Roles of Krüppel‐like factor 5 in kidney disease

Abstract Transcription factor Krüppel‐like factor 5 (KLF5) is a member of the Krüppel‐like factors’ (KLFs) family. KLF5 regulates a number of cellular functions, such as apoptosis, proliferation and differentiation. Therefore, KLF5 can play a role in many diseases, including, cancer, cardiovascular disease and gastrointestinal disorders. An important role for KLF5 in the kidney was recently reported, such that KLF5 regulated podocyte apoptosis, renal cell proliferation, tubulointerstitial inflammation and renal fibrosis. In this review, we have summarized the available information in the literature with a brief description on how transcriptional, post‐transcriptional and post‐translational modifications of KLF5 modulate its function in a variety of organs including the kidney with a focus of its importance on the pathogenesis of various kidney diseases. Furthermore, we also have outlined the current and possible mechanisms of KLF5 activation in kidney diseases. These studies suggest a need for more systemic investigations, particularly for generation of animal models with renal cell‐specific deletion or overexpression of KLF5 gene to examine direct contributions of KLF5 to various kidney diseases. This will promote further experimentation in the development of therapies to prevent or treat various kidney diseases.

. 5,7 KLF5 has three highly conserved C2H2 type zinc-finger domains in its C-terminal known to bind GC-rich regions in DNA, leading to modulation of their gene targets. 8 KLF5 regulates a number of cellular functions and can play a part in a variety of diseases, such as cancer, cardiovascular disease and gastrointestinal disorders. 8 Recently, KLF5 was identified in the kidney and was shown to regulate podocyte apoptosis, renal cell proliferation, tubulointerstitial inflammation and renal fibrosis. [9][10][11][12][13] In this review, we have summarized the current evidence confirming an important role for KLF5 in various kidney diseases and we have discussed the possible mechanisms regulating KLF5 expression transcriptionally or translationally. Moreover, we have outlined the possible pathogenic pathways that are involved in the kidney diseases, including, acute kidney injury (AKI) and chronic renal fibrosis related to nephropathy and nephritis. The mechanistic studies that precisely define KLF5 regulation of kidney diseases are not completed known.
Therefore, in the current review, we have included mechanisms that regulate KLF5 stability and/or activity in other organs and cell types in addition to the kidney. These studies will drive further research in delineating the mechanistic role of KLF5 in kidney diseases. These studies collectively suggest that KLF5 may serve as a new therapeutic target in the treatment of kidney disease.

| P OS T-TR ANS L ATIONAL , TR ANSCRIP TIONAL , P OS T-TR ANSCRIP TIONAL REG UL ATION OF KLF5
Post-translational, transcriptional and post-transcriptional regulation are main regulatory mechanisms of KLF5 expression. Acetylation, phosphorylation, ubiquitination and sumoylation are some posttranslational modifications that regulate stability and activity of a variety of proteins, including KLF5. 14 Transcriptional regulation is the modifications that occur when DNA is transcribed into RNA, including control at the level of transcription apparatus, transcription factors and chromatin. 15 These aforementioned modifications of KLF5 often occur by recruitment of modifier proteins (co-activators or co-repressors) by KLF5 and can regulate KLF5-mediated target gene expression. Post-transcriptional regulation are the modifications that occur after DNA is transcribed into RNA and before RNA is translated into protein, including modifications of RNA splicing, nuclear export, RNA stability and miRNA-mediated regulation of protein expression. 16,17 Although, the role for KLF5 in mediating kidney disease is established, the studies involving KLF5 transcriptional, post-transcriptional and post-translational modifications in the kidney are limited. Therefore, in order to better understanding the role of KLF5 in the pathogenesis of various kidney diseases and to drive future studies in the kidney, we have summarized how KLF5 is regulated post-translationally, transcriptionally and post-transcriptionally in the various cells and tissues, including in the kidney.

| Acetylation
Acetylation and deacetylation occur via addition or removal of an acetyl group (CH 3

