Latent transforming growth factor beta binding protein 4: A regulator of mitochondrial function in acute kidney injury

Abstract Recently, latent transforming growth factor beta binding protein 4 (LTBP4) was implicated in the pathogenesis of renal damage through its modulation of mitochondrial dynamics. The seminal article written by Su et al. entitled “LTBP4 (Latent Transforming Growth Factor Beta Binding Protein 4) Protects Against Renal Fibrosis via Mitochondrial and Vascular Impacts” uncovers LTBP4's renoprotective role against acute kidney injury via modulating mitochondrial dynamics. Recently, LTBP4 has emerged as a driver in the mitochondrial‐dependent modulation of age‐related organ pathologies. This article aims to expand our understanding of LTBP4's diverse roles in these diseases in the context of these recent findings.


| INTRODUC TI ON
Acute kidney injury (AKI) remains a global issue that can easily develop into chronic kidney disease (CKD; Jankowski et al., 2021).CKD can result in an increased risk for cardiovascular disease, in a way accelerating the aging of cardiac tissue (Jankowski et al., 2021).The transition from AKI to CKD is a critical tipping point for patients on the road to end-stage kidney failure (Fiorentino et al., 2018;Jankowski et al., 2021).AKI is defined as an abrupt decline in kidney function, lasting from hours to days (Fiorentino et al., 2018;Heung & Chawla, 2014).Once AKI has occurred, there are two possible fatesresolution or progression to CKD (Fiorentino et al., 2018;Heung & Chawla, 2014).While recurrent bouts of AKI independently increase the probability of patients developing CKD and progressing to endstage kidney failure (Fiorentino et al., 2018;Heung & Chawla, 2014), CKD sensitizes patients to further AKI (Hsu & Hsu, 2016).Since CKDassociated kidney damage is irreparable (Jankowski et al., 2021), there is a requirement for renal replacement therapy.As such, there is an urgent demand for effective therapeutic strategies that prevent or even stop the detrimental transition from AKI to CKD.However, to progress in the field, the driving mediators of this transition must first be understood.
Transforming growth factor-β (TGF-β) plays a pivotal role as a profibrotic growth factor in the transition from AKI to CKD (Su et al., 2021).While initially activated during AKI to facilitate kidney repair, prolonged TGF-β signaling drives cellular responses that lead to CKD onset and progression (Juban et al., 2018;Su et al., 2021).
As such, research endeavors aimed at targeting TGF-β signaling will provide valuable insight into the therapeutic feasibility of halting the AKI to CKD transition.
Latent transforming growth factor beta binding protein 4 (LTBP4) has recently emerged as a possible therapeutic target.
Recent reports identify LTBP4 as a novel regulator of TGFβ-1 and, thus, wider cytokine receptor signaling (Su et al., 2015(Su et al., , 2021)).This extracellular matrix glycoprotein is implicated in the pathogenesis of a plethora of fibrotic diseases (Su & Urban, 2021).Previously, Su and colleagues discovered that LTBP4 is upregulated in CKD and participates in resolving renal fibrosis (Su et al., 2021).Now, in a recent Circulation Research article, Su and colleagues expand these findings to AKI.Here, investigators demonstrated that LTBP4, via a mitochondrial-dependent mechanism, possesses a protective function in AKI (Su et al., 2023), further supporting that LTBP4 is renoprotective.Specifically, in loss-of-function LTBP4 mouse models, ischemia-reperfusion injury (IRI) increased dynamin-related protein 1 (DRP1), stimulated mitochondrial fission, and altered mitochondrial structure (Su et al., 2023).The resulting mitochondrial dysfunction, oxidative stress, and inflammation were implicated in the enhanced severity of fibrosis and the accelerated progression from AKI to CKD.Furthermore, this study revealed that targeting DRP1 was beneficial in slowing the progression to CKD and uncovered a potential therapeutic target for kidney disease (Su et al., 2023).However, this broad-reaching study has specific implications for our current understanding of oxidative stress, mitochondrial dynamics, and other potential pathways in which LTBP4 may be involved.

