Exploring the Functionality of Krüppel-Like Factors in Kidney Development, Metabolism, and Diseases.

1. Introduction

The kidneys are intricate organs that have a vital function in maintaining an overall balance of waste and fluids in the body [1]. They preserve the body's electrolyte and acid-base balance by filtering and reabsorbing substances such as ions, glucose and amino acids from the bloodstream. In addition, they eliminate waste products produced by cellular metabolism and xenobiotics [2]. They additionally perform endocrine functions such as hormone production e.g., erythropoietin, 1-α hydroxylase, and renin, which are involved in erythropoiesis, calcitriol synthesis, and blood pressure regulation, respectively [3]. It is important to note that to maintain their proper cellular function, particular responses are determined by effectors such as transcriptions factors who respond to external signals resulting in the modulation of gene expression, thereby tightly controlling physiological function [4]. Amongst the responses from different transcription factors, in recent years the Krüppel-like factor (KLF) family of transcription factors have acquired much attention for their roles in development and functional maintenance of the kidney [5].

Briefly, the Krüppel-like factor (KLF) family is a 17-member family of transcription factors characterized by three zinc fingers (Cys2/His2) with highly conserved C-terminal domains [6]. Regarding renal function KLFs have been associated with several biological processes including differentiation, terminal maturation, as well as the maintenance of the structure and function of the glomerular filtration barrier, protecting glomerular endothelial cells and podocytes from inflammatory damage and the development of fibrosis [7]. Members such KLF10 as have been associated with the promotion of inflammation and cellular proliferation by positively regulating pro-inflammatory cytokines and activating pathways that induce cellular growth [8].

The structural variations in their regulatory domains determine the variety of roles that KLFs play in renal biological processes. Through their interactions with various coactivators and/or corepressors, these domains enable them activation or repression of the promoter activity of their target genes [9]. Interestingly, various dietary, hormonal, and neural signals can modulate KLF expression, particularly when in an altered state. This dysregulation in their expression affects a variety of functions in which they are involved. For this reason, the dysregulation of KLFs can lead to serious health issues [10].

This article aims to provide an overview, based on a comprehensive review, of the most relevant findings related to the involvement of KLFs in various biological processes of the kidney. We emphasize KLFs during renal development, their role in regulating metabolic processes of essential macronutrients such as glucose and lipids, as well as their implications in the onset and progression of significant renal diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), and diabetic kidney disease (DKD), highlighting those KLFs that exhibit renal-protective effects.

2. Krüppel Like Factors

Krüppel-like factors make up a family of zinc-finger motif DNA-binding transcription regulators. Their name derives from the initial discovery of Krüppel protein in Drosophila melanogaster in 1993. A protein which participates in regulating fly embryo segmentation [11]. In humans, the KLFs are consisting of 17 members, which, as mentioned, exhibit three highly conserved Cys2His2-type zinc finger motifs in their carboxyl-terminal regions. Each zinc finger maintains a fixed length of 23 or 21 amino acids for their motifs [12]. The DNA-binding domain structure, evolutionarily conserved, shows structural homology with Specificity Proteins (SP), which leads to considering KLFs as a subgroup of the SP/KLF family [13]. These motifs allow binding to both the GC-rich proximal promoter regions and the CACCC elements (GT boxes) within the promoter regions of multiple genes [14].

The phylogenetic features that mark the evolutionary distance amongst KLF family members, coupled with specific structural traits in their less conserved amino-terminal regions, result in the division of KLFs into three distinct groups [15]. The first group is composed of KLF3, KLF8, and KLF12, which harbor a Pro-X-Asp-Leu-Ser (PXDLS) motif, where X represents a hydrophobic amino acid. This motif facilitates interaction with the amino-terminal substrate binding domain (SBD) of C-terminal binding proteins (CtBP), aiming to repress transcription [9,15,16,17]. The second group includes KLF1, KLF2, KLF4, KLF5, KLF6, and KLF7, who, except for KLF7, along with KLF8 from group 1 and KLF15 (the latter by homology), possess a transcriptional activation domain (TAD) in their N-terminal regions [9,16]. Both KLF1-2, KLF4-6, and KLF13 (the latter from group 3), can interact with histone acetyltransferase enzymes such as cAMP response element-binding protein (CREB) binding protein (CBP), p300, and p300/CBP-associated factor, facilitating chromatin remodeling and promoting transcriptional activity in DNA regions regulated by KLFs [9,16,18,19]. The binding of KLF1, KLF4, KLF5, and KLF11 (the latter from group 3) to histone deacetylase enzymes suppresses their transcriptional activity. Therefore, this second group of KLFs can modulate both activation and re-pression of gene expression at the transcriptional level depending on a biological context and the gene regulatory region through which they are operating [20]. Lastly, in the third group, members KLF9, KLF10, KLF11, KLF13, KLF14, and KLF16 contain a Cabut domain in their N-terminal section that encompasses a Sin3 interaction domain (SID) and their activities as transcriptional repressors [7]. Both KLF15 and KLF17 are not classified within these three phylogenetic groups because their protein interaction domains have not yet been determined. Finaly, researchers have identified that various KLFs also contain nuclear localization signals (NLS) and nuclear export signals (NES) which regulate their subcellular localization, as illustrated in Figure 1.

3. KLFs in Kidney Physiology

The kidneys play essential roles in maintaining homeostasis in the body, mediated by hormonal signaling processes and their interaction with transcription factors [8,21]. One family of transcription factors involved in regulating renal function is the KLFs [8,22,23], which are expressed in various parts of the nephron, contributing to both its structure and cellular composition [24]. The subsequently these two fundamental aspects of kidney's cellular biology provide a better understanding of the role of KLFs and their influence on determining cellular function and behavior.

Kidneys are divided into 2 specific regions: the renal cortex and medulla. Between these two regions are the nephrons, which are considered the functional and structural units of these organs, composed of the glomerulus and the tubular system [1,25].

Surrounded by a cup-shaped structure called Bowman's capsule, the glomerulus is where blood filtration occurs. The parietal epithelial layer is composed of parietal cells that play a structural maintenance role [1], while the visceral layer is formed by podocytes, which are perivascular cells enveloping the outer layer of the basal membrane of the glomerular capillaries (GC), together forming the glomerular filtration barrier (GFB) [26,27]. The integrity of this barrier, maintained by tight junctions between epithelial and endothelial cells, is the mechanism that ensures proper glomerular filtration. Occludin is one of the junction proteins expressed in podocytes, and its expression is downregulated by KLF2 to prevent its overexpression from altering the structure of the glomerular basement membrane (GBM) [28].

KLF4 collaborates with histone deacetylases (HDACs), specifically HDAC1 and HDAC3, to upregulate the expression of E-cadherin, podocin, and nephron [29]. E-cadherin is another adhesion molecule [30], while podocins and nephrin are crucial for the formation and maintenance of the slit diaphragm (SD), a specialized adhesion structure of podocytes [29,31,32]. KLF4 also induces the expression of cytokeratin 8 (K8) and K18, which, like podocins, participates in cytoskeletal organization [29]. Together, the expression of these proteins maintains the structure and functionality of podocytes by modulating cellular adhesion and polarity.

Maintaining cell adhesion and polarity is crucial to prevent acquisition of mesenchymal characteristics. KLF4 is also involved in downregulating mesenchymal markers, such as vimentin and α-smooth muscle actin (α-SMA) in podocytes [29]. Vimentin is expressed in mesenchymal cells, which generally lack both intercellular adhesion and polarity, providing resistance to migration-related stress [33]. In contrast, α-SMA contributes to motility and contraction of the cytoskeleton [34], allowing greater cell mobility and the ability to migrate to sites of injury or inflammation. Therefore, KLF4 prevents structural damage and the progression of diseases by inhibiting epithelial-to-mesenchymal transition (EMT).

In GC, which are made up of fenestrated endothelial cells (ECs) possessing transcellular pores of 60 to 100 nm in diameter, selective permeability for molecules according to their size and charge is achieved [1,35]. KLF2 contributes to regulating the size and distribution of these pores, preventing uncontrolled solute permeability by inhibiting the phosphorylation of the myosin light chain, thus avoiding the contraction of the cytoskeleton that would reduce the size of the ECs and lead to the formation of gaps between them [28]. Another way to maintain proper glomerular filtration is through the modulation of angiogenesis mediated by VEGF-A, where KLF2 downregulates its expression. This prevents the excess of unnecessary or dysfunctional blood vessels that could disrupt hemodynamic balance and affect blood pressure [36].

The role of KLF4 in ECs is related to mediating inflammation by downregulating adhesion molecules, such as VCAM1 induced by TNF-α. This is achieved by inhibiting the expression of the p65 subunit of NF-κB, which is required for the activation of this transcription factor and its binding to the VCAM1 promoter. In this way, KLF4 modulates the adhesion and recruitment of lymphocytes to endothelial cells, preventing chronic inflammation [37,38].

The GC also depends on mesangial cells to support their structure, as they lack an interstitial tissue lining [39]. These cells collaborate in regulating blood pressure and fluid balance because of their plasticity, which gives them the ability to produce renin and adapt to environmental changes [40]. In these cells, KLF4 attenuates the expansion of the mesangial matrix and their proliferation through the negative regulation of the mTOR pathway by downregulating the expression of phosphorylated (p) mTOR and pS6K proteins. This protects the kidney from mesangial cells, which produce extracellular matrix by secreting type IV and type V collagen and fibronectin from the mesangial matrix, from producing it in excess, which could lead to the development of fibrosis-related diseases [41].

Between the parietal layer and the visceral layer of Bowman’s capsule, a urinary space known as the "Bowman space" is formed. This space represents the beginning of the urinary system and is contiguous with the proximal convoluted tubule (PCT), in the renal cortex [42]. The PCT, which is the first segment of the nephron's tubular system, receives glomerular ultrafiltrate through epithelial cells (EpC) that allow permeability to both water and luminal fluid, reabsorbing glucose, amino acids, and minerals such as phosphate, chloride, and bicarbonate, as well as secreting hydrogen ions and toxins produced by cellular metabolism and xenobiotics present in the filtrate [2]. The expression of KLF4 and KLF11 mitigates inflammation and fibrosis by decreasing the expression of cytokines MCP-1, MIP-3α, and IL-8 induced by TGF-β1. This is possibly related to the phosphorylation of KLF4 that induces SMAD and p38/MAPK signaling pathways in vascular smooth muscle cells (VSMCs) [43,44] or through binding to p65 and inhibiting NF-kB signaling. The decrease of these inflammatory cytokines limits the production of collagen type I and tissue fibrosis [45].

KLF15 in these EpC can decrease the expression of these fibrogenic components by negatively regulating the MAPK pathways, which, when activated, contribute to the production of TGF-β1 and other pro-fibrogenic factors. Thus, KLF15 also modulates the fibrogenic response and helps prevent the accumulation of an extracellular matrix [46].

The specific expression of genes within each segment of the renal tubule determines its respective role [24], unfortunately, given what we know, the expression and function of KLFs in the subsequent segments of the nephron's tubular system remain incompletely understood. Following the proximal PCT, there are several segments, one of which is the loop of Henle. The Henle's loop aids in the concentration of urine by passively reabsorbing water into the medullary interstitium. It reabsorbs approximately 25% of sodium chloride, chloride, and potassium, all of which are later expelled alongside hydrogen ions. The distal convoluted tubule performs the functions of reabsorbing sodium, potassium, and chloride, besides secreting hydrogen and potassium ions. Finally, the cortical collecting tubule, a specific region of the collecting duct, connects to a collecting duct that drains into the renal papillae. This segment reabsorbs the same electrolytes as the distal convoluted tubule [1,42].

Therefore, the main function of KLFs in the kidneys can be briefly summarized as the preservation of structure, cell adhesion, the glomerular filtration barrier, and regulating the extracellular matrix (ECM) and inflammation of the cell lineage that composes them. This has the effect of modulating glomerular filtration, secretion, and elimination of unnecessary toxins from the body. Figure 2 illustrates the basic structure of the nephron, along with the location of its main cell groups that integrate the specific gene expression of KLFs within it. Table 1 summarizes the specific functions of each of the KLFs involved in the kidney's functional regulation processes.

3.1. Klf in Kidney Development

The process of organogenesis involves the division and organization of cells to establish the foundational structures of the embryo. Cell cycle arrest plays a crucial role in this process as it enables the progression towards terminal differentiation, which is a vital step in acquiring specialized functions [47].

The intricate progression of the cell cycle and cellular differentiation at a molecular level is regulated by signaling pathways and transcription factors [48]. This principle highlights the importance of the KLF family in contributing to kidney development. In embryonic cell cycle regulation, in vitro studies suggest Klf5 promotes podocyte survival by blocking the mitogen-activated protein (MAP) kinase ERK/p38 pathway, as well as decreasing the expression of apoptosis-related proteins such as Bax, caspase-3, caspase-8, and caspase-9, while increasing the expression of the antiapoptotic protein Bcl-2, which inhibits both cell cycle arrest and apoptosis by preventing Bax from translocating to the mitochondria [49,50,51]. In contrast, the inhibition of Klf5, along with other proteins such as Stat5a/b and ICAM-1 through pharmacological agents, has been associated with a positive regulation of the Bax/Bcl-2 ratio. This relationship regulates apoptosis; specifically, the inhibition of Klf5 induces an increase in the expression of Bax, which translocate to the mitochondria, promoting the release of cytochrome c and consequently the activation of caspase-3 and PARP, which are key proteins in the apoptotic process. The expression of Bcl-2, is not significantly affected during KLF5 downregulation [52]. KLF6 has been associated with maintaining mitochondrial function and preventing cell death in podocytes. This is because of KLF6's ability to bind to the promoter region of the cytochrome c oxidase assembly gene (SCO2) and positively regulate it. Continuous expression of SCO2, a metallo-chaperone, is crucial for transporting copper ions to electron carriers in the mitochondrial electron transport chain. This process, including cytochrome c, is vital for preventing the activation of the intrinsic apoptotic pathway in podocytes. By doing so, it avoids any harm to the glomerulus and maintains its filtration capacity [23].

The complete maturation of the nephrons occurs postnatally and is essential for the kidney to develop its maximum urinary concentrating ability. Various mechanisms are outlined below to illustrate how both KLF12 and KLF15 are fundamental in this process. KLF15 acts as a negative regulator of the chloride channel ClC-K1, which is expressed in the EpC thin ascending limb of the loop of Henle during postnatal development. This KLF15/ClC-K1 repression prevents the formation of channels that facilitate the passage of chloride into the urine, avoiding dysregulation in the organism's electrolyte balance, since the concentration of chloride in urine directly affects its osmolarity [22]. KLF12 is also overexpressed between 15 and 22 days after birth in the EpC of the medullary collecting ducts (IMCD) undergoing maturation. The specificity of KLF12 expression was determined by comparing it with the expression of aquaporin 2 (AQP2), which is upregulated in the IMCD and shares similar DNA-binding sites to those of the KLFs. The co-localization of KLF12 and AQP2 suggests KLF12 plays a key role in the positive regulation of AQP2 expression and its target gene, the urea transporter (UT-A1) [53,54]. This positive regulation of KLF12/AP-2/UT-A1 influences the transport of water and urea into the IMCD, resulting in the accumulation of urea necessary for proper urine concentration [55]. Figure 3 illustrates the involvement of KLFs in regulating renal organogenesis and postnatal maturation of nephrons.

KLF15 has been also associated with podocyte differentiation, since it upregulates the expression of nephrin, podocin, synaptopodin, and Wt1; all essential genes for maintaining a differentiated phenotype and preventing the loss and detachment of podocytes [26,56]. Meanwhile, KLF4 regulates the differentiation of specific nephron segments and individual cell types by cooperating with p53 and CREB, as evidenced by the p53-CRE-KLF binding sites in the promoter regions of renal function genes, such as AQP2, bradykinin receptor B2 (B2R) and epithelial sodium channel (ENaC) during terminal nephron differentiation [8,48,57].

To summarize, KLFs are vital for the proper development, structure, and function of the glomerular filtration barrier. They safeguard endothelial cells (ECs), facilitate the differentiation of podocytes, maintain their specialized integrity, and regulate cell cycle and apoptosis.

3.2. Klf in Kidney Metabolism

Metabolic processes enable the kidney to use, produce, and reabsorb nutrients, fulfilling its own energy demands while also ensuring homeostasis. To regulate these metabolic processes effectively, transcription factors and coactivators are involved in modulating the expression of genes that code for the enzymes involved. This ensures the proper occurrence of these processes.

Transcription factors such as KLFs have been closely associated with regulating metabolism, especially in the hepatic context [58,59,60,61,62,63,64], where they regulate genes involved in lipid metabolism such as CREB, Carbohydrate Response Element Binding Protein (ChREBP), Sterol Regulatory Element Binding Protein 1 (SREBP-1), Peroxisome Proliferator-Activated Receptor (PPAR). Moreover, they further regulate genes involved in glycolytic metabolism such as Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1 Alpha (PGC-1α) [58,65,66,67] because they share the same DNA binding sites in conserved CACCC sequences and GC-rich elements [68].

The variations in metabolic activity among KLFs remain unexplored. However, in vitro studies have shown that KLF6 overexpression in proximal tubular cells (Hk-2) exposed to high glucose concentrations increases the expression of the protein that interacts with thioredoxin (Txnip) [69]. This protein is particularly intriguing due to its involvement in glucose oxidation, which is vital for energy acquisition. One of the most significant factors is related to the glucose transporter GLUT1. It is regulated by Txnip and could influence its localization by directly binding to the transporter. This binding induces internalization through clathrin-coated pits [70]. Therefore, its expression suggests that peripheral glucose uptake and utilization of this substrate could be compromised, leading to metabolic disturbances in conditions of high and persistent glucose consumption, such as in diabetes mellitus. Thus, silencing KLF6 may represent a therapeutic target, as its inhibition significantly attenuates Txnip expression and mitigates metabolic consequences.

Within the Hk-2 cellular model, KLF14 is an important player in lipid metabolism. The lack of KLF14 has been linked to a decrease in mitochondrial activity by reducing the expression of PPARα, resulting in the suppression of target genes involved in fatty acid transport. One example of such a gene is FATP1, which is found in the cell membrane and helps in the absorption and incorporation of lipids. Furthermore, it inhibits the expression of CPT1, an enzyme that facilitates the transfer of acyl groups from acyl-CoA to carnitine in the intermembrane space of the mitochondria, leading to the production of acyl-carnitine esters. By utilizing this process, the inner mitochondrial membrane allows for the transportation of long-chain fatty acids. In the next step, CPT2 takes over and transfers the acyl groups from acyl-carnitine back to CoA, ultimately leading to the restoration of acyl-CoA. Subsequently, this acyl-CoA can be metabolized through a series of enzymatic reactions within the β-oxidation pathway of fatty acids [71,72].

Thus, the downregulation of PPARα causes the accumulation of lipids inside cells, a process that can occur in a wide range of renal cell types, including mesangial cells, podocytes, and proximal PCT [73]. The latter cells, which rely on fatty acid oxidation for energy production, are the ones that this applies to [74]. Renal damage can occur when lipids accumulate in cells that are not meant for storage. The build-up of this substance promotes lipoperoxidation, which generates reactive oxygen species (ROS), leading to oxidative stress and contributing to the development of different diseases like acute kidney injury (AKI) and fibrosis [75]. Nevertheless, the overexpression of KLF14 counteracts these effects by enhancing mitochondrial activity, reducing lipogenesis, and decreasing lipid accumulation. In experiments conducted on living subjects, it has been found that KLF15 can positively regulate both CPT1 and Acyl-CoA Acyltransferase 2 (ACAA2) by tightly binding to the PPARα binding sites [76]. The closeness of DNA suggests that KLF15 and PPARα may coordinate in governing the expression of these genes.

Thus, it is crucial to have high levels of both KLF14 and KLF15 to ensure sufficient energy supply, specifically to the proximal tubular cells, by utilizing lipids through fatty acid oxidation. Figure 4 illustrates the implication of KLFs in renal metabolism both under healthy conditions and their effect on states of dysregulation.

3.3. Klfs in Kidney Disease

When the expression of KLFs is dysregulated, it can disrupt various physiological processes in which they are involved. This disruption can cause poor management of inflammation, tissue repair, regeneration, and other cellular adaptations to stress.

Throughout this review, it has been discussed that KLFs demonstrate distinct patterns of expression in different renal cells, greatly influencing their various functions in maintaining overall balance within the body [77]. The dysregulation of KLFs is associated with kidney diseases, including CKD, AKI, and DKD, where the pathophysiology affects the balance of glomerular, tubular, and inflammatory functions, and this variability is commonly observed. Take into consideration that specific KLFs have protective properties for the kidneys and blocking them frequently leads to the deterioration and advancement of the disease. Conversely, the disease can also be exacerbated by the excessive expression of other KLFs.

It is important to mention that AKI, which is characterized by a sudden decrease in kidney function, has the potential to progress to chronic injury and eventually develop into CKD [78]. In this context, KLF2, KLF4, KLF9, KLF10, and KLF15 have beneficial effects [36,79,80,81,82] while KLF5 promotes cell proliferation, tubular damage, and inflammation [83]. Various pathological conditions, such as diabetic nephropathy, which is characterized by albuminuria and progressive renal insufficiency [84], precipitate the development of CKD. Here, the KLFs that stand out are primarily KLF3 and KLF6, which induce inflammation and fibrosis and facilitate epithelial-mesenchymal transition, respectively. Ultimately, all these diseases converge on a common outcome of renal fibrosis, characterized by excessive deposition of ECM [85,86]. The activity of each KLF in these three diseases is detailed in Table 2.