CO-) on a protein. Changes in protein acetylation
can alter its function. 18,19 KLF5 is a pro-proliferative transcription factor, known to promote cellular proliferation by inhibiting p15 or cyclin-dependent kinase inhibitor 2B (CDKN2B) expression.
However, in the presence of transforming growth factor beta (TGFβ), p300 was recruited to the KLF5-SMAD complex, which acetylated KLF5. Acetylation of KLF5 altered binding of other factors to p15 promoter resulting in induction of p15 expression and inhibition of cellular proliferation in various cell types including, human embryonic kidney cell line (HEK293) cells. [20][21][22] Acetylation of KLF5 by p300 and deacetylation by histone deacetylase 1 (HDAC1) and histone chaperone TAF1/SET is also reported in vascular smooth muscle cells (VSMCs) and in human HeLa cells. 23,24 Acetylation of TA B L E 1 Comparing basic features of KLF5 between human and mouse (based on Ensembl) KLF5 at K369 by p300 enhanced its transactivation activity, 23 while HDAC1 mediated KLF5 deacetylation inhibited its transactivation activity. 24

| Phosphorylation/ubiquitination
Resveratrol induced KLF5 phosphorylation in renal HEK293 cells and prevented association of KLF5 with c-Myc. 25 Moreover, KLF5 phosphorylation could modulate its activation status as KLF5-Ser153 phosphorylation by protein kinase C at the CREB binding protein (CBP) interaction site promoted its transactivation function in human epithelial cells (HEC-1B), 26 while angiotensin II (Ang II)induced extracellular signal-regulated kinase (ERK)-mediated KLF5 phosphorylation promoted interaction of KLF5 and c-Jun, resulting in the suppression of p21 expression in VSMCs. 27 In addition, KLF5 phosphorylation could also modulate its stability, as GSK-3β induced phosphorylation of KLF5, was shown to promote FBW7mediated KLF5 degradation. 28 A pulse-chase experiment revealed that KLF5 was a rapid turnover protein with a half-life of approximately 2 hours. KLF5 was ubiquitinated and degraded at the proteasome in epithelial cells. 29 WWP1 E3 ubiquitin ligase bound via its WW domain to the PY motif in the transactivation domain on KLF5, resulting in the ubiquitination and degradation of KLF5. 30 In contrast, ubiquitin independent proteasomal degradation of KLF5 was also documented. 31

| Sumoylation
Sumoylation is a protein modification, which can alter protein stability, protein function/activity, protein cellular localization and appropriate protein targeting. 14 Sumoylation occurs when a small ubiquitin-related modifier (SUMO) is incorporated in a protein. This modification can alter the function of metabolic enzymes and/or the metabolic pathways by modulating functions of key transcription factors. 32 KLF5 is a critical regulator of energy metabolism and its sumoylation is documented. 33,34 Sumoylation of KLF5 acts as a molecular switch that controls its association with either transcriptional activation or transcriptional repression complexes. 34 KLF5 regulated lipid metabolism by activating peroxisome proliferator-activated receptorδ (PPARδ) pathway.
KLF5 was basally sumoylated, which promoted its association with repressor complexes, and inhibited expression of lipid oxidation genes. Upon agonist binding to PPARδ, KLF5 became desumoylated resulting in its association with an activation complex, which induced expression of lipid metabolism genes. Thus, KLF5 sumoylation status controlled its switch between transcriptional repression to activation. 35 In addition, KLF5 sumoylation also controlled its nuclear localization. 33 Du et al further identified K151 and K202 as sumoylation sites on KLF5. KLF5 sumoylation facilitated its nuclear localization and function by inactivating its nuclear export signal. 33

