts, as determined by expression of smooth muscle actin (α-SMA); in apoptosis, as determined by expression of cleaved caspase-3 (CC3); in senescence, as determined by expression of the p21 cell cycle inhibitor; or in the expression of E-cadherin (a marker of EMT, associated with tissue fibrosis), compared with age-matched Tert+/+ mice (Extended Data Fig. 1e–h).
The incidence of kidney fibrosis increases with age; therefore, it is likely to be caused by the combination of molecular and cellular aging events, such as the presence of short telomeres14 together with exogenous damage to the kidney. Thus, we next challenged the kidneys of Tert+/+ and G3 Tert−/− mice with FA, previously described to induce kidney fibrosis at a high dose of 250 mg kg−1 body weight27. FA-induced nephropathy is widely used to study interstitial kidney fibrosis4,27,28. In particular, FA administered intraperitoneally in mice leads to rapid appearance of FA crystals in tubules, followed by severe nephrotoxicity.
To this end, we first subjected Tert+/+ and G3 Tert−/− mice aged 8–9 weeks to increasing doses of FA (50, 100, 125 and 250 mg kg−1 body weight; Fig. 1a) and selected the highest dose of FA that did not induce kidney fibrosis in wild-type mice. Wild-type mice treated with 50, 100 and 125 mg kg−1 body weight appeared well and did not show any signs of kidney pathology as determined by normal kidney appearance (Fig. 1b), normal creatinine and blood urea nitrogen (BUN) in the blood (Fig. 1c,d), as well as by absence of fibrosis as indicated by Masson’s Trichrome staining (Fig. 1e) and absence of tubular injury by PAS + D staining (Fig. 1e). In contrast, the majority of wild-type mice (75%) treated with an FA dose of 250 mg kg−1 body weight died at day 2 after treatment due to FA-induced acute renal failure (data not shown). Thus, 125 mg kg−1 was the maximum tolerated dose of FA that did not cause lethality in wild-type mice with normal telomere length. We reasoned that this dose of FA, although not sufficient to induce kidney fibrosis in Tert+/+ mice, may synergize with short telomeres in telomerase-deficient mice to induce kidney fibrosis.
Indeed, we treated G3 Tert−/− mice with the same doses of FA and observed that, even after the 125 mg kg−1 dose, the kidneys presented a pale color (Fig. 1b), suggestive of kidney fibrosis. Indeed, G3 Tert−/− mice treated with increasing doses of FA showed increased creatinine (Fig. 1c) and BUN (Fig. 1d) levels in the blood at a 125 mg kg−1 FA dose, indicative of kidney failure, as well as increased fibrosis as detected by Masson’s trichrome staining (Fig. 1e) and increased renal tubular injury as determined by increased PAS + D staining (Fig. 1e). FA doses below 125 mg kg−1 did not induce a fibrotic phenotype in G3 Tert−/− mice (Fig. 1e). Thus, we selected a dose of 125 mg kg−1 body weight of FA for further experiments.
Severe kidney dysfunction in telomerase-deficient mice treated with a sublethal dose of folic acid
To address the role of short telomeres in kidney fibrosis, we subjected Tert+/+ and G3 Tert−/− mice at 8–9 weeks of age to a dose of 125 mg kg−1 body weight of FA, which does not induce fibrosis in wild-type mice (Fig. 2a). Blood was collected at days 2, 7 and 14 from the submandibular vein. At day 14, mice were euthanized and kidneys were harvested for further analysis. As expected, the kidneys from FA-treated G3 Tert−/− mice appeared pale and damaged compared to similarly treated wild-type kidneys (Fig. 2b,c). To confirm kidney damage, we analyzed the urinary parameters and observed a marked increase in 24-h urinary albumin excretion and urinary albumin to creatinine ratio in FA-treated G3 Tert−/− mice compared to FA-treated Tert+/+ and untreated Tert+/+ mice and G3 Tert−/− mice (Fig. 2b).
We also examined BUN and creatinine in all mouse cohorts. Administration of a high dose of FA is known to induce a transient elevation of BUN and creatinine levels at 48 h after injection followed by subsequent renal dysfunction accompanied with interstitial fibrosis29. Two days after the injection of a low dose of FA (125 mg kg−1 body weight), however, both the untreated and FA-treated wild-type mice (Tert+/+) mice showed normal BUN and creatinine levels, indicating normal kidney function (Extended Data Fig. 2a,b). Consistently, we did not observe any reduced mobility or increased mortality of the wild-type mice (Tert+/+) treated with a low dose of FA (125 mg kg−1 body weight). Similarly, untreated G3 Tert−/− mice showed normal creatinine and BUN levels and normal viability at day 2. As expected, FA-treated G3 Tert−/− mice showed significant increases in both BUN and creatinine levels on days 2, 7 and 14, with creatinine levels progressively increasing after day 2, indicating renal dysfunction as early as after 2 d of FA administration (Extended Data Fig. 2a,b). Further biochemical parameters in the blood of Tert+/+ and G3 Tert−/− mice indicated renal damage (Supplementary Table 1). Alkaline phosphatase and amylase levels are commonly elevated in patients with CKD and those requiring dialysis30 and are indicators of renal damage31. Impaired kidney function can result in hyperamylasemia32. At days 7 and 14, FA-treated G3 Tert−/− mice showed significantly higher levels of alkaline phosphatase and amylase compared to levels in FA-treated Tert+/+ and untreated Tert+/+ mice and G3 Tert−/− mice (Supplementary Table 1). Hypercalcemia33, hyperphosphatemia34, hypernatremia35 and hyperkalemia36 are common complications in patients with CKD, particularly in those with end-stage renal disease. We observed increased calcium, phosphorus and sodium levels in FA-treated G3 Tert−/− mice at days 2, 7 and 14, whereas potassium levels were increased only at day 14 compared to those in FA-treated Tert+/+ and untreated Tert+/+ mice and G3 Tert−/− mice (Supplementary Table 1). As an indicator of tubular function, we measured blood glucose, globulin, total protein and albumin levels. At days 2, 7 and 14, the blood glucose, globulin and total protein levels were significantly elevated in FA-treated G3 Tert−/− mice compared to FA-treated Tert+/+ and untreated Tert+/+ mice and G3 Tert−/− mice, whereas albumin remained unchanged (Supplementary Table 1).
Telomerase-deficient mice show collagen deposition and activated myofibroblasts in the kidney after a sublethal dose of folic acid
It has been previously shown that FA-induced renal injury concurs with development of segmental interstitial fibrotic lesions approximately 2 weeks after treatment37. In agreement with this, at 14 d after treatment, we observed interstitial fibrotic areas as indicated both by Masson’s trichrome and Sirius red staining in the kidneys of FA-treated G3 Tert−/− mice, while no fibrosis was detected in the kidneys from G3 Tert−/−, Tert+/+ and FA-treated Tert+/+ mice (Fig. 2c), again indicating that short telomeres sensitize to kidney fibrosis after a subpathological dose of FA that does not induce fibrosis in wild-type mice. FA-treated G3 Tert−/− mice also developed significant tubulointerstitial damage as indicated by increased PAS + D staining, including tubular dilatation, atrophy and loss of epithelial differentiation after injury, compared to normal kidney histology in the FA-treated Tert+/+ and untreated Tert+/+ mice and G3 Tert−/− mice (Fig. 2c). Next, we performed immunohistochemistry for α-SMA, fibronectin and collagen type VI and double immunofluorescence with α-SMA and vimentin to detect interstitial myofibroblasts (Fig. 3a). Again, we only detected increased staining for α-SMA, fibronectin, collagen type VI and vimentin in the FA-treated G3 Tert−/− mice, compared to undetectable staining in FA-treated Tert+/+ and untreated Tert+/+ mice and G3 Tert−/− mice (Fig. 3a).
EMT involves a change from the apical–basolateral polarity of the epithelial cells to the front–rear polarity of the mesenchymal cells and the expression of mesenchymal markers, such as fibroblast-specific protein-1, vimentin, N-cadherin and α-SMA. These changes induce enhanced migratory capacity, invasiveness, elevated resistance to apoptosis and increased production of ECM components38. To demonstrate that the proliferating cells observed in FA-treated G3 Tert−/− mice are indeed EMT cells and not tubular cells, we performed double immunofluorescence staining for Ki67 and α-SMA. We found that 36% of proliferating cells were myofibroblasts (α-SMA+Ki67+) in FA-treated G3 Tert−/− mice compared to untreated G3 Tert−/− mice (Fig. 3b). Increased fibrosis in the FA-treated G3 Tert−/− mice but not in similarly treated wild-type or untreated mice was also confirmed by quantitative PCR (qPCR) to determine the mRNA levels of key fibrotic genes, including Acta2 (encoding α-SMA) and Vim (encoding vimentin), Col1a1, Col3a1 and Col4a1 (encoding collagen type I alpha-1, -3 and -4 chains, respectively) and Fn1 (encoding fibronectin 1). These genes were significantly upregulated in the kidneys of FA-treated G3 Tert−/− mice compared to FA-treated Tert+/+ and untreated Tert+/+ mice and G3 Tert−/− mice (Fig. 3c).
Increased kidney apoptosis and senescence in telomerase-deficient mice after a sublethal dose of folic acid
Short telomeres have been shown by us and others to induce a persistent DNA damage response at chromosome ends, leading to cell cycle arrest or apoptosis39,40. In particular, short telomeres induce the p53 and p21 cell cycle inhibitors22,41. Interestingly, p21 is known to be an activator of TGFβ37, thus providing a potential mechanism by which short telomeres, even in the absence of fibrosis, may contribute to activation of EMT programs.
In this regard, we studied whether kidney dysfunction and increased fibrosis in G3 Tert−/− mice treated with FA was accompanied by increased cellular senescence or apoptosis, two well-known cellular responses to telomere dysfunction with aging14. We observed that FA treatment induced a significant increase in levels of CC3 (apoptosis marker), p21 and p53 cell cycle inhibitors, and γ-H2AX (DNA damage marker) in G3 Tert−/− mice, compared to undetectable levels in FA-treated Tert+/+ and untreated Tert+/+ mice and G3 Tert−/− mice (Fig. 4a–d). Our model shows that these events produce increased turnover of kidney cells leading to telomere shortening. In support of this, we observed an increase in the transcript levels of the cell cycle regulators encoding genes CCnd1, Ccnd2, Ccnb1 and Ccne1 (Extended Data Fig. 3a), suggesting that a prominent profibrotic phenotype can result in G2/M arrest. Finally, we confirmed the presence of shorter telomeres in the kidneys of G3 Tert−/− mice compared to those of wild-type mice as determined by telomere quantitative fluorescence in situ hybridization (Q-FISH) directly on kidney sections (Fig. 4e). Treatment with a low dose of FA induced a clear telomere shortening in both genotypes (Fig. 4e).
Short telomeres sensitize to tubular damage and immune infiltration in kidney
Next, we set to study the pathways by which short telomeres induce kidney injury. The kidney injury molecule-1 (KIM-1, also known as hepatitis A virus cellular receptor (HAVCR) 1; encoded by the gene Havcr1) is a type 1 transmembrane protein that is undetectable in healthy kidneys but greatly induced after injury42, localizing to the apical surface of the surviving proximal tubular cells (PTCs)43. Inflammation is proposed as a key driver of kidney fibrosis42, and the neutrophil gelatinase-associated lipocalin (NGAL) gene product (lipocalin-2 (Lcn2) or siderocalin)44 is induced during acute kidney injury (AKI)44. Thus, we used Havcr1 and Lnc2 as biomarkers of CKD due to tubulointerstitial damage. We found that Havcr1 and Lcn2 were upregulated by 134- and 206-fold, respectively (Fig. 5a) in FA-treated G3 Tert−/− mice compared to basal expression in FA-treated Tert+/+ and control, untreated Tert+/+ mice and G3 Tert−/− mice. Next, we studied the expression of molecules involved in immune infiltration, also associated with tissue fibrosis. We observed a 16-fold upregulation of Emr1 mRNA expression (the mouse gene encoding the F4/80 antigen) in FA-treated G3 Tert−/− mice (Fig. 5a). We also examined the expression of the pan-macrophage marker F4/80 and T cell markers (CD3e, CD4 and CD8a) by immunohistochemistry. We observed increased macrophage colonization (F4/80-positive area) and increased T cells in the kidneys of FA-treated G3 Tert−/− mice compared to those of FA-treated Tert+/+ mice or those of untreated Tert+/+ and G3 Tert−/− mice (Fig. 5b).
Activation of epithelial-to-mesenchymal transition-related pathways in G3 Tert −/− mice
Recruitment of macrophages in tissue fibrosis is known to involve TGFβ, also important for the EMT process45 associated with kidney fibrosis46. Thus, we next sought to determine whether the presence of short telomeres is associated with changes in the expression of genes involved in EMT. To this end, we studied the expression of EMT and EMT-related pathways such as TGFβ signaling, a major environmental stimulus that induces EMT in adult epithelia, by performing RNA sequencing (RNA-seq) in the kidneys of 10-week-old Tert+/+ and G3 Tert−/− mice either untreated or treated with low-dose FA (125 mg kg−1 body weight). Gene-set enrichment analysis (GSEA) of untreated Tert+/+ versus untreated G3 Tert−/− mice showed that the EMT (normalized enrichment score (NES) = 3.37) and the TGFβ (NES = 1.9) pathways were upregulated in telomerase-deficient mice compared to wild-type mice (Fig. 6a,b), suggesting that short telomeres may contribute to a higher basal activation of some EMT genes. The transcriptional upregulation of EMT genes was more pronounced in FA-treated Tert+/+ and FA-treated G3 Tert−/− animals (NES > 6), compared to that of untreated animals. These findings were confirmed in untreated younger Tert+/+ versus G3 Tert−/− mice at 7 weeks of age, as well as in older mice (47 weeks of age; Extended Data Fig. 4a,b). Thus, short telomeres are associated with changes in the expression levels of some but not all genes involved in an EMT in kidney epithelial cells, and are therefore not sufficient to activate the classical EMT program or induce fibrosis on their own. Interestingly, treatment with FA induced an enrichment of the EMT and the TGFβ pathways in both genotypes, Tert+/+ and G3 Tert−/−. This enrichment was higher in FA-treated G3 Tert−/− kidneys compared to FA-treated Tert+/+ kidneys (NES = 2.42), thus supporting the idea of a higher EMT activation in response to FA in telomerase-deficient mice (Fig. 6a,b).
