Mitochondria-derived vesicles and their potential roles in kidney stone disease

Recent evidence has shown significant roles of mitochondria-derived vesicles (MDVs) in mitochondrial quality control (MQC) system. Under mild stress condition, MDVs are formed to carry the malfunctioned mitochondrial components, such as mitochondrial DNA (mtDNA), peptides, proteins and lipids, to be eliminated to restore normal mitochondrial structure and functions. Under severe oxidative stress condition, mitochondrial dynamics (fission/fusion) and mitophagy are predominantly activated to rescue mitochondrial structure and functions. Additionally, MDVs generation can be also triggered as the major MQC machinery to cope with unhealthy mitochondria when mitophagy is unsuccessful for eliminating the damaged mitochondria or mitochondrial fission/fusion fail to recover the mitochondrial structure and functions. This review summarizes the current knowledge on MDVs and discuss their roles in physiologic and pathophysiologic conditions. In addition, the potential clinical relevance of MDVs in therapeutics and diagnostics of kidney stone disease (KSD) are emphasized.


Introduction
Vesicular transport is a regulatory mechanism in all living cells. The cell-derived vesicles are originated from various cellular organelles, including mitochondria. Several lines of evidence have demonstrated essential roles of mitochondria-derived vesicles (MDVs) in mitochondrial quality control (MQC) system [1][2][3][4]. This control system is crucial for mitochondrial homeostasis and cell survival regulation [5,6]. As such, MDVs formation in the MQC system is recognized as the first-line and vital regulatory mechanism in both physiologic and pathologic conditