Part 1 Heme Oxygenase 1: A Defensive Mediator in Kidney Diseases

Part 1

Anne Grunenwald ¹, Lubka T. Roumenina ¹ and Marie Frimat ^2,3,*

Abstract: The incidence of kidney disease is rising, constituting a significant burden on the healthcare system and making the identification of new therapeutic targets increasingly urgent. The heme oxygenase (HO) system performs an important function in the regulation of oxidative stress and inflammation and, via these mechanisms, is thought to play a role in the prevention of non-specific injuries following acute renal failure or resulting from chronic kidney disease. The expression of HO-1 is strongly inducible by a wide range of stimuli in the kidney, consequent to the kidney’s filtration role which means HO-1 is exposed to a wide range of endogenous and exogenous molecules, and it has been shown to be protective in a variety of neuropathological animal models. Interestingly, the positive effect of HO-1 occurs in both hemolysis- and rhabdomyolysis-dominated diseases, where the kidney is extensively exposed to heme (a major HO-1 inducer), as well as in non-heme-dependent diseases such as hypertension, diabetic nephropathy, or progression to end-stage renal disease. This highlights the complexity of HO-1’s functions, which is also illustrated by the fact that, despite the abundance of preclinical data, no drug targeting HO-1 has so far been translated into clinical use. The objective of this review is to assess current knowledge relating to HO-1’s role in the kidney and its potential interest as a nephroprotection agent. The potential therapeutic openings will be presented, in particular through the identification of clinical trials targeting this enzyme or its products.

Keywords: heme-oxygenase-1; heme; kidney; hemolysis; rhabdomyolysis; toxicity; ischemia-reperfusion

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1. Introduction

The number of people worldwide with chronic kidney disease (CKD), acute kidney injury (AKI), or requiring renal replacement therapy exceeds 850 million [1]. The forecasted incidence is a cause for concern, with CKD projected to become the world’s fifth leading cause of death by 2040 [2]. It is generally accepted that kidney diseases are a significant public health issue and that the need for a multimodal approach to curb this presumed evolution is urgently needed [3]. Of key importance is the identification of novel therapeutic targets, both to limit the impact of acute renal events and to ameliorate poor outcomes in CKD, across the range of underlying etiologies. Several candidates for nephroprotection are currently being studied [4,5]. The heme oxygenase (HO) system is one of the targets that could be used to protect kidney structures from damage caused by oxidative stress, limiting inflammation and consequently accelerated aging.

HO was discovered in 1968 by Tenhunen and colleagues, who first described its ability to catalyze the breakdown of heme (Fe-protoporphyrin IX) to free iron, carbon monoxide (CO), and biliverdin, which is rapidly converted to bilirubin [6]. Two main isoforms of HO were described: inducible HO-1 and constitutive HO-2. Initially merely considered a recycling system for heme from aged red blood cells, HO has since been attributed a number of cytoprotective properties via its metabolites and downstream signaling, expanding its definition from that of a “molecular wrecking ball” to a “mesmerizing” trigger of cellular events, to quote Maines et al. [7]

Driven by its potential antioxidant and anti-inflammatory capacities, HO-1 is the subject of growing interest for its role in different human pathologies. Among the studied organs, the kidney has often been highlighted over the last 30 years, notably through the identification of a role for HO-1 in various renal, metabolic and vascular diseases of both an acute and chronic nature [8–11]. HO-1 is essential for efficient kidney function, as illustrated by kidney damage in humans with HO-1 deficiency and by numerous animal studies (described below). The kidney’s physiological functions specifically influence HO-1 regulation. Indeed, renal tissue is very susceptible to hypoxia [12], a major driver of HO-1 expression, and is particularly exposed to toxic molecules because of its filtration and reabsorption functions. This particularity of being a “detoxification” organ, shared with the liver, means the kidney is highly exposed to damage-associated molecular patterns (DAMPs) and stress mediators. Interestingly, the kidney possesses a capacity for the synthesis of albumin, alpha protein, and haptoglobin under ischemic or toxic ischemic damage, underlining the parallel with the liver [13,14]. Renal cells, therefore, require adaptation strategies to preserve their functions and integrity in such a harsh environment. The kidney is a major site of exposure to heme in the event of extravascular hemolysis or rhabdomyolysis, and hence also for the induction of HO-1 expression. Under these conditions, HO-1 overexpression is easily explained by the need to catabolize the free heme overload. However, HO-1’s involvement in kidney diseases goes beyond the framework of heme-related diseases, illustrating the complexity of its mechanisms of action which extend beyond heme catabolism alone [15].