| Mitochondrial changes
Increasingly, reviews have focused on mitochondria as a target to prevent the shift from AKI to CKD (Ishimoto & Inagi, 2016).Recent findings have shown that decreased Sirtuin 3, a regulator of mitochondrial function, levels result in renal fibrosis concomitant with increased DRP1 and other fission protein levels, while fusion proteins are decreased in their expression (Cheng et al., 2022).This highlights a model in which CKD is induced by mitochondrial dysfunction, which may involve an interplay between altered mitophagy, biogenesis, dynamics, contact sites, and biogenesis, as previously reviewed (Jiang et al., 2020).While Sirtuin 3 is one promising mitochondrial target for reducing CKD, further targets are needed given the diverse roles of Sirtuin 3 in mitochondrial metabolism (Cheng et al., 2022).
A key finding of the study by Su and colleagues is the role of LTBP4 in mitochondrial dynamics (Figure 1).Specifically, during AKI in loss of murine Ltbp4S, or mice lacking the short isoforms of LTBP4, there is an uptick of DRP1 to cause increased mitochondrial division and altered structure, as determined through western blotting and mRNA analysis.This results in impaired mitochondrial function and reduced angiogenesis resulting in increased severity of fibrosis, marked by increases in kidney injury molecule-1, with loss of LTBP4 in IRI.Notably, as past studies have indicated that a loss of ATP generation is a hallmark of AKI, this suggests pathology is mediated by DRP1-dependent mitochondrial fission and dysregulation, reaffirming previous findings that show CKD development arising from mitochondrial fission (Cheng et al., 2022;Jiang et al., 2020).
Ltbp4s-deficiency also causes oxidative stress, inflammation, and mitochondrial dysfunction, which together accelerate CKD development.To understand the therapeutic value of this, the authors applied, Mdivi-1, a mitochondrial fission inhibitor, which was observed to be more effective in Ltbp4s-deficient mice through SOD1dependent antioxidative activity.Notably, blocking DRP1 protected against negative effects, including the accelerated progression from AKI to CKD caused by a loss of LTBP4, suggesting the role of DRP1 as a target for AKI.
While the study by Su et al. suggests changes in mitochondrial dynamics upon loss of LTBP4, it is unclear how the 3D structure of mitochondria is affected and the effectors of mitochondrial dynamics.3D reconstruction of mitochondrial structure can offer valuable insight into how mitochondrial connectivity (Vue, Garza-Lopez, et al., 2023;Vue, Neikirk, et al., 2023) and cristae quality changes (Crabtree et al., 2023).One outstanding question from the study is whether Mdivi-1 restores function more through modulation of reactive oxygen species (ROS) or mitochondrial structure, which may be better examined by considering the functional roles of altered mitochondrial 3D structure post-Mdivi-1 treatment.
Interestingly, across the aging process, there is a loss of DRP1, which can also cause mitochondrial dysfunction (Sharma et al., 2019).Paradoxically, in other models, the loss of DRP1 can extend the longevity (Sharma et al., 2019).This suggests that there may be differential functions of DRP1 in certain cases that govern its effectiveness and necessity.Importantly, in the context of cardiac pathology, Mfn1 and Mfn2 knockout alongside that of DRP1 slowed disease progression more than the sole KO of one alone (Sharma et al., 2019).Su and colleagues did not look at changes in other mitochondrial fusion and fission proteins, which may better offer an understanding of mitochondrial dynamics than levels of DRP1 alone.Additionally, while Su et al. show that LTBP4 is increased in AKI without sex-dependent differences, it remains unclear if LTBP4 expression can be increased to prevent dysfunction associated with AKI through concomitant increased DRP1 expression.Alternatively, can other pathways affect DRP1 expression, thus serving therapeutic roles for in preventing the AKI to CKD shift?For example, recent research shows that in skeletal muscle (Yan et al., 2022) and brain tissue (Gusdon et al., 2017), age-related loss of DRP1 may be moderated by exercise regimens.

| Inflammation and oxidative stress
This study introduces a greater nuance to our understanding of LTBP4's roles in oxidative stress and inflammation.Currently, many of the studies on LTBP4 are focused on its role in Duchenne muscular dystrophy (DMD) and other hereditary diseases (Su & Urban, 2021).This limits understanding of LTBP4's roles in the kidney beyond the findings by Su et al., but it does offer areas of potential research that may be further elucidated in the context of LTBP4's mechanistic roles in the shift of AKI to CKD (Figure 2).It is possible that fibrosis observed due to LTBP4 is due to macrophages, which in DMD occurs alongside LTBP4 expression to cause inflammation (Juban et al., 2018).Notably, DMD is exacerbated by oxidative stress, an age-related product, while NF-kB and regeneration are both negatively correlated with age, emphasizing the age-dependence of this progressive muscle degeneration disorder (Messina et al., 2011).Notably, in DMD, while typically movement is lost within the first 15 years of life, increased LTBP4 levels alongside inhibitors of oxidative stress are associated with increased ambulatory time (Weiss et al., 2018).Beyond this, LTBP4 genotypes are associated with ambulatory time (Flanigan et al., 2013), suggesting that LTBP4 expression can have important implications for age-related diseases.Depending on the form of LTBP4, preventing proteolytic cleavage and the positive form of LTBP4 can be selected for through antibodies, underscoring the ability of LTBP4 to be targeted in therapies (Demonbreun et al., 2021).
Still, based on the study by Su et al., there remain numerous avenues to explore regarding the roles of LTBP4, especially in the context of aging.Inflammation caused by AKI, potentially related to ROS (Tomsa et al., 2019), is highlighted in the article by Su and colleagues.Notably, one of the effects of the loss of LTBP4 is reactive oxygen species accumulation (Su et al., 2023), which is also a hallmark of the aging process (Sharma et al., 2019).It remains unclear, however, if ROS accumulation in tissue systems is partially fueled by age-related loss of LTBP4.Notably, TGFβ1 initiation is induced by the mitochondrial ROS (Hsieh et al., 2018).Similarly, although DMD is a different model, it displays isoform-dependent responses to LTBP4 suggesting this as a potential area that may be further elucidated.The relationship between LTBP4 and oxidative stress species across aging remains unclear, especially if LTBP4 expression may inversely reduce ROS levels.Nonetheless, studies in DMD may be considered in a renal context to consider ROS-dependent pathways that can be targeted in both respective pathological states.