Upregulation of key EMT transcription factors in telomerase-deficient mice with short telomeres
TGFβ1-induced EMT is mediated by ZEB1 and SNAIL in a Smad-dependent manner45. TWIST, SNAIL and ZEB1 are transcription factors that regulate the EMT transcriptional program. Activation of Twist, Snail or Zeb1 is sufficient to induce a mesenchymal phenotype4. Thus, we set to determine the mRNA expression levels of Tgfb1, Snail1, Snail2, Twist1, Zeb1 and Zeb2 by PCR with reverse transcription (RT–PCR). We observed enhanced expression of Tgfb1 by 32-fold in FA-treated G3 Tert−/− mice compared to other mouse cohorts (Fig. 6c). Snail1 and Snail2 were also upregulated by sixfold in FA-treated G3 Tert−/− mice (Fig. 6c). Twist1 was upregulated by sevenfold, whereas Zeb1 and Zeb2 were upregulated by four- and five-fold, respectively, in FA-treated G3 Tert−/− mice (Fig. 6c). Thus, all the major EMT players were upregulated in the kidneys of telomerase-deficient mice with short telomeres subjected to a sublethal dose of FA, compared to wild-type mice subjected to the same dose, suggesting that short telomeres may be contributing to EMT changes after FA treatment. Lysyl oxidase (LOXL2) is required and sufficient for hypoxic repression of E-cadherin, which mediates cellular transformation and induces EMT47. The expression of Loxl2 was upregulated by 20-fold (Fig. 6c) in FA-treated G3 Tert−/− mice. One of the hallmarks of EMT is the downregulation of E-cadherin isoforms such as Cdh1 and expression of mesenchymal markers45. In agreement with this, the expression of the E-cadherin Cdh1 was downregulated by twofold in FA-treated G3 Tert−/− mice, as determined both by mRNA levels and by immunohistochemistry, compared to the other mouse cohorts (Fig. 6c,d). SMAD3 is the key effector in TGFβ signaling. We found that Smad3 was upregulated by twofold in FA-treated G3 Tert−/− mice (Fig. 6c,d).
To understand how progenitor and stem cells may be affected by short telomeres during FA-induced kidney fibrosis, we studied the expression of sex determining region Y box 9 (Sox9), Wilms’ tumor (Wt1), paired box 2 (Pax2), spalt like transcription factor 2 (Sall2), activin A receptor type 2B (Acvr2b, a Tgfb superfamily member) and Klotho (Kl). SOX9 is known to be important in kidney development in mouse48 and human49 studies and required for renal fibrosis50. This precedes the expression of Wt1, Pax2 and NOTCH signaling within the epithelium. Wt1 maintains a switch between the mesenchymal and epithelial cell states and is needed to induce mesenchymal-to-epithelial transformation (MET), as well as playing a key role in the progression of nephrogenesis51. PAX2, a nuclear transcription factor, is associated with MET and it is required for kidney cell differentiation52. Re-expression of Wt1 and Pax2 in the tubular epithelial cells plays an important role in the promotion of EMT, and there may be therapeutic value in silencing Pax2 and Wt1 to prevent or reverse renal fibrosis53. In this regard, we observed a significant increase in the transcript levels of Sox9, Pax2, Wt1 and Acvr2b expression in Tert−/− mice exposed to FA compared to all other groups, suggesting that under those conditions short telomeres sensitized the kidneys to undergo an EMT (Extended Data Fig. 5a). Sox9 immunostaining was significantly increased in FA-treated G3 Tert−/− mice compared to untreated and treated Tert+/+ mice and G3 Tert−/− mice (Extended Data Fig. 5b). Sall2 is negatively regulated by Wt1 (ref. 54) and Klotho (Kl) is an antiaging protein predominantly produced in the kidney55 that regulates telomerase activity56. A low Kl level may be a pathological intermediate for exacerbation of kidney damage. Interestingly, we found that Sall2 and Kl expression were downregulated in FA-treated G3 Tert−/− mice (Extended Data Fig. 5a).
Persistent expression of NOTCH signaling pathways within the epithelium results in interstitial fibrosis57. NOTCH is a strong regulator of SNAIL1 and SNAIL2 (ref. 58). Thus, we analyzed the expression of Notch receptors (Notch1, Notch2 and Notch3), Notch ligand Jagged 1 (Jag1) and mitochondrial transcription factor A (Tfam) as a direct Notch target59 important for kidney function. We found a threefold increase in Notch1 and Notch2 and a fourfold increase in Notch3 and Jag1 in FA-treated G3 Tert−/− mice compared to FA-treated Tert+/+ and untreated Tert+/+ mice and G3 Tert−/− mice (Extended Data Fig. 6). We also found a fivefold upregulation of Tfam in FA-treated G3 Tert−/− mice compared to FA-treated Tert+/+ and untreated Tert+/+ mice and G3 Tert−/− mice (Extended Data Fig. 6).
Trf1 deletion-induced telomere dysfunction triggers renal fibrosis associated with activation of EMT
To assess the contribution of dysfunctional telomeres to the induction of renal fibrosis, we used a second model of telomere dysfunction by deleting TRF1, one of the components of the shelterin telomere protective complex. In particular, we used a mouse model previously described by us60 in which treatment with tamoxifen led to deletion of Trf1 in all kidney cells (Fig. 7). Tamoxifen diet was maintained until the humane end point, and Trf1 deletion was confirmed by RT–qPCR (Fig. 7a). Mice deleted for Trf1 developed kidney fibrosis, as indicated by increased collagen deposition (Masson’s Trichrome) and fibrotic lesions (Sirius red staining) compared to the Trf1+/+ mice (Fig. 7b), as well as by activation of myofibroblasts (SMA staining; Fig. 7b). We also observed a three- to four-fold increase in mesenchymal markers (Acta2 and Fn1) and a two- to six-fold increase in EMT markers (Tgfb1, Snail1, Snail2, Twist1, Zeb1 and Zeb2) in Trf1flox/flox mice compared to Trf1+/+ mice (Fig. 7c,d), suggesting that dysfunctional telomeres may also contribute to transcriptional changes associated with EMT.
Telomerase overexpression rescues EMT changes associated with short telomeres
To further explore a contribution of short telomeres in the activation of EMT programs in the kidney, we addressed whether they could be rescued by expression of the catalytic subunit of telomerase or TERT61. To this end, we first isolated kidney epithelial cells, in particular PTCs, from 10- to 11-week-old Tert+/+ and G3 Tert−/− mice. PTCs were transduced with either an empty vector or with a vector expressing Tert (Methods). Telomerase overexpression was confirmed by qPCR (Fig. 8a). Consistent with telomerase overexpression, both wild-type and G3 Tert−/− cells showed a significant increase in telomere length after 1 week in culture (Fig. 8b). Before transduction, at day 8 in culture, the G3 Tert−/− cells presented a clear mesenchymal morphology as indicated by a spindle-like shape that was not observed in wild-type cells, which presented a cobblestone-like morphology (Extended Data Fig. 7a). G3 Tert−/− cells transduced with the empty vector showed a complete myofibroblast phenotype characterized by the loss of E-cadherin, enhanced SMA staining and positivity for Snail1 and TGFβ markers (Fig. 8c and Extended Data Fig. 7b,c), suggesting that 100% of the cells underwent the EMT. In contrast, control Tert+/+ cells transduced with the empty vector showed a mixture of epithelial cells (E-cadherin-positive, Snail1- and TGFβ-negative and SMA- negative cells) and myofibroblasts (E-cadherin-negative, Snail1- and TGFβ-positive and SMA-positive cells; Fig. 8c and Extended Data Fig. 7b,c), indicating some evidence of EMT. Interestingly, Tert+/+ cells transduced with mTERT, showed a reverse of phenotype, including evidence of MET, restoration of the cobblestone-like phenotype in culture, expression of E-cadherin and loss of expression of SMA, TGFβ and Snail1 (Fig. 8c,d and Extended Data Fig. 7b,c). Importantly, G3 Tert−/− cells transduced with mTERT also showed a reverse of the mesenchymal phenotype with loss of SMA, TGFβ and Snail1 expression (Fig. 8c and Extended Data Fig. 7b,c). Indeed, G3 Tert−/− cells transduced with mTERT showed decreased expression of many ECM (Acta2, Vim, Col3a1 and Col4a1) and EMT (Tgfb1, Snail1, Snail2 and Zeb1) genes compared to G3 Tert−/− cells transduced with the empty vector (Fig. 8d), confirming the restoration of the epithelial features upon TERT expression and telomere elongation. Thus, our results show that mTERT overexpression and telomere elongation is sufficient to rescue the EMT phenotype and restore the MET phenotype in kidney tubular cells. This culture system provides a potent tool to investigate EMT driven by TERT.
EMT is a cellular plasticity process by which epithelial layers lose their integrity concomitant with a loss of cell polarity and cell-to-cell interactions mediated by loss of E-cadherin3. The resulting cells express mesenchymal properties, including expression of vimentin and α-SMA. EMT is orchestrated by a number of transcription factors (EMT-TFs) and has key roles both during normal development3 and in pathological conditions such as cancer and various tissue fibrotic diseases, including kidney fibrosis4,62.
As both cancer and tissue fibrosis have been associated with aging, it is of particular relevance to understand how known molecular and cellular mechanisms of aging14 may impact the expression of EMT-TFs and the origin of EMT pathological processes.
Accumulation of short and dysfunctional telomeres associated with cell division during the life span of an organism is considered one of the primary hallmarks of aging as it triggers persistent DNA damage that it is sufficient to impair the regenerative capacity of adult stem cell compartments10,13. Indeed, accelerated telomere shortening owing to telomerase deficiency both in mice24,25 and humans8 leads to premature loss of the regenerative capacity of tissues, including development of tissue fibrosis, of which pulmonary fibrosis is one of the most prevalent8,22. In support of short telomeres being an important factor contributing to tissue fibrosis, we previously showed that pulmonary fibrosis associated with short telomeres in a mouse model of telomerase deficiency could be reverted by telomerase activation and elongation of short telomeres23.
Here, we generated a mouse model of fibrosis in the kidney associated with short telomeres by challenging telomerase-deficient mice with short telomeres to a low, sublethal dose of FA, a damaging agent to the kidney, which does not induce fibrosis in similarly treated wild-type controls. Telomerase-deficient mice treated with a sublethal dose of FA show all the hallmarks of the human disease including severe kidney dysfunction, as indicated by elevated blood levels of creatinine and urea. In addition, mice with dysfunctional telomeres owing to deletion of Trf1 encoding for a shelterin protein spontaneously develop kidney fibrosis, highlighting the importance of proper telomere function in the protection of fibrotic pathologies. Thus, the new mouse models generated here may represent good tools to understand the role of short and dysfunctional telomeres and resulting DNA damage in the molecular events associated with fibrosis.