The aim of this review is to summarize the large amount of experimental and clinical data describing the importance of HO-1 in the kidney, without forgetting that HO-2 is also significant: constitutively expressed in the kidney under homeostatic conditions, it is in the first line of defense against ischemia and other insults, and its renal cytoprotective effects have been demonstrated in heme protein- and ischemia-induced AKI [16]. Compared to the literature about HO-1, however, there are very few studies examining the functional significance of HO-2 in kidney diseases; therefore, this review will focus on HO-1. After a general introduction to the characteristics of HO-1 in the kidney, we will describe the role of this enzyme in different renal cell populations and in kidney diseases. Given its primary function of recycling heme, we will approach these pathological states according to whether or not there is a massive release of free heme. Finally, current clinical applications will be presented, revealing the disparity between the profusion of available experimental data and the relative paucity of therapeutic applications.

2. About Heme Oxygenase-1 in Kidney

2.1. Why Is HO-1 Particularly Important for the Kidney?

Over the past few decades, the key role of the HO-1 system in the pathophysiology of kidney diseases has been supported by several studies. In the two HO-1-deficient patients reported up to date, the kidney was among the damaged organs. Both patients had hematuria and proteinuria. Kidney biopsies revealed increased mesangial proliferation and focal thickening of the capillary loops by light microscopy, and endothelial detachment in the glomerular capillary by electron microscopy [17,18]. Renal injuries were also present in both animal models invalidated for HMOX1, with some differences apparent between the mouse [19] and rat models [20]. In Hmox1-/- mice, lack of functional heme oxygenase was responsible for enhanced susceptibility to oxidative stress [19], decreased hemophagocytosis of senescent red blood cells by tissue macrophages, increased hemolysis, and redistribution of iron from splenic and hepatic macrophages to hepatocytes and renal proximal tubule cells [21]. Similarly, Hmox1-/- rats exhibited anemia, splenomegaly, and prominent interstitial inflammatory cell infiltrates and fibrosis scarring in their kidneys. While glomerular lesions were anecdotal in the mouse model, Atsaves et al. found an increase in mesangial matrix and focal and segmental glomerulosclerosis lesions in Hmox1-/- rats. These morphological findings were associated with increased blood urea nitrogen, serum creatinine, and albuminuria, but interestingly there was no increase in iron deposits in the glomeruli, tubules, or interstitium [20].

Spontaneous renal damage in both human and animal models invalidated for HMOX suggests that HO-1 is important for efficient renal function. The renal protective role of this enzyme has been confirmed in multiple models of renal challenge in HO-1 invalidation/inhibition or overexpression conditions [22]. However, the histological differences cited above illustrate the difficulties of transposing results from one species to another. Differences in the regulation of HO-1 between humans and mice are well described, motivating the generation of human hHO-1 BAC transgenic mice [23]. In this model, HO-1 is overexpressed, and it remains unknown as to whether this higher background expression may obscure subtle damage over long periods of time, or even have adverse effects itself.

A better understanding of how HO-1 functions in the kidney, and how its expression or function is controlled, is essential for our comprehension of kidney physiology and pathology but it is (as always) important to remember the limits of any study in its interpretation and translation: we will attempt to sustain this critical viewpoint throughout this review.