| Methylation
Adding methyl groups (-CH 3 ) to DNA is the progress of DNA methylation, which usually leads to transcriptional silencing. 36 KLF5 methylation was reported in several cells/tissues and diseases. To date, a single study which demonstrated that in clear cell renal cell carcinoma (ccRCC), KLF5 protein expression was lower in tumour tissues as compared to adjacent normal renal tissues and was also lower in different ccRCC (A498, RCC4 and 786-O) cell lines compared to immortal renal HEK-293T cells. 37 The reduced expression of KLF5 was found to be associated with increased methylation of CpG loci in the promoter of KLF5 gene in the ccRCC or cell lines compared to normal renal tissue or normal cell lines. Furthermore, treatment of ccRCC cells with 5-Aza-CdR, a DNA methyltransferase (DNMT) inhibitor, up-regulated the expression of KLF5 and repressed ccRCC cell growth, suggesting that hypermethylation might contribute to the down-regulation of KLF5 in ccRCC. 37 This epigenetic modification of KLF5 was found in renal tumour tissues and cell lines. This study indicated that KLF5 was expressed in renal tissue and cells and its expression was controlled by its DNA methylation levels of gene promoter. Similarly, in dermal fibroblasts of systemic sclerosis, CpG methylation of KLF5 promoter contributed to down-regulation of KLF5 protein. 38 Hypermethylation of KLF5 intron 1 was also associated with decreased KLF5 expression in acute myeloid leukaemia and was related to poor overall survivial. 39

| Effects of miRNAs, activators, repressors on KLF5 gene expression
Identifying pathogenic roles of microRNAs (miRNAs) could have an important clinical impact for treating and preventing kidney diseases. Eventually, this could lead to novel and specific therapies and diagnostic tools for kidney diseases. Several studies have suggested that microRNAs, namely, miR-145, miR-152, miR-10b-3p, miR-448-3p, miR-375 and miR-9, can suppress the expression of KLF5 by directly interacting with its 3'-untranslated regions. [40][41][42][43][44][45][46] MiR-145-5p can also target and inhibit KLF5 expression. 47 MiR-145 was detected in urinary exosomes of type 1 diabetic patients and in experimental models of diabetes, and its expression was increased in the glomeruli of diabetic animals. 48 Moreover, MiR-145 inhibited KLF5-NFκB-inflammation pathway in lipopolysaccharide (LPS) treated macrophages. 40,41 Additionally, smooth muscle enriched long noncoding RNA competitively bound to miR-10b-3p and exerted an inhibitory effect on miR-10b-3p-KLF5 pathway in atherosclerosis model. 42 MiR-152 targeted KLF5 and suppressed the inflammatory responses in atherosclerosis model. 43 Furthermore, miR-488-3p also targeted KLF5 and promoted an anti-inflammatory pathway in macrophages in a rat model of intracranial aneurysm. 44 In the progression of oral squamous cell carcinoma, KLF5 regulated genes involved in proliferation and apoptosis. MiR-375 repressed KLF5 activation and resulted in abrogation of cellular proliferation and induction of cell apoptosis. 45 KLF5 expression and activation was inhibited in VSMCs treated with high glucose and miR-9 mimic, while, in the presence of miR-9 inhibitor, the expression of KLF5 was increased compared to cells treated with high glucose alone.
Moreover, expression and activation of KLF5 was associated with proliferation and migration of VSMCs. 46 KLF5 could also be regulated by many other activators and repressors which are related to kidney disease. As we know, Ang II and TNFα are important mediators in kidney disease. Ang II can lead to hemodynamic effects and activate inflammation/fibrosis pathways in kidney disease. 49 TNFα is a pro-inflammatory cytokine in kidney disease. 50 It was reported that human VSMCs treated with either Ang II or TNFα induced KLF5 expression along with a unique inhibitor of apoptosis protein-survivin. Overexpression of survivin also led to up-regulation of KLF5 in human VSMCs. 51 Moreover, CCAAT/enhancer-binding proteins (C/EBP) β and δ were shown to induce KLF5 expression during adipocyte differentiation of mouse embryonic fibroblasts 52 and C/EBPβ was also a regulator of kidney disease. Overexpression of C/EBPβ in the kidney of ischaemia reperfusion-injured mice aggravated the kidney injury, it increased the level of blood urea nitrogen (BUN) and creatinine. 53 In addition, Egr-1, a reported factor that promoted kidney injury, was demonstrated to activate KLF5 in VSMCs. 54,55 Furthermore, IL-1β, HIF-1α and C3a, known regulators of kidney disease, could induce KLF5 expression. 56,57 In contrast, overexpression of C/EBPα inhibited KLF5 and decreased invasiveness of the human colon cancer cells (SW480 cells). 58 Similarly, C/EBPα was shown to have a protective effect in the podocytes of mice subjected to Adriamycin-induced kidney injury as knockout of C/EBPα aggravated Adriamycin-induced kidney injury. 59 All-trans retinoid acid was shown to repress KLF5 expression in intestinal epithelial cells (IEC6) 60 and was shown to have an anti-inflammatory effect on diabetic kidney disease. 61 Moreover, sex hormones were known to influence kidney disease. 62 It was reported that androgen-induced KLF5 expression in human breast cancer cell lines and a prodrug of 17β-oestradiol inhibited KLF5-NFκB inflammatory pathway in the Alzheimer's Disease mouse model. 63,64 In addition, oncogenic regulator protein SET inhibited KLF5 activation by binding to its DNA-binding domain (DBD). SET binding prevented KLF5 DBD acetylation by its coactivator/acetylase p300. In the absence of SET, p300 acetylated DBD of KLF5 and activated its transcription. 23 The Human Protein ATLAS demonstrated that the SET protein was expressed in many tissues including the kidney. Additionally, overexpression of SET in human embryonic kidney 293T cells promoted the cell proliferation. 65

| E VIDEN CE FOR THE ROLE S OF K LF5 IN MODEL S OF K IDNE Y D IS E A S E
The earliest study on the role of KLF5 in the kidney was found in 2004. 66 To date, several lines of evidence have identified the role of KLF5 in various animal and cell models of kidney diseases, for which the key information is summarized in Table 2. From these studies, it is appreciated that KLF5 is mainly expressed in the nucleus of renal collecting duct epithelial cells of normal mice. [11][12][13] However, KLF5 expression is also induced or increased in other renal cells, such as proximal tubule cells, distal tubule cells and mesangial cells of mice under different pathogenic conditions (Table 2). [10][11][12][13]67 Renal fibrosis and dysfunction are the two common hallmarks of progressive renal diseases.

Unilateral ureteral obstruction (UUO) is a model of progressive
renal fibrosis in rodents and is known to mimic accelerated human chronic obstructive nephropathy with primary injury in renal tubules caused by obstructed urine flow. 68 Three studies have shown increased expression of KLF5 mRNA and/or protein in the unilateral obstructed kidney compared to contralateral unobstructed kidney of mice with UUO. 12,13,69 When the C57BL/6 mice subjected to UUO, the expression of KLF5 was induced in renal tubule cells of the unilateral obstructed kidney (both cortex and medulla) as compared to the control kidney, which were detected by Western blot and immunohistochemistry staining, along with increased proximal tubular cell proliferation and possible progression of kidney fibrosis. 13 Furthermore, immunofluorescent staining revealed the staining of KLF5 was mainly in proximal tubules of fibrotic kidneys, but not in endothelial cells and fibroblasts. 13  reported that KLF5 expression was increased in both the renal cortex and medulla regions, but not in endothelial cells and fibroblasts, these results were consistent with what was demonstrated in UUOinduced renal fibrosis and dysfunction. 13 Increased KLF5 expression was also observed in diabetic kidney and diabetic kidney disease is associated with morphological changes including expansion of mesangial matrix and tubular interstitial space, as well as podocyte damage and glomerular basement membrane thickening. 10,70 Western blot analysis demonstrated increased expression of KLF5 protein in the renal cortex of db/db mice (9-weeks of age), compared to control mice. 10 As summarized in Table 2, increased renal expression of KLF5 was seen in other animal models of various aetiological kidney diseases, for instance, in the renal medulla of spontaneously hypertensive rats, in which the mRNA expression of renal KLF5 was increased along with the progression of renal epithelial-mesenchymal transition 71 ; in the kidneys of mice irradiated at the renal region with single dose of 16 Gy, in which, renal tissues were collected at the 10 and 20-week time-point after irradiation and showed a significant increase in KLF5 mRNA. 66 In addition to KLF5's role in modulating chronic kidney injury, KLF5 was also shown to regulate acute renal pathogenesis such as the nephritis (

| THE CURRENT AND P OSS IB LE FUN C TIONS , REG UL ATORY MECHANIS MS OF KLF5 AC TING ON VARI OUS KIDNE Y DISE A SE S
The studies in the kidney, demonstrate that KLF5 modulates kidney diseases by regulating a variety of cellular responses including apoptotic cell death, inflammation, cell proliferation and fibrosis, making KLF5 a plausible therapeutic drug target to treat kidney diseases.
In the following sections, we have summarized the role of KLF5 in modulating apoptosis, cell proliferation, inflammation, oxidative stress, obesity/diabetes, fibrosis, stemness and differentiation in these acute and chronic renal diseases, as potential mechanisms contributing to these kidney diseases. The summarized potential mechanistic pathways of KLF5 in these kidney diseases are illustrated in Figure 1, including the roles of KLF5 in various cell types in the kidney.

| KLF5 and apoptosis
Apoptosis is usually the cellular response to injury or its microenvironment alterations and is associated with the activation of cell death mediators and an inactivation of pro-survival factors. Renal

Disease models Regulation of KLF5 expression The role of KLF5 in kidney Refs
Peripheral blood mononuclear cells from children with nephrotic syndrome.
↓in the peripheral blood mononuclear cells of children with nephrotic syndrome KLF5 mRNA level decreased in the peripheral blood mononuclear cells from children with nephrotic syndrome as compared to control individuals.  (Table 2). In contrast to this in vitro study, an in vivo study showed an opposite, pro-apoptotic role for KLF5 on renal cell apoptosis. 12 These authors

| KLF5 and proliferation
Dysregulated proliferation of renal mesangial cells and tubular cells leads to glomerulosclerosis and tubulointerstitial fibrosis in kidney. 78 The role of KLF5 in proliferation of renal mesangial cells and tubular cells has been identified. 10,13 As we know, mesangial expansion is one of the important features in diabetic kidney disease. 70 Therefore

| KLF5 and inflammation
Inflammatory response is widespread in acute and chronic kid- Thus, increased KLF5 expression may regulate tubulointerstitial inflammation. 72  injury. 12 Moreover, chemotactic peptides S100A8/S100A9 were induced by KLF5 and exogenous administration of recombinant S100A8/S100A9 peptides into mouse kidneys promoted an accumulation of CD11b + F4/80 lo cells as detected by flow cytometry.
Collectively, KLF5-S100A8/S100A9-M1-macrophages pathway promoted renal inflammation in UUO-induced kidney injury. 12 Thus, the above studies demonstrate a role for KLF5 in modulating inflammation in the kidney. However, the precise mechanisms underlying these effects are not known. In addition to the kidney, the role of KLF5 on regulating inflammation is widely studied in other organs and the possible mechanisms mediating these effects have been discussed below. These mechanisms may provide some future directions in delineating mechanisms by which KLF5 modulates renal inflammation. KLF5 was also shown to directly regulate the transcriptional activation of these pro-inflammatory genes. 83,[86][87][88] In addition, proinflammatory TNFα was also shown to induce KLF5 expression in human VSMCs. 51

| KLF5 and oxidative stress
Although oxidative stress promotes inflammation, apoptosis and fibrosis in kidney disease, 90 38 These studies collectively suggested a pro-as well as an anti-fibrotic role for KLF5.

| KLF5, stemness and differentiation
The Although a precise role for KLF5 in generation of adipose-derived mesenchymal stem cells is unknown, KLF5 has been identified as an important regulator of adipocyte differentiation. 52,112 In AKI, renal tubule cells have the capacity to regenerate and repair itself by renal cell structural remodelling. One such mechanism of renal regeneration involves dedifferentiation and proliferation of surviving renal tubule cells after AKI. 113,114 Interestingly, KLF5 was reported to promote dedifferentiation and regeneration of crypt cells in intestinal epithelium after radiation injury as KLF5 can modulate proliferation and stemness. 115 However, the direct role for KLF5 in regulating renal cell dedifferentiation remains to be determined.

| CON CLUS I ON S AND PER S PEC TIVE S
The current evidence has shown that KLF5 is an important modu- These studies may identify KLF5 as a new therapeutic target for the treatment of fibrotic kidney disease associated with diabetes.

ACK N OWLED G EM ENTS
The

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.