Emerging role of tumor suppressor p53 in acute and chronic kidney diseases

Abstract

p53 is a major regulator of cell cycle arrest, apoptosis, and senescence. While involvement of p53 in tumorigenesis is well established, recent studies implicate p53 in the initiation and progression of several renal diseases, which is the focus of this review. Ischemic-, aristolochic acid (AA) -, diabetic-, HIV-associated-, obstructive- and podocyte-induced nephropathies are accompanied by activation and/or elevated expression of p53. Studies utilizing chemical or renal-specific inhibition of p53 in mice confirm the pathogenic role of this transcription factor in acute kidney injury and chronic kidney disease. TGF-β1, NOX, ATM/ATR kinases, Cyclin G, HIPK, MDM2 and certain micro-RNAs are important determinants of renal p53 function in response to trauma. AA, cisplatin or TGF-β1–mediated ROS generation via NOXs promotes p53 phosphorylation and subsequent tubular dysfunction. p53-SMAD3 transcriptional cooperation downstream of TGF-β1 orchestrates induction of fibrotic factors, extracellular matrix accumulation and pathogenic renal cell communication. TGF-β1-induced micro-RNAs (such as mir-192) could facilitate p53 activation, leading to renal hypertrophy and matrix expansion in response to diabetic insults while AA-mediated mir-192 induction regulates p53 dependent epithelial G₂/M arrest. The widespread involvement of p53 in tubular maladaptive repair, interstitial fibrosis, and podocyte injury indicate that p53 clinical targeting may hold promise as a novel therapeutic strategy for halting progression of certain acute and chronic renal diseases, which affect hundreds of million people worldwide.

Introduction

Fibrotic disorders, marked by the excess accumulation of extracellular matrix molecules (ECM), lead to tissue scarring and end stage disease regardless of the organ system and account for nearly 50% of all mortalities worldwide [1]. The incidence of chronic kidney disease (CKD), which impacts 10–15% of the world population, is expected to increase in the coming decades given the rapid rise in risk factors, such as diabetes and hypertension [2–4]. An exuberant wound healing response following injury (e.g., ischemia, diabetes, hypertension, exposure to toxins) promotes the progressive loss of organ function resulting in end-stage disease [2–12]. After mild injury, kidney repair can restore renal structure and function. Maladaptive repair mechanisms in response to severe or persistent injury, however, promotes the progression of fibrosis and CKD [13–15]. The resulting G₂/M arrest of tubular epithelial cells due to renal injury under these circumstances triggers the secretion of profibrotic factors such as TGF-β1 and CTGF, which promote fibroblast proliferation [13]. Fibrogenesis, a highly dynamic and complex process characterized by renal tubular cell injury, inflammatory cell infiltration, myofibroblast activation, and excess ECM deposition, is the common mechanism that culminates in kidney failure [2–15]. Effective therapies to slow or prevent the progression of chronic renal disease remain a huge unmet clinical need despite tremendous advances in the underlying mechanisms [5, 6]. Therefore, exploring novel targets for suppressing or reversing fibrosis progression is of paramount importance.

TGF-β is the major mediator of fibrosis in the kidney and many other organs. Irrespective of the initial etiology (e.g., diabetes, obstruction, hypertension, aristolochic acid and ischemia reperfusion), activation of TGF-β1 contributes to renal injury progression and fibrosis [4–28]. TGF-β1 not only activates/phosphorylates p53 but also utilizes p53 in concert with SMAD3 transcription factors in orchestrating the fibrotic phenotype in several renal cell types [27, 28]. p53 tissue-specific knockout mice and pharmacological inhibition, illustrates the integral role of p53 in the progression of renal lesions. This review summarizes recent findings identifying p53 as an effector of both acute and chronic nephropathies and addresses the potential targetability of p53 driven pathways in mitigating kidney failure.

p53 Regulation

p53 is a prominent tumor suppressor given its central role in DNA damage repair, growth arrest, senescence, and apoptosis [29]. In unstressed cells, p53 levels remain low due to its interactions with the ubiquitin ligase, MDM2, which promotes p53 degradation. Upon activation by a wide variety of stress signals (i.e., genotoxic damage, oncogene expression), p53 acquires a homotetramer transcriptional conformation that impacts different phenotypes depending on the subset of genes expressed [29]. The transcriptional activity of p53 is regulated by various post-translational modifications [29, 30]. p53 is stabilized by phosphorylation at p53^Ser15 by several protein kinases (e.g., ATM, ATR, Chk2) which prevent p53 interaction with MDM2 and subsequent degradation [30, 31]. p53 phosphorylation at the Ser15 site acts as a nucleation event that permits subsequent phosphorylation at its serine/threonine residues (in the N-terminus) and lysine acetylation (mainly in the C-terminus) [30, 32]. In response to genotoxic stress, for example, ATM-dependent p53^Ser15 phosphorylation promotes the recruitment of histone acetyltransferases (e.g., p300, CBP, PCAF) leading to the acetylation of lysine residues in the DNA-binding and carboxy-terminal domain of p53, modulating its DNA binding ability [32, 33].

ATM plays a central role in DNA damage repair and is required for maintaining genomic stability by stalling the transition through the G1/S and G2/M checkpoints [32, 33]. Double stranded DNA breaks promote ATM phosphorylation at Ser1981 (pATM^Ser1981), a marker of ATM autophosphorylation and activation [33]. ATM is then recruited to the sites that need to be repaired through the Mre11/RAD50/NBS1 (MRN) complex [33]. Recent studies, however, identified a role for ATM independent of DNA damage. H₂O₂ treatment, for example, promotes the phosphorylation of ATM (that is independent of MRN complex activation and γ-H2AX) leading to phosphorylation of Chk2 and p-p53^Ser15 [34] indicative of redox control of this tumor suppressor. ATM-dependent p53^Ser15 phosphorylation, further, leads to the stabilization and activation of p53 and subsequent transcriptional induction of target genes such as p21, resulting in growth arrest [28].

p53 involvement in acute and chronic renal diseases

Multiple preclinical studies confirmed the therapeutic value of targeting p53 in both acute and chronic renal diseases. p53 is phosphorylated and/or upregulated in several models, including ischemia–reperfusion injury (IR) and aristolochic acid nephropathy (AAN), as well as in response to obstructive (UUO), diabetic and nephrotoxin-induced injury (e.g., cisplatin). Intravenous administration of p53 short interfering RNA (siRNA), moreover, mitigated tubular trauma and afforded renoprotection from ischemic- and cisplatin-induced kidney damage in mice [35] suggesting a key role for p53 in disease progression. Cisplatin treatment in mice leads to renal tubular senescence and fibrosis which is likely mediated by oxidative stress induced p53 activation [36]. Pharmacological blockade of p53 (via Pifithrin-α administration) reduced tubular cell apoptosis induced by cisplatin. Mitochondrial dysfunction consequent to cisplatin exposure increases reactive oxygen species production. Administration of the antioxidant N-acetylcysteine (NAC) attenuated cisplatin-induced premature senescence, reduced p53 and p21 levels and blunted expression of genes characteristic of the senescence-associated secretory phenotype (SASP) [37]. Progression of AAN in mice is also mediated via p53 dependent mechanisms since p53 knockout mice are significantly protected from AA-induced acute tubular injury and disease progression compared to wild type counterparts [38].

p53 is also required for transition of acute kidney injury (AKI) to chronic kidney disease (CKD). A seminal study demonstrated that tubular epithelial cells undergo G2/M cell cycle arrest in response to ischemia–reperfusion-, aristolochic-acid- and obstruction-induced nephropathies and secrete fibrotic factors such as TGF-β1 and CTGF, which promote fibroblast growth [13]. Pifithrin-α administration in mice, indeed, ameliorated the development of chronic renal disease following AKI, suggesting that p53 is a major mediator of fibrosis progression [13]. Renal proximal tubule cell (PTC)-specific p53 knockout mice, indeed, confirmed the functional role of p53 in the AKI to CKD transition. Ischemic injury-induced activation of TGF-β1, tubular injury/cell cycle arrest, and interstitial fibrosis evident in wildtype mice were significantly reduced in animals with PTC-specific p53 ablation [39, 40]. Similarly, UUO-driven G₂M arrest and fibrosis are also attenuated in mice with PTC-specific p53 ablation or in animals receiving Pifithrin-α prior to ureteral ligation, confirming a critical role for p53 activation in obstructive renal disease [41]. Suppression of kidney p53 expression via tail vain injection of p53 shRNA post-UUO attenuated renal G2/M arrest and p21 expression and reduced the extent of Sirius Red staining compared to mice receiving control constructs, further confirming role of p53 in pathogenic tubular proliferative defects associated with progressive obstructive nephropathy [42].

Ongoing diabetic kidney disease is, in fact, accompanied by increased expression and phosphorylation of p53 in both tubules and podocytes. Nuclear staining of p53 is robustly increased in patients with diabetic and IgA nephropathy compared to control patients with minimal change disease, further supporting p53 activation in human progressive kidney injury [41]. Mice treated with the diabetic causative agent, streptozotocin and diabetic prone db/db mice displayed increased renal p53, BAX and Puma expression as well as epithelial apoptosis [43, 44]. Reduction of oxidative stress by catalase overexpression not only reduced p53, BAX and Puma upregulation but also suppressed the extent of diabetic renal injury [43, 44]. Human renal allograft rejection is associated with tubular injury, progressive fibrosis, as well as p53 and p21 induction. Allograft p53 and p21 expression positively correlate with creatinine levels and negatively correlate with estimated glomerular filtration rate (eGFR) [42], further highlighting clinical relevance of p53 hyperactivation in transplant failure. The extent of p53 involvement in progressive human nephropathies, however, requires further investigation given the widespread p53 activation in different animal models of renal diseases.

Exacerbation of ischemia reperfusion injury in the context of diabetes (as seen in the akita/streptozotocin mouse model), furthermore, leads to a p53-dependent rise in serum creatinine levels, more severe tubular injury, and increased apoptosis [45]. CKD-mediated AKI, induced by dual diabetic and ischemic insults, can also be mitigated by pifithrin-α treatment and PTC p53 ablation [45]. Global p53 knockout mice, in contrast, develop more severe acute kidney injury compared to wild type animals in response to ischemia–reperfusion likely due to increased inflammation, suggesting that p53, in certain conditions, may also exert renal protective effects [46] (Fig. 1).

Fig. 1.

Causative Role of p53 in fibrotic disease. In response to various renal insults (e.g., obstruction, diabetes, FSGS, HIV and nephrotoxins) p53 is phosphorylated and/or overexpressed in the damaged kidney, leading to epithelial cell death/cell cycle arrest and a secretory associated senescent phenotype, fibrotic factor (CTGF, PAI-1 and TGF-β1) expression/secretion, myofibroblast accumulation, inflammation. Collective maladaptive repair responses, orchestrated by p53 results in tissue fibrosis and end stage renal disease

Regulators of p53 activation/induction during kidney disease

TGF-β1

A prominent TGF-β family member involved in tissue fibrosis, TGF-β1 binds the TGF-β1 receptor type II, leading to the formation of a heterocomplex with the ALK5/TGF-β1 receptor type I. Activated ALK5, in turn, phosphorylates receptor-SMADs (R-SMADs; SMAD2, SMAD3), and interaction with the common shuttle SMAD4 facilitates R-SMAD/SMAD4 translocation to the nucleus where they act as transcription factors, causing expression of TGF-β1 target genes (reviewed in 22). The SMAD proteins have weak DNA binding affinity suggesting additional co-factors (e.g., Snail, YAP/TAZ, Catenin, AP-1, c-Jun) are necessary for specific and optimal target gene expression (20–25). Non-canonical (non-SMAD) signaling pathways (e.g., NOX, EGFR, MAPK, YAP/TAZ, Rac-GTPases, p53) activated by TGF-β1 cooperate with SMAD3 in orchestrating subsequent renal fibrotic responses (e.g., PAI-1, fibronectin, collagen-1, CTGF induction) [16–28, 47–50] (Figs. 1, 2).

Fig. 2.

TGF-β1 and the nephrotoxin cisplatin utilize distinct p53 mediated pathways to mediate progression of renal injury. TGF-β1 initiates both canonical SMAD3 and non-canonical pathways (which involves NOX mediated ATM phosphorylation and subsequent activation of p53, which then cooperates with SMAD3) to promote fibrotic genes, epithelial cell cycle arrest and epithelial-fibroblast crosstalk leading to tubulointerstitial fibrosis (red arrows). Ciplatin phosphorylates p53 via NADPH (oxidase)/ROS dependent activation of ATR/ChK kinases. Subsequent p53 dependent induction of BAX and puma lead to tubular apoptosis/necrosis and acute kidney injury (AKI), which could transition to CKD if AKI is severe or persistent (black arrows)

Crosstalk between TGF-β1 and p53 signaling pathways are evident during development and in pathological conditions. In Xenopus development, p53 is required for maximal induction of mesodermal genes by integrating the downstream signaling of Activin (a TGF-β1 superfamily member) and fibroblast growth factor (FGF) [51]. p53^{Ser6 or Ser9} phosphorylation promotes binding of SMAD2 to p53, and SMAD2/p53 functional cooperation is necessary for mesoderm developmental gene expression (52). Several recent studies demonstrate p53 and SMAD3 crosstalk during the progression of several renal disorders, consistent with their activation in the injured kidney [27, 28]. p53, rapidly phosphorylated (at Ser15 and Ser9) in response to TGF-β1 stimulation, functions as a transcriptional regulator of cytokine signaling via SMAD3 interaction in HK-2 renal epithelial cells and NRK-49F renal fibroblasts [27]. Many fibrotic genes such as PAI-1, CTGF, fibronectin and TGF-β1, in fact, possess p53 binding elements in their promoter regions [30, 53–55]. p53 as well as SMAD3 are, indeed, engaged in the PAI-1 promoter in response to TGF-β1 stimulation in renal epithelial cells [27]. ATM, rapidly phosphorylated (activated) by TGF-β1 stimulation, is necessary for p53^Ser15 and ^Ser9 phosphorylation and subsequent pro-fibrotic gene transcription/ expression [28]. Moreover, p22^Phox and p47^Phox NOX subunits and Rac-1 GTPase serve as upstream mediators of ATM- and p53-dependent signal propagation by TGF-β1 [28, 50]. Redox control of the ATM-p53 axis is critical not only for fibrotic factor induction but induction of renal epithelial growth arrest by TGF-β1 (28;50) (Fig. 2). NOX/Rac, ATM, p53, SMAD3 and fibrotic target genes such as PAI-1 and CTGF are rapidly induced during obstructive nephropathies [27, 28, 50] (Fig. 2). p53 involvement in TGF-β1 signaling may extend beyond transcriptional regulation which is further discussed in the micro-RNA regulation of p53.

Oxidative stress-induced kinases

Kinase-dependent activation of p53 mediates the DNA damage response and epithelial cell growth arrest which are important contributors to tubular apoptosis in acute renal injury. In this context, upstream kinases, specifically ATR and ATM, orchestrate p53 activation and subsequent renal epithelial injury. ATR and Chk2 are activated in the kidney following cisplatin treatment in mice [36, 56–58]. Blockade of ATR, using a dominant negative mutant in renal epithelial cells, inhibits cisplatin-mediated p53 phosphorylation and apoptosis. Chk2 phosphorylation, downstream of ATR in response to cisplatin treatment, is required for p53 mobilization [56–58] (Fig. 2).

Aristolochic acid nephropathy and ischemic reperfusion injury promotes the phosphorylation of ATM in tubular epithelial cells in vivo [13]. Similarly, AA-treated epithelial cells increased ATM, Chk2 and p53 activation [13, 59] and promoted p53-dependent G2/M cell cycle arrest, which was prevented by ATM shRNA knockdown [59]. AA-induced G2/M cell cycle arrest was blocked by the antioxidant N-Acetyl cysteine (NAC) as well as by pharmacological inhibitors of ATM and Chk2. NAC treatment reduced AA-dependent phosphorylation of cell cycle regulators ATM, Chk2 and p53 and inhibited p21 induction as well as tubular apoptosis, suggestive of redox regulation ATM-p53 axis in epithelial cell cycle arrest [59].

p53-mediated induction and apoptosis, also evident in db/db diabetic mice, can be suppressed by overexpressing the antioxidant enzyme catalase in proximal tubular cells suggesting ROS as a mediator of p53-dependent diabetic phenotypes [43, 44]. Although, the upstream signaling leading to p53 activation was not investigated in this scenario, it is likely that free radical generation mediates p53 activation and apoptosis. Indeed, glucose stimulation (a hyperglycemic scenario) suppressed AMPK expression with subsequent NOX4 dependent p53 overexpression leading to podocyte injury and proteinuria [60].

Ataxia telangiectasia and Rad3-related protein (ATR)

DNA strand breakage frequently accompanies kidney injury [61, 62]. Sensor kinases (ATM, ATR, DNA-PK) activate the DNA damage response (DDR) pathway upregulating the G1/s and G2/M cell cycle checkpoint kinases Chk1 and Chk2 [63, 64]. When DNA damage is severe, however, repair may be compromised leading to cell cycle arrest at the G1/S or G2/M boundaries. G2/M stalling following injury is associated with proinflammatory and profibrotic genomic programming resulting in the rapid progression of renal disease [13]. Recent findings suggest that ATR activation protects the injured proximal tubular epithelium by limiting the extent of maladaptive repair thereby attenuating the subsequent fibrotic response [65]. Mice with a proximal tubular deletion of the Atr gene subjected to renal ischemia–reperfusion or UUO developed more severe kidney pathology and fibrosis compared to Atr^+/+ controls correlating with an increased incidence of G₂/M-arrested cells, expression of components of the senescence-associated secretory phenotype, functional impairment, and eventual onset of CKD. It was concluded that, as part of the maladaptive repair response, the inadequate recovery of injured but surviving epithelial cells to replace dead or dying elements coupled with G₂/M arrest results in a persistent state of tissue injury and subsequent chronic disease [65].

Cyclin G1

Fibrosis and acquisition of a senescence-like phenotype are frequent sequalae of renal injury regardless of etiology [10–15]. Proximal tubular epithelial cells are particularly susceptible to growth arrest in G₂/M upon severe acute kidney injury, a cell cycle state associated with a JNK-dependent increased expression of profibrotic factors resulting in maladaptive repair [13]. Similar to senescent cells, G₂/M-stalled cells form target of rapamycin (TOR)-autophagy spatial coupling compartments (TASCCs) which, in turn, induces expression of a profibrotic senescence-associated secretory phenotype (SASP) [66–69]. The SASP is a collection of soluble factors secreted by senescent cells that exert a complex paracrine impact on various cells in the immediate microenvironment [66–69]. p53 is a major contributor to G₂/M arrest in the injured kidney likely via upregulation of cyclin G1 [13, 69]. Increased cyclin G1 appears to promote proximal tubular epithelial cell dedifferentiation, G₂/M residence, TASCC formation and secretion of profibrotic factors (e.g., CTGF/CCN2, TGF-β1). Inhibition of TASCC formation in proximal tubular cells attenuated production of profibrotic effectors, and proximal tubular-specific deletion of TASCC components reduced the development of renal fibrosis [69]. Collectively, these data suggest that the cyclin G1/TASCC pathway may constitute a new target for the therapy of kidney disease.

Murine double minute-2 (MDM2)

p53 is a critical regulator of podocyte death as knockdown of Mdm2, an E3 ligase that mediates p53 degradation, in podocytes increases p53 levels, leading to p53-dependent podocyte loss in vitro. Cell death and progression of glomerular injury induced by podocyte specific Mdm2 ablation in mice can be suppressed in double-transgenic animals by dual p53 and Mdm2 silencing in podocytes [70]. A recent study demonstrated that MDM2 expression is dramatically attenuated in patients with diabetic nephropathy [71]. Similarly, liver-specific ablation of Mdm2 leads to hepatocyte apoptosis and fibrosis via p53 dependent CTGF upregulation suggesting that p53-driven mechanisms orchestrate tissue injury and aberrant fibrotic tissue repair in multiple organs [72].

Snail1

The transcription factor Snail1 is a potent inducer of epithelial to mesenchymal transition (EMT) via downregulation of E-cadherin expression and upregulation of vimentin during embryogenesis and tumorigenesis [73]. Highly upregulated during renal disease progression in humans and mice, Snail1 is a major inducer of renal epithelial cell dedifferentiation (partial EMT) which promotes tubular cell G2/M arrest and subsequent CKD progression [74, 75]. Accordingly, kidney tubular-specific depletion of Snail1 in mice ameliorates UUO-induced epithelial dedifferentiation, G2/M arrest, and fibrosis [74, 75]. Snail1 expression appears to be upstream inducer of p53 during CKD progression as suppression of Snail1 expression via delivery of shRNA plasmids in mice attenuated UUO-driven renal p53 expression, G2/M arrest, and fibrosis progression [42]. Precise mechanisms of Snail1 regulation of p53, however, are currently not clear.

Homeo-domain interacting protein kinase 2 (HIPK2)

Elevated HIPK2 expression is causatively linked to renal fibrosis in several models including human immunodeficiency virus (HIV)-expressing transgenic mice (Tg26 mice; a model for HIV-1-associated nephropathy), UUO and folic acid nephropathy [76]. HIV infection via redox sensitive pathways induces HIPK2 protein expression and promotes p53 as well as SMAD3 signaling activation likely via HIPK2 dependent mechanisms, leading to epithelial apoptosis and plasticity in vitro and in vivo [76]. However, precise mechanisms of p53 regulation by HIPK2 kinase remain to be elucidated.

Numb

Numb positively regulates p53 levels by entering in a tricomplex with p53 and the E3 ubiquitin ligase MDM2, thereby preventing ubiquitination and degradation of p53 [77]. While expressed in normal kidneys, Numb mRNA and protein levels are further induced as early as day 3 post-UUO, which correlated with G2/M growth arrest of the tubular epithelial cells. Overexpression of Numb in cultured HK2 cells lead to G2/M arrest, as well as p53, p21 and fibrotic gene induction [78]. Inhibition of p53 using pifithrin-α reversed the pathologic consequences of Numb overexpression. Proximal tubule-specific ablation of Numb in mice also led to reduced fibrosis, and attenuated p53, p21, TGF-β1 and CTGF expression compared to control animals following ureteral ligation [78]. These data conclusively demonstrate that tubular Numb expression contributes to renal p53 signaling amplification and fibrosis during progressive CKD.

Hypoxia-inducible factor 1-alpha (HIF1-α)

Hypoxia plays an important role in the development of renal fibrosis as rarefication of peritubular microvessels occurs regardless of injury type [79]. The transcription factor HIF1-α is activated in hypoxic conditions and induces p53 protein accumulation and stabilization [80]. HIF1-α, indeed, is upregulated in the kidney such as diabetic nephropathy and UUO, and pharmacological inhibition or genetic silencing of HIF1-α in these models attenuates fibrogenesis [81, 82]. p53 upregulation by HIF1-α orchestrates hypoxia-induced G2/M growth arrest in tubular epithelial cells and fibrosis in vivo [83]. HIF1-α, indeed, binds to the hypoxia response element (HRE3) region of the p53 promoter to activate p53 transcription, and HIF1-α silencing prevents hypoxia-induced p53 expression [83]. p53 expression alone is sufficient to suppress CDK1 and cyclinB1/D1 and initiate G2/M arrest in vitro. Furthermore, pharmacological inhibition of p53 using pifithrin-α during UUO results in marked downregulation of CDK1/cyclinB1/D1 and fibrotic matrix molecules [83]. This demonstrates that HIF1-α is a novel regulator p53 induction during renal injury.

Notch

Notch proteins are upstream regulators of p53 in podocyte pathologies. In this regard, podocyte specific in vivo overexpression of intracellular domain of Notch1 (ICN1), which is elevated during diabetic renal injury and FSGS, leads to p53 dependent cell death, proteinuria, and glomerulosclerosis. Inhibition of Notch signaling by pharmacological or molecular approaches reversed these phenotypes [84, 85].

Phosphate tensin homologue (PTEN)

Tumor suppressor PTEN expression is dramatically decreased in tubular interstitial regions during renal injury correlating with fibrosis in several animal models including ischemia reperfusion-, UUO-, AAN- and STZ-driven renal injury [86–88]. Silencing of PTEN in human renal tubular epithelial cells leads to cell cycle arrest, dedifferentiation as well as production/secretion of profibrotic factors including PAI-1 and CTGF [87, 88]. PTEN loss activates p53, and stable silencing of p53 in epithelial cells rescues PTEN loss-induced growth arrest as well as suppresses fibrotic gene expression (e.g., PAI-1, fibronectin and CTGF). p53 mediated PAI-1 induction is a critical regulator of growth arrest consequent to PTEN loss [88]. These studies are consistent with the recognition that PAI-1 is a critical downstream target of p53 in the induction of replicative senescence [89] and that cell cycle arrest induction by TGF-β1 requires p53-PAI-1 signaling axis in human renal epithelial cells [28, 50].

Plasminogen activator inhibitor-1 (PAI-1)

A recent study, furthermore, demonstrates that PAI-1 is a novel regulator of renal tubular p53 expression. Sustained PAI-1 expression in HK-2 cells promotes epithelial dedifferentiation (loss of E-Cadherin and gain of vimentin), G2/M cell cycle arrest, apoptosis, induction of matrix molecules (CTGF, fibronectin and collagen-1) and robust upregulation of p53 protein [90]. Stable depletion of p53 in PAI-1-tranduced renal cultures led to a reduction in pro-fibrotic factors and p21 expression and attenuation of PAI-1-induced renal epithelial cell death compared to double transgenic controls [90]. While mechanisms are still unclear, these studies suggest that PAI-1-directed renal p53 upregulation is causatively linked to tubular maladaptive repair/dysfunction.

MicroRNA involvement in renal p53 signaling

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally by binding the 3′-untranslated region (3′-UTR) of mRNAs with high specificity and are important regulators of renal pathology (reviewed in [91]). Two miRNA families, miR-192 and miR-30, may impact the expression/activation of p53 in a positive or negative manner, respectively. TGF-β, p53 and miR-192 expression increased in the cortex of diabetic mice correlating with glomerular expansion and fibrosis [92–94]. Administration of an miR-192 locked nucleic acid construct (to silence miR-192 expression) blocked p53 expression in STZ-treated mice [93]. Further, genetic deletion of miR-192 in STZ-induced diabetic mice exhibited a reduction in fibrosis, hypertrophy, proteinuria, and albuminuria consistent with attenuated p53 and TGF-β expression [93, 94]. miR-192 knockout mice had an increase in the miR-192 target, Zeb2, which negatively regulated the expression of p53 and subsequent induction of collagen, fibronectin and TGF-β (Fig. 3). Moreover, TGF-β increased the promoter activity of p53 in a miR-192-dependent manner in mouse mesangial cells [93]. Interestingly, aristolochic acid treatment increased the expression of miR-192 in proximal tubular cells. The overexpression of miR-192 in PTCs was sufficient to promote G2/M growth arrest [59], while attenuating the expression of MDM2 resulted in an increase of p53 activation. Collectively, these studies suggest that p53-dependent G2/M growth arrest and pro-fibrotic gene induction is miR-192-dependent warranting investigation of anti-miRNA therapy to target p53-dependent kidney injury and disease progression.

Fig. 3.

micro-RNA and p53 interactions during renal tissue injury progression. Multiple micro-RNAs regulate p53 during renal injury, providing additional targets to suppress p53 mediated tubulointerstitial and glomerular disease progression. p53-driven upregulation of miR-34a and miR-199a-3p downstream of TGF-β1 represses klotho and SOCS7, respectively, and induces fibrosis (red arrows). Additionally, miR-192 induction downstream of aristolochic acid (AA) treatment leads to p53 mediated epithelial cell cycle arrest while STZ-mediated diabetic injury induced miR-192 expression regulates p53 dependent matrix expansion and fibrosis. Furthermore, miR-214-mediated downregulation of ULK1 expression represses renal autophagy, resulting in increased ECM accumulation/fibrotic factor expression (green arrows). miR-30 downregulation evident during focal glomerular segmental sclerosis (FSGS) facilitates p53 activation with subsequent induction of glomerular injury (blue arrows). Cisplatin-driven renal injury-induced miR-375 expression (in a p53 dependent manner) promotes tubular cell death (black arrows). On the other hand, miR-17–5 upregulation by p53 downstream of ischemia–reperfusion injury downregulates death receptor 6 and exerts anti-apoptotic effects (purple arrows)

Another recent study documented the emerging role of p53 directed upregulation of micro-RNA-199a-3p downstream of TGF-β1 in promoting renal fibrosis. Here, induction of miR-199a-3p downregulates the expression of SOCS7, which in turn activates the JAK/STAT3 pathway to promotes renal fibrosis [41]. These findings highlight not only the therapeutic potential of inhibition of p53 activation but also targeting upstream regulators of p53 activation (e.g., ATM-NOX axis) as well as downstream mediators (such as miR-199a-3p or JAK/STAT3) in suppressing renal fibrosis driven by TGF-β1.

p53 activation during cisplatin nephropathy induces expression of miR-375, which in turn downregulates hepatocyte nuclear factor 1 homeobox β (HNF1-β) and promote apoptosis [95]. However, not all micro-RNAs induced by p53 consequent to acute injury exert detrimental effects. During the progression of ischemia–reperfusion (IR) injury, activation of miR-17-5p downstream of p53 attenuated the death receptor 6 expression and subsequent apoptosis [96] (Fig. 3).

Podocytes from normal kidneys express all members of the miR-30 family. Patients with focal segmental glomerulosclerosis (FSGS) had reduced levels of the miR-30 family [97]. Rats treated with puromycin aminonucleoside (PAN), to recapitulate FSGS, had attenuated miR-30 expression correlating with proteinuria and podocyte injury. Exogeneous transfer of miR-30a to podocytes of PAN-treated rats exhibited reduced Notch1 and p53 activation and ameliorated proteinuria and podocyte apoptosis [97] (Fig. 3). Similarly, TGF-β transgenic mice had decreased miR-30 correlating with increased podocyte damage and glomerulosclerosis. In vitro, TGF-β downregulated miR-30, and increased activation of p53 by TGF-β were reduced in miR-30 overexpressing podocytes [98]. TGF-β-dependent apoptosis, moreover, was blocked in miR-30 overexpressing podocytes. Interestingly, glucocorticoid treatment sustained miR-30 expression alleviating podocyte damage, which potentially targets the p53-dependent pathological phenotype [97].

p53 also induces miR-34a expression during renal fibrosis progression. Patients with renal fibrosis as well as UUO-injured mice display increased renal tubular miR-34a levels. miR-34a null mice, indeed, developed less fibrosis following UUO compared with wildtype mice [99], confirming its pathogenic role in CKD. shRNA-mediated repression of p53 prior to TGF-β1 stimulation in HK2 cultures attenuated miR-34a levels. Additionally, miR-34a repression mitigated TGF-β1-driven genetic responses [99], strongly suggesting that p53-mediated miR-34a upregulation downstream of TGF-β1 promotes fibrogenesis.

Recent studies also highlight emerging role of p53-mediated miRNAs such as miR-214 in renal autophagy and fibrosis progression. Autophagy is a conserved lysosome-dependent degradation of unnecessary or dysfunctional cellular components. Although, autophagy is dispensable for kidney development, autophagy is tightly regulated, enabling cells to adapt to cellular stress and maintaining kidney homeostasis. Dysregulation of autophagic mechanisms contributes to pathogenesis of AKI and maladaptive repair processes that drive CKD progression and aging [100]. Impaired autophagy is evident in DKD models and in human diabetic kidneys. In fact, proximal tubule-specific ablation of autophagy-related gene 7 (Atg7) leads to decreased autophagy, renal hypertrophy, tubular injury, inflammation, fibrosis, and increased albuminuria in STZ-induced DKD highlighting its protective role in renal disease [101]. Mechanistically, p53-dependent miR-214 upregulation reduced the expression of unc-51–like autophagy-activating kinase 1 (ULK1) [102]. miR-214 ablation in proximal tubules restored ULK1 expression and autophagy and improved renal function. Furthermore, proximal tubule p53 depletion attenuates miR-214 induction promotes increased ULK1 levels, autophagy and ameliorates STZ-driven DKD [102]. Human renal biopsies revealed a strong correlation between p53/miR-214 axis and renal fibrosis.

Discussion and therapeutic significance of p53 inhibition in acute renal injury and progressive CKD

Gene targeting and pharmacological blockade studies demonstrated the critical role of p53 in tubular and podocyte injury and progressive nephropathies. The recognition that p53 is a critical orchestrator of ischemic, diabetic, obstructive and podocyte injury and subsequent transition from AKI to CKD makes p53 an attractive target for suppressing progressive renal disease of numerous etiologies. Furthermore, demonstration that TGF-β1, a pro-fibrotic cytokine that mediate fibrosis in regardless of the initial causative injury, utilizes p53 as a critical regulator of fibrotic gene induction and epithelial growth arrest further validates p53 as a viable target for suppressing TGF-β1 driven maladaptive responses.

An important consideration surrounding p53 inhibition as an anti-fibrotic therapy would be minimizing potential adverse responses associated with the prolonged p53 deficiency (e.g., predisposition to cancer or inflammation) [29–31, 46], which could interfere with the potential benefits of p53 targeting to suppress progressive renal injury and CKD, for which there are no effective therapies. Temporary and reversible inhibition of p53, in this regard, may not only minimize acute tissue damage (e.g., epithelial cell apoptosis/ growth arrest) but also reduce the progression of acute renal injury to maladaptive tissue repair response [13, 35]. In this regard, a recent study demonstrates that ammonium-functionalized carbon nanotube (fCNT)–mediated p53 siRNA delivery is well tolerated in mice and primates and administration of fCNT–mediated (p53 + meprin) siRNA significantly reduced cisplatin induced kidney injury, fibrosis, and immune cell infiltration in rodents [103]. Furthermore, intravenous administration of QPI-1002, a synthetic siRNA against p53 in a phase II clinical trial (in 332 patients undergoing kidney transplantation) presented 15% risk reduction in delayed graft function in comparison to placebo control group in a randomized and double-blind study (by Quark Pharmaceuticals Inc). In a Quark-sponsored Phase III trial (NCT03510897), 1043 cardiac surgery patients at high risk for AKI were treated with a single IV dose of QPI-1002 or placebo to assess the primary endpoint of reduction in major kidney adverse events post-90 days of surgery [104]. In July 2021, the study was expectedly terminated early due to the study results not meeting its efficacy outcome at the 90-day time point; however, further details of this trial have not been released to date.

Gene silencing of p53 and Pifithrin-α treatment blocked TGF-β1 induced p53 dependent fibrotic gene expression (e.g., PAI-1, CTGF, fibronectin, collagen-1 and TGF-β1) in the kidney cells [27, 28]. Thus, targeting p53 may not only suppress TGF-β1 secretion by G2/M arrested proximal tubules during AKI progression to CKD [13] but also TGF-β1-mediated induction of fibrotic phenotype [27, 28]. Indirect approaches targeting micro-RNAs that modulate p53 activation (e.g., mir-192) and inhibition of oxidative stress-related pathways (such as NOXs) serve as alternative ways to suppress p53 driven injury progression and fibrosis.

Funding

Supported by NIH Grant GM057242 to PJH and a Capital District Medical Research Institute Grant to RS.

Data availability

Enquiries about data availability should be directed to the authors.

Declarations

Conflict of interest

The authors have not disclosed any competing interests.