2.2. Regulation of HO-1 Expression in Kidneys

HO-1 protein is ubiquitous, being anchored in the membrane of the endoplasmic reticulum and also localized in mitochondrial nuclei and caveolin, while the HO-1 gene (HMOX1), which encodes HO-1, is located on Chromosome 22. Many stimuli can modulate the transcription of HMOX1, reflecting the broad spectrum of DNA-binding motifs within its promoter and the large number of signaling pathways that lead to its transcription. The main regulator of the HMOX1 gene is nuclear erythroid factor 2 (Nrf2), whose activation depends on its interaction with the Kelch-type, ECH-associated protein (Keap1). The Keap–Nrf2 interaction promotes the degradation of Nrf2 (healthy state), while destabilization of this complex releases Nrf2 which translocates into the nucleus and upregulates certain genes, including HO-1 (stressed state). There are many other activators or repressors of HO-1 forming a complex biomolecular network (detailed in previous reviews [7,10]). Details on these are beyond the scope of this review and we will here focus upon the renal-specific features of HO-1 expression.

In the kidney, HO-1 protein levels are undetectable under homeostatic conditions, except in the tubules where it nevertheless remains low [24]. Many stress conditions are known to up-regulate the renal transcription HO-1, including oxidative stress, heat shock, hypoxia, heavy metals, and toxins (reviewed in Bolisetty et al. [8]). Under these conditions, HO-1 expression remains heterogeneous among the different renal compartments. The kidney consists of two main areas: the output cortex and the medulla in the innermost region. The medulla contains most of the length of the nephrons, the functional components of the kidney that filter fluid from the blood. The renal cortex includes the glomeruli, those “tufts” of capillaries where plasma is filtered through the glomerular basement membrane. The filtered fluid then flows along the convoluted proximal tubule to the loop of Henle, and then to the convoluted distal tubule and collecting ducts, which drain into the ureter (Figure 1). Glomerular filtration and reabsorption of solutes (the kidney’s two major functions) are supported by renal blood flow, which is among the highest in the body, with kidneys receiving about 20 to 25% of cardiac output. Compared to the cortex, blood flow to the renal medulla is relatively low. This disparity is required to enable an effective urine concentrating mechanism and also explains the particular susceptibility of the medulla to decreased blood flow. Under stress conditions, HO-1 is thus more highly expressed in the medulla than the cortex [25]. Its expression is very strong in tubules (especially proximal tubules), but minimal or absent in glomeruli, as illustrated in Figure 2 in both hemolytic patients and heme-injected mice. However, it has been shown that prior induction of HO-1 within the glomeruli prevented subsequent development of nephrotoxic glomerulonephritis in a Lewis rat model, but the major producer of HO-1 in the glomeruli was the infiltrating macrophages, not the intrinsic glomerular cells [26]. This lower capacity of glomeruli to express HO-1, which is a key cytoprotective mechanism, could explain glomeruli’s particular sensitivity to stressors in certain pathologies [27].

Taken together, these data show that HO-1 levels are normally undetectable in the kidney but that HO-1 is highly inducible in tubules during pathologies, notably via its main transcription factor Nrf2. The capacity of glomeruli to induce HO-1 is lower, however.

2.3. HO-1 and Nephroprotection

Like HO-2, HO-1 drives the NADPH-dependent addition of an oxygen molecule to the porphyrin ring of heme, thus catalyzing the oxidation of heme and the equimolar release of biliverdin, free iron, and carbon oxide (CO). All these events are cytoprotective by removing free heme, regulating iron efflux, and increasing bilirubin and CO levels. Indeed, the noxious effects of free heme are well described, particularly at the vascular site via direct toxicity, but also through the stimulation of pro-inflammatory signaling pathways (such as DAMPs) and the activation of the complement system [28]. Similar to endothelial cells, such adverse effects are reported with other cell types when massively exposed to heme. This is the case for human skeletal muscle fibers, in which exposure to heme induces contractile dysfunction [29], and renal tubular cells [30]. In addition, iron is notably toxic even at low concentrations. It participates in the Fenton reaction, producing hydroxyl, and can result in cell death by ferroptosis, a cell-death pathway characterized by an iron-dependent decrease in glutathione (GSH) levels and the accumulation of lipid hydroperoxides to lethal levels [31]. Thus, it has been reported that the cytoprotection of HO-1 was due, at least in part, to an increased iron flux [32], which following its release is either stored in ferritin or exported from macrophages to plasma for re-use via the transmembrane ferrous exporter ferroportin. CO counteracts vasoconstriction and has powerful anti-oxidant properties, as does biliverdin, which is enzymatically converted to bilirubin via biliverdin reductase [33].

Transposing these properties to the renal level, it is easy to understand research interest in harnessing this molecule in order to treat kidney disease. The positive effects have been described for almost 30 years, following the work of Nath et al., who reported that HO-1 conferred protection in a rat model of glycerol-induced, acute renal failure. In their model, a single prior injection of hemoglobin rapidly induced HO-1 messenger RNA and protein within the kidney, which prevented progression to renal failure [34]. The nephroprotective effects of HO-1 and its metabolites have since been reported in different models of renal disease [8,35]. The potential importance of HO-1 in the pathogenesis of kidney diseases is also suggested by the link between polymorphisms in the HO-1 gene and renal outcome. Individuals with shorter (GT)n repeats in the HO-1 promoter region have a higher transcriptional activity and thus higher HO-1 levels than individuals with longer (GT)n repeats [36]. Interestingly, long (GT)n repeats have been associated with a worse prognosis in several contexts, namely, a higher risk for AKI after cardiac surgery or decreased renal function after kidney transplantation [37,38]. However, there is no evidence of a protective effect of short repeat (GT)n for graft or recipient survival following renal transplant [39], underlining the complexity of the mechanisms involved. The levels of HO-1 expression may also vary with age. The renal expression of gene HO-1 in control C57Bl6 mice were significantly decreased at 18 compared with 3 months of age [40]. In another study, protein HO-1 expression was not significantly different at baseline between 6–8-week-old mice and 1-year-old mice, but the older animals’ ability to upregulate HO-1 in response to ischemia-reperfusion injuries (IRI) was impaired (especially in the medulla), and they exhibited worse renal function compared with young animals [41]. These results echo the renal sensitivity of elderly humans to nephrotoxic agents.

To summarize, the preventive or genetic induction of HO-1 expression has been shown in many studies to confer renal protection. A decrease in renal HO-1 expression may contribute to the kidney’s sensitivity to certain pathologies.

3. HO-1 in Renal Cell Populations

Comparative analyses have revealed that HO-1 is not expressed to the same extent in the distinct cell populations of the kidney for a given stimulus (Figure 1). During intravascular hemolysis or in the presence of HO-1-inducing stress stimuli or drugs in the circulation, the endothelial cells are the first cells to be challenged by these systemic stressors. If the stressor is filtered in the glomeruli, then the podocytes are exposed and may be damaged, as seen in diabetic nephropathy. Further, the stressor could encounter mesangial cells which have a secretory and immunomodulatory role and proliferate during HO-1 deficiency. Finally, the stressor may reach the tubular epithelium which is the most- studied cell type in relation to HO-1 (the proximal and distal tubules must be considered separately with respect to the epithelium). Tubular epithelial cells have the most potent capacity to overexpress HO-1 and suffer the most severe injury in case of its deficiency or during intravascular hemolysis and rhabdomyolysis. This section describes the key functions of the main kidney cell types and the specific role of HO-1 within them.

3.1. Endothelial Cells

The endothelium is comprised of a monolayer of endothelial cells (EC) which accomplish critical functions in the blood vessels. They cover the interior of blood vessels and perform barrier functions, but they also serve as an exchange interface with adjacent tissues, a sensor for tissue and intravascular stress, and as a source of defense mediators [42]. As such, EC are the first line of cells to encounter circulating stressors and HO-1 expression is essential for their survival, with HO-1-deficient patients and murine models exhibiting extensive endothelial damage [19,43]. The pro-survival effects of HO-1 in EC occur in every organ and there exists a large body of work regarding its induction by various molecules [44,45]. According to its localization (in different organs, in vessels or capillaries), EC structure, specialization and functions vary. Thus, EC from different vascular beds have dynamic expression profiles (both spatially and temporally) and this might explain their selective involvement in different disease processes [42].

In the kidney, three main types of the endothelium can be distinguished: glomerular, peritubular capillary, and medium/large vessel endothelium [46], each with different levels of HO-1 expression or function. Glomerular EC is “fenestrated” and covered by a glycocalyx [47] responsible for restricting macromolecule passage to the urinary chamber [48]. Despite the glycocalyx, small hydrophobic molecules (e.g., heme) can enter cell membranes and activate proinflammatory and prothrombotic pathways, including TLR-4 signaling, thus promoting blood cell adhesion and vaso-occlusion (Schaer et al., 2013). Peritubular capillaries’ EC is also fenestrated and lays upon a thin stroma. They transport reabsorbed components, sustaining tubular epithelial cell function [46]. Finally, the endothelia of both medium and large vessels are contiguous, with interconnected EC. The structural and functional diversity of EC in the kidney as a function of their localization makes them differentially sensitive to specific stressors, such as toxic or proinflammatory filtrated molecules for glomerular EC, hypoxia for peritubular capillaries, or changes in shear stress, with correspondingly specific responses to such assaults [46].

The main factors affecting HO-1 expression in EC are hypoxia, shear stress, the presence of heme, and other circulating stressors (ammonia [49], S-adenosyl methionine [50], etc.), circulating substances (statins [51], resveratrol [52], etc.) or drugs. Oxygen partial pressure decreases along the peritubular capillary, notably under stress conditions with excessive O2 consumption. Interestingly, HO-1 expression has been reported to decrease upon hypoxia while HO-2 expression was preserved in macrovascular human umbilical vein EC (HUVEC) [53]. However, HO-1 expression is inducible in microvascular peritubular capillaries’ EC under different conditions that may provoke hypoxia (heme, Angiotensin II, etc.). Physiological shear stress is a major determinant of EC survival, as shown in vivo in human carotid atherosclerotic plaques [54] and rabbit carotids with reduced blood flow [55]: it also induces the expression of anti-inflammatory factors (in vitro in HUVEC and in vivo in rabbit aorta [56]) and vasodilatory ones (in vitro and in vivo in mice [57]). Interestingly, HO-1 expression is induced by shear stress, in vitro in macrovascular (aorta) and microvascular (HMEC) human EC [58], and this expression was reported in rats to be directly related to the level of shear stress: higher expression at high flow levels (in arteries), but requiring other factors for HO-1 to be expressed at low flow levels (in microvessels) [59]. This phenomenon has so far only been reported in intestinal vessels. However, if these results are verified in other organs, it may explain decreased HO-1 induction in capillaries such as glomerular EC under stress [27]. EC is highly exposed to cytotoxic hemolysis-derived products and endothelial responses to these insults reflect the heterogeneity of EC. Microvascular EC, and particularly glomerular EC, are less prone to up-regulate HO-1 compared with macrovascular EC, both in vitro and in a mouse model of hemolysis [27]. This renders them particularly susceptible to injury through differences in their complement C3 regulation and heme degradation as compared with macrovascular EC [27]. Little is known about the HO-1 induction capacity of the peritubular capillaries’ EC.

Renal macrovascular EC remains poorly studied with respect to HO-1 expression and function. The most widely used model in the study of macrovascular EC is the human umbilical vein EC (HUVEC). Exposure to heme in HUVEC is responsible for rapid signal transduction, mobilization of the endothelial Weibel Palade bodies, and NF-kB activation, thus conferring them a pro-inflammatory and pro-thrombotic phenotype [60–62]. However, longer exposure allows them to develop adaptation mechanisms and become highly resistant to oxidative-stress-mediated injuries and lipid peroxidation products. These include the induction of ferritin and HO-1 [63], though HO-1 induction in glomerular EC in vitro and in mouse models of hemolysis is much weaker [27,64]. HO-1 induction in macrovascular EC notably modulated complement activation [27,65], thrombomodulin expression in hemolysis conditions [27] and in the context of septic kidney injury [66], and expression of adhesion molecules associated with EC activation [67].