| Tissue dependency across aging
Another remaining question is does LTBP4 protect against tissue damage in other regions?A key factor of the aging heart is cardiac fibrosis (Biernacka & Frangogiannis, 2011), which is modulated by TGF-β activation and previously has been linked to LTBP2 activity (Shi et al., 2021).It remains unclear if, like LTBP2 (Shi et al., 2021), increased LTBP4 levels can "accelerate" cardiac fibroblast apoptosis, thus potentially slowing aging.Indeed, given that LTBP4 may impair progression to CKD (Su et al., 2023), this may already serve to slow renal aging.Notably, one prior study found that increased expression of LTBP4 in muscle dystrophy resulted in increased muscle mass and strength in a myostatin-dependent manner (Lamar et al., 2016).While further research is needed, sarcopenia, or the age-related loss of muscle mass, has been linked to myostatin levels, establishing LTBP4 as a potential therapy for sarcopenia (Yasar et al., 2022).Another recent study in lung tissue shows that across aging, LTBP4 is increased (Koloko Ngassie et al., 2023).
Notably, in lung tissue, antioxidant protein Sestrin 2 can restore against pathology caused by knockout of LTBP4, suggesting that lung remodeling is similarly protected against LTBP4 through pathways involving modulating the oxidative stress response (Tomasovic et al., 2017).Finally, a study in adult murine adipose stem cells demonstrates that while aging alone did not increase LTBP4, the confluence of aging and obesity increased LTBP4 expression (Xie et al., 2023).Notably, DRP1 is affected by both aging (Sharma et al., 2019) and diet, with high-fat diets having modulated DRP1-dependent responses to mitophagy in cardiac tissues (Tong et al., 2023).Together, these pluralistic studies suggest that in other tissues, LTBP4 may still be reliant on factors exposed by Su and colleagues, including oxidative stress and mitochondria, underscoring the importance of understanding if there may be tissue-differential effects of LTBP4 expression across aging.

| Mechanistic understanding
One potential avenue to explore the age-related effects of LTBP4 is through alternative signaling pathways it may be engaging it.
Mammalian/mechanistic target of rapamycin (mTORC) signaling is commonly understood to decrease across the aging process (Papadopoli et al., 2019), yet interestingly, in chronic infections, mTORC signaling is inhibited by TGF-β to preserve metabolism (Gabriel et al., 2021).While a fundamentally different model, how LTBP4 may similarly play roles in cell proliferation and metabolism, in melanoma increased levels of LTBP4 are associated with cancer inhibition (na Wang et al., 2020).Notably, this occurs through modulation of the Hippo-Yes-associated protein (YAP) pathway (na Wang et al., 2020).
The Hippo pathway has an evolving understanding of the regulation of aging that has previously been reviewed, including through affecting ROS and mTORC pathways (Yeung et al., 2019).Indeed, LTBP4 expression may be increased in aging (Koloko Ngassie et al., 2023;Lausecker et al., 2022), which may concomitantly result in increased Hippo pathway activation.This suggests a potential role of TGF-β in age-related mTORC changes, yet LTBP4 remains poorly investigated as an effector in this age-related change.While it is unclear if these F I G U R E 2 Figure showing pathways with and without latent transforming growth factor beta binding protein 4 (LTBP4), as well as potential implications in aging.Across aging, there is an increase of LTBP4, while a similar increase is also seen in acute kidney injury.Increases in LTBP4 can cause factors including an increase in muscle mass and activation of the Hippo-pathway, which may interact with mammalian/mechanistic target of rapamycin (mTORC).In contrast, decreases in LTBP4 can result in mitochondrial fission, muscle dystrophy, fibrosis, generation of reactive oxygen species, and inhibition of angiogenesis.While these occur in tissues outside of the kidney, they may be relevant to mechanistic insight of LTBP4.

F
Figure showing pathways with and without latent transforming growth factor beta binding protein 4 (LTBP4), in acute kidney injury.With increased LTBP4 expression (left), there are reductions in mitochondrial fission leading to healthy kidneys.With decreased expression (middle), increases in mitochondrial fission result in the development of chronic kidney disease.With decreased expression, however, inhibition of mitochondrial fission (right) can prevent the development of chronic kidney disease.