Interestingly, we find here that short telomeres lead to changes in the expression of genes involved in EMT, although these changes were not sufficient to induce kidney fibrosis. These changes were further exacerbated in the telomerase-deficient mice treated with FA that develop kidney fibrosis. This is supported by increased expression of Snail1, Snail2, Zeb1, Zeb2 and Twist1 transcription factors that results in epithelial cells turning into myofibroblasts. Myofibroblasts deposit ECM containing collagen leading to the development of renal fibrosis.
Supporting evidence that short telomeres contribute to EMT changes, we show that expression of the mouse telomerase TERT catalytic subunit and the subsequent elongation of telomeres are sufficient to revert EMT programs and to restore an epithelial phenotype in kidney cells in culture.
As short telomeres accumulate with aging in an organism, it is tempting to speculate that pathological EMT programs associated with aging such as cancer and different types of tissue fibrosis may originate, at least in part, from the presence of short telomeres.
Mice and animal procedures
Tert heterozygous mice generated as previously described63 were backcrossed to a >98% C57BL/6 background. Tert+/− mice were intercrossed to generate first generation (G1) homozygous Tert−/− knockout mice. G3 Tert−/− mice were generated by successive breeding of G2 Tert−/− mice. Tert+/+ and G3 Tert−/− male and female mice of pure C57BL/6 background were treated with FA (F7876; Sigma-Aldrich). Male and female G3 Tert−/− mice (aged 7, 27 and 47 weeks) were euthanized to analyze the kidney phenotype for signs of fibrosis and for RNA-seq analysis.
Trf1lox/lox mice64 were crossed with mice expressing CreERT2 recombinase driven by the tamoxifen-inducible ubiquitin C (UBC) promoter65 to generate Trf1lox/lox;hUBC-CreERT2 or Trf1+/+;hUBC-CreERT2 mice. These mice were fed ad libitum with a long-term tamoxifen-containing diet, starting at 10 weeks of age.
All mice were generated and maintained at the Animal Facility of the Spanish National Cancer Research Centre (CNIO) under specific pathogen-free conditions with a 12-h light/dark cycle. Mice were housed in plastic cages containing wood shavings or chipped wood bedding with food and water available ad libitum in accordance with the recommendation of the Federation of European Laboratory Animal Science Associations. All animal procedures were approved by the CNIO-ISCIII Ethics Committee for Research and Animal Welfare (CBA 03_2019-v2).
Folic acid dose titration and inoculation
A single intraperitoneal injection of FA with different doses (50, 100, 125 and 250 mg kg−1 body weight) in vehicle (0.2 ml of 0.3 mmol l−1 NaHCO3) or vehicle alone was administered to 6- to 8-week-old Tert+/+ and G3 Tert−/− mice. Blood was collected at days 7 and 14 and was analyzed by VetScan Comprehensive Diagnostic Profile kit. Mice were euthanized at day 14 after FA injection, and the kidneys were collected from FA-treated or vehicle-treated animals and analyzed by immunohistochemistry for renal fibrosis.
A single intraperitoneal injection of FA with a low dose of 125 mg kg−1 body weight or 0.3 M NaHCO3 (200ul) was inoculated to 6- to 8-week-old Tert+/+ and G3 Tert−/− mice. Blood collected at days 2, 7 and 14 and was analyzed by VetScan Comprehensive Diagnostic Profile kit. Mice were euthanized at day 14, and kidneys were perfused with cold PBS and harvested.
Kidneys were fixed in 4% formaldehyde and embedded in paraffin. Paraffin sections (5-μm thick) were stained with Masson’s trichrome, Sirius red and PAS + D using standard procedures. Percentages of fibrotic areas were quantified using the National Institutes of Health (NIH) ImageJ program.
Primary cell culture
Primary mouse PTCs were isolated from 10- to 11-week-old Tert+/+ and G3 Tert−/− mice as described previously66. mTert-pBabe-puro was a gift from M. Alvarez and J. Bidwell (Addgene plasmid no. 36413). Retroviral plasmid vectors (mTert-pBabe-puro) and the packaging plasmids (PLC Eco and pCMV-VSV-G) were co-transfected into the packaging cell line 293T. Viral supernatants were collected 48 h later, centrifuged to remove cell debris, strained through 0.45-μm filters (Millipore) and used to infect isolated PTCs from Tert+/+ and G3 Tert−/− mice. Stable cell lines were selected with 2 μg ml−1 puromycin on day 10 until day 14. Cells were collected for RNA extraction and immunofluorescence.
Immunohistochemical and immunofluorescence staining
Kidney tissues were fixed in 4% formaldehyde and embedded in paraffin. Paraffin-embedded kidney tissue sections at a thickness of 5–7 μm were subjected to immunohistochemical staining. Primary antibodies (and their dilutions) were: rat monoclonal to p21 (HUGO-291H/B5; CNIO histopathology core unit; 1:400), rat monoclonal to p53 (POE316A/E9; CNIO histopathology core unit; 1:400), rat monoclonal to p21 (HUGO-291H/B5; CNIO histopathology core unit; 1:400) rat monoclonal to CD8a (AM-OTO94A; CNIO histopathology core unit), mouse monoclonal to phospho–Histone H2AX (Ser 139; 05-636; Millipore; 1:400), mouse monoclonal to E-cadherin (610182; BD Biosciences; 1:400), rat monoclonal to F4/80 (MCA497; AbD Serotec; 1:400), rabbit polyclonal activated caspase-3 (9661; Cell Signaling Technology; 1:400), rabbit monoclonal to CD3e (99940; Cell Signaling Technology; 1:400), rabbit monoclonal to CD4 (25229; Cell Signaling Technology; 1:400), rabbit polyclonal to Collagen type VI (ab6588; Abcam; 1:400), rabbit polyclonal to fibronectin (ab2413; Abcam; 1:400) and rabbit polyclonal to Sox9 (AB5535; Millipore; 1:400).
For immunofluorescence, mouse monoclonal antibody to α-SMA-Cy3 (C6198; Sigma; 1:400), rabbit monoclonal to vimentin (5741; Cell Signaling Technology; 1:200), rabbit monoclonal to Ki67 (12202; Cell Signaling Technology; 1:400), rabbit polyclonal TGFβ (3711S; Cell Signaling Technology; 1:200), rabbit polyclonal SNAIL + SLUG (ab180714; Abcam; 1:200) and rat monoclonal E-cadherin (DECMA-1; ab11512, Abcam; 1:200) were used. Images were obtained using a confocal ultra-spectral microscope (Leica TCS-SP5). The percentages of positively stained areas by immunohistochemistry and immunofluorescence were quantified using NIH ImageJ (v1.52n).
Gene expression analysis and real-time PCR examination
Total RNA was isolated from kidney tissues and PTCs using TRIzol reagent (Takara), according to the manufacturer’s instructions. cDNA was synthesized with 1 μg of total RNA, cDNA synthesis mix (BioMake), and oligo-dT primers. Gene expression was measured by a real-time PCR assay (BioMake) and a 7900HT real-time PCR system (Applied Biosystems). The relative amount of mRNA to the internal control was calculated as 2∆CT, in which ∆CT = ∆CTexperimental − ∆CTcontrol. Genes and primers are listed in Supplementary Table 2 (F: forward primer; R: reverse primer).
For RNA-seq experiments, total RNA samples (300 ng) were used. RNA quality scores were 6.3 on average (range 4.7–8.0) when assayed on a PerkinElmer LabChip analyzer. Sequencing libraries were prepared with the QuantSeq 3′ mRNA-Seq Library Prep Kit (FWD) for Illumina (Lexogen; 015), following the manufacturer’s instructions. Library generation was initiated by reverse transcription with oligo-dT priming, and a second-strand synthesis was performed from random primers by a DNA polymerase. Primers from both steps contained Illumina-compatible sequences. cDNA libraries were purified, applied to an Illumina flow cell for cluster generation and sequenced on an Illumina NextSeq 550 (with v2.5 reagent kits), following the manufacturer’s protocols. Read adaptors and poly(A) tails were removed with the command ‘bbduk.sh’, following the Lexogen recommendations. Processed reads were analyzed with the Nextpresso pipeline67 as follows. Sequencing quality was checked with FastQC v0.11.7 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were aligned to the mouse reference genome (GRCm38) with TopHat (v2.0.10)68 using Bowtie (v1.0.0)69 and SAMtools (v0.1.19)70 (--library-type fr-secondstrand in TopHat), allowing three mismatches and twenty multihits. Read counts were obtained with HTSeq-count (v0.6.1)71 using the mouse gene annotation from GENCODE (GRCm38; vM20 Ensembl 95). GSEAPreranked72 was used to perform GSEA for several gene signatures on a preranked gene list, setting 1,000 gene-set permutations. Only those gene sets with significant enrichment levels (FDR q value < 0.25) were considered.
Q-FISH determination on paraffin-embedded tissue sections was performed as described previously41. After deparaffinization, tissues were postfixed in 4% formaldehyde for 5 min, washed three times for 5 min each in PBS and incubated at 37 °C for 15 min in pepsin solution (0.1% porcine pepsin, Sigma; 0.01 M HCl, Merck). After another round of washes and fixation as mentioned above, slides were dehydrated in an ethanol series (70%, 90% and 100%; 5 min each). After 10 min of air drying, 30 l of telomere probe mix (10 mM Tris-Cl (pH 7), 25 mM MgCl2, 9 mM citric acid, 82 mM Na2HPO4, 70% deionized formamide (Sigma), 0.25% blocking reagent (Roche) and 0.5 μg ml−1 telomeric PNA probe (Panagene) were added to each slide. A coverslip was added and slides were incubated for 3 min at 85 °C, and for a further 2 h at room temperature in a wet chamber in the dark. Slides were washed twice for 15 min each in 10 mM Tris-Cl (pH 7), 0.1% BSA in 70% formamide under vigorous shaking, then three times for 5 min each in TBS 0.08% with Tween 20 and then incubated in a DAPI bath (4 μg ml−1 in PBS; Sigma) before mounting samples in Vectashield medium (Vector). Confocal images were acquired as stacks every 1 μm for a total of 5 μm using a Leica SP5-MP confocal microscope, and maximum projections were done with LAS-AF software. Telomere signal intensity was quantified using Definiens software.
Statistics and reproducibility
No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported previously4,46. Mice were randomly allocated within the groups, and investigators were blinded to group allocation. Our sample sizes corresponded to the number of mice used for each experiment, as indicated. The quantitative analyses of immunohistochemistry stainings were performed in the whole scanned kidney section comprising 10–15 areas. For immunofluorescence analysis, 10–20 images were collected from each individual. Data distribution was assumed to be normal, but this was not formally tested. No data points or mice were excluded from the analyses. Results of statistical analyses are expressed as means ± s.e.m. Statistical analyses of immunohistochemical/immunofluorescence quantifications and of qPCR and Q-FISH analyses were performed using one-way ANOVA with post hoc Tukey’s test in Prism (GraphPad). Two-way ANOVA with post hoc Bonferroni’s test was used to analyze the blood and urine parameters. Statistical significance was defined as P < 0.05.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
RNA-seq data have been deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus under accession GSE162447 (subseries GSE162445 and GSE162446). The Molecular Signatures database used in the study is available at https://www.gsea-msigdb.org/gsea/msigdb/. All other data supporting the findings of this study are available from the corresponding author upon request.
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Research in the Blasco laboratory is funded by the Spanish State Research Agency, Ministry of Science and Innovation, cofounded by the European Regional Development Fund (AF2017-82623-R and SAF2015-72455-EXP), the Comunidad de Madrid Project (S2017/BMD-3770), the World Cancer Research Project (16-1177), the European Research Council (SHELTERINS GA882385) and the Fundación Botín (Spain).
The authors declare no competing interests.
Peer review information Nature Aging thanks Zhaoyong Hu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Telomerase-deficient mice with short telomeres do not spontaneously develop renal fibrosis.
a, Scheme of the experimental approach. b-f, Representative images and quantification of Masson trichrome (b), Sirius Red (c), PAS-Diastase (d), SMA (e) and E-cadherin (f) in Tert+/+ and G3Tert−/− mice. g-h, Representative images and quantification of p21 (g) and CC3 (h) immunohistochemistry stainings in Tert+/+ and G3Tert−/− mice. Insets show amplified images. A t-test two tailed was used for statistical analysis. Data is presented as mean values +/- SEM. The number of mice analyzed per genotype is indicated.
Blood creatinine (a) and blood urea nitrogen (BUN) (b) levels of untreated and FA treated Tert+/+ and G3Tert−/− mice. Blood samples were collected at days 2, 7 and 14. Mice were sacrificed at day 14. a, Two-way Anova with post hoc Bonferroni test was used for statistical analysis. Data is presented as mean values +/- SEM. The number of mice analyzed per genotype is indicated. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.
Relative expression of CCnd1, CCnd2, CCnb1 and CCne1 in Tert+/+, FA-treated Tert+/+, G3Tert−/− and FA-treated G3Tert−/− mice 14 days after administration of a low dose of FA. One-way Anova was used for statistical analysis. Data is presented as mean values +/- SEM. The number of mice analyzed per genotype is indicated. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.
a, Gene expression data obtained by RNA-seq of kidney samples from 7- and 47-week old Terc+/+ and G3 Tert−/− mice. b, Gene Set Enrichment Analysis (GSEA) plots for the EMT pathway. False Discovery Rates (FDR) are indicated. Samples correspond to kidneys of five independent Tert+/+, G3 Tert−/− mice.
a, Relative expression of Sox-9, Wt-1, Pax-2, Sall2, Acvr2b, Klotho in in Tert+/+, FA-treated Tert+/+, G3Tert−/− and FA-treated G3Tert−/− mice 14 days after administration of a low dose of FA. b, Representative images and quantification of Sox-9 immunohistochemistry staining. The insets show amplified images. One-way Anova was used for statistical analysis. One-way Anova was used for statistical analysis. Data is presented as mean values +/- SEM. The number of mice analyzed per genotype is indicated. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001
Relative expression of Notch1, 2, 3, Jagged1 and Tfam in Tert+/+, FA-treated Tert+/+, G3Tert−/− and FA-treated G3Tert−/− mice 14 days after administration of a low dose of FA. One-way Anova was used for statistical analysis. Data is presented as mean values +/- SEM. The number of mice analyzed per genotype is indicated. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.
a,b, Microscopic bright field images of proximal tubule cell (PTC) culture at day 8 (a) and day 14 post-transduction with either the empty or the mTert containing vector (b). c, Representative images of Immunofluorescence for E-Cadherin, SMA and Tgfβ1. Twenty micrographs were captured from each condition.
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Saraswati, S., Martínez, P., Graña-Castro, O. et al. Short and dysfunctional telomeres sensitize the kidneys to develop fibrosis. Nat Aging 1, 269–283 (2021). https://doi.org/10.1038/s43587-021-00040-8
Nature Reviews Nephrology (2021)
Genome Instability & Disease (2021)