Taken together, these data indicate that the kidney endothelium is strongly influenced by toxins and stressors as a result of its filtration function. Such stressors seem to trigger strong adaptation mechanisms in macrovascular EC which allow them to resist insults to a certain level, mainly by the upregulation of HO-1 and its associated genes. While there is a marked lack of data in the literature, available studies suggest that, in contrast to macrovascular EC, this HO-1 upregulation is less pronounced in the glomerular microvascular endothelium, making it particularly sensitive to environmental stress.

3.2. Podocytes

Podocytes are hyper-specialized cells that maintain the glomerular filtration barrier through the synthesis of glomerular basement membrane (GBM) components and the formation of the slit diaphragm by attaching their foot processes to the glomerular endothelial cells [68] (Figure 1). Their strategic localization, enabling their filtration function, makes them particularly sensitive to mechanical, oxidative, and immunological stress. Podocytes have unique adaptive responses, including the upregulation of vimentin and desmin [69], or antioxidant proteins such as Sirtuin1 and metallothionein. Sirtuin upregulates Nrf2, thus enhancing HO-1 expression. [70,71] Interestingly, sirtuin-1 expression (together with Nrf2 and HO-1) has been reported as being increased by exposure to advanced glycation end-products [71], and HO-1 was shown to be specifically enhanced in podocytes in diabetes patients [72]. Although less studied, the expression of HO-1 has also been described under hemolytic conditions [27,73], with heme inducing HO-1 expression in an Nrf2-dependent manner in podocytes. Interestingly, Nrf2-deficient mice spontaneously developed proteinuria with foot process effacement decreased synaptopodin and nephrin expression, and podocyte apoptosis [73].

Though the literature on HO-1 in podocytes is not abundant, what evidence there is suggests a protective physiological role for the Nrf2/HO-1 axis by preserving pedicels and inhibiting the death and detachment of podocytes. In pathological contexts, HO-1 is overexpressed as a defense mechanism but could also be a byproduct of exhausted resistance mechanisms resulting from overwhelming stress, in which context it is insufficient to prevent podocyte injury.

3.3. Mesangial Cells

Mesangial cells, together with the mesangial matrix they produce, form the mesangium, a support tissue for the glomerulus floculus. Among mesangial cells are immune cells, monocyte/macrophage-like (5 to 15%), and contractile cells, which are similar to smooth muscle cells (85 to 95%) and provide structural support and contraction [74,75]. Heme promotes the proliferation of smooth muscle cells [76] and its receptor TLR4 is expressed on mesangial cells [77], but the role of HO-1 in these cells is poorly studied. Nitric oxide (NO) has been shown to induce HO-1 in mesangial cells [78], HO-1-deficient patients have a mild mesangial proliferation [17,18,43], and HO-1 has been shown to modulate mesangial cell proliferation via p21 upregulation [79]. Further work is needed to determine whether the large body of knowledge accumulated for macrophages is applicable for the macrophage-like mesangial cells.

3.4. Tubular Cells

If HO-1 expression was described in tubules some time ago, it is only recently that its regulation has been described as being segment specific; HO-1 also plays distinct roles in different segments of the tubule in order to maintain renal functions [80].

3.4.1. Proximal Tubule

The proximal tubules are the cells in the kidney which show the highest capacity for the overexpression of HO-1. Indeed, proximal tubular epithelial cells have been shown to be especially sensitive to oxidative stress in vitro [81]. They rely strongly upon HO-1 for protection from, and adaptation to, stressors, as evidenced by the fact that tubular injury is the cardinal pathologic feature in human HO-1 deficiency [82]. The main function of the renal proximal tubule is the uptake of water, ions, amino acids, and filtered proteins. In the absence of glomerular damage, filtered proteins are mostly of a size below 68kDa. Internalization of proteins, including heme-associated ones such as myoglobin or hemoglobin, but also hemopexin, occurs via the megalin and tubulin receptors [83]. This reabsorption function requires a large amount of energy (ATP) provided by mitochondrial beta-oxidation [84]. However, severe ATP depletion results in mitochondrial injury, causing further depletion of energy stores and the formation of reactive oxygen species [85]. This high metabolic de- mand produces a state of relative hypoxia in the kidney medulla, and the proximal tubules are particularly sensitive to ischemia/reperfusion and heme excess [86,87]. The sensitivity to heme is also explained by the constitutive expression of TLR4 (a heme receptor) by the proximal tubules [61], and TLR4 expression is increased in renal ischemia-reperfusion injury and septic injuries [77].

While HO-1 is expressed only weakly in the kidney under normal conditions, it is strongly inducible in proximal tubules under different forms of stress [88], proteinuria among them [80]. The interplay among the uptake of heme-containing proteins, osmotic pressure driving heme influx, TLR4 expression, and increased synthesis of heme in proximal tubules may together explain this specific, strong upregulation of HO-1. It has been suggested that HO-1 expression in tubular epithelial cells depends more upon basolateral exposure to heme (from the peritubular capillaries under hemolytic conditions) than intraluminal heme content. However, under conditions of hypoxia and of the altered polarity of renal EC, HO-1 upregulation has been reported to depend more upon apical exposure to heme [89]. In tubular epithelial cells, HO-1 upregulation by heme is dependent on Nrf2 stabilization [90]. HO-1 has a dual role in tubular EC, being simultaneously an anti-oxidative and a regulator of cell death. Indeed, HO-1 is responsible for the upregulation of p21, a cyclin-dependent kinase inhibitor responsible for stopping cell cycle progression, which reduces apoptosis in tubular EC [91,92]. Furthermore, HO-1 expression decreases ferroptosis in proximal tubular cells, a non-apoptotic regulated cell death associated with accumulation of reactive oxygen species (ROS) derived from lipid peroxidation [93]. Finally, HO-1 has been shown to inhibit autophagy in proximal tubular cells [94].

Altogether, proximal tubular cells are massively exposed to HO-1 inducers in pathological contexts (filtration of hemoglobin, myoglobin, hemopexin-heme complexes; ischemia, filtration of toxic products or drugs, etc.) and rely upon HO-1 for their protection, but HO-1 is also a marker of tubular stress and injury when their capacity for adaptation is overwhelmed.

3.4.2. Distal Tubule

Distal tubular EC along the medulla, notably in the thick, ascending limb of the loop of Henle (mTAL) and the distal convoluted tubule (DCT), are involved in the concentration and dilution of urine and the maintenance of homeostatic salt and solute levels by expressing different channels specific to their localization and cell subtype. During water reabsorption, the concentration of toxins (notably heme) becomes much higher in the distal nephron segments. Because of the low partial pressure of oxygen along the peritubular capillaries, these distal tubular EC are particularly vulnerable to hypoxia and rely more on anaerobic, glycolytic ATP production than proximal cells. Distal tubules have a greater ability to survive and adapt to hypoxic stress [95], being less sensitive to cell death, especially after ischemic injury [96].

Distal tubules, like proximal tubules, only weakly express HO-1 at a basal level; upon stimulation, however, its induction by heme has been called into question [88]. (Interestingly, HO-2 may be enhanced by heme in distal tubules [97].) However, HO- 1’s role in distal tubules remains to be fully elucidated as its expression has also been associated with improved renal function after ischemia/reperfusion in cadaveric donor transplantation [98], as well as in CKD [99]. This may be related to the regeneration function of distal tubular epithelial cells, but further studies are needed to better describe the relationship between HO-1 and distal tubular epithelial cells’ functions.

To conclude, while data regarding distal tubules are lacking, some evidence suggests that enhanced HO-1 expression could confer protection to distal tubules’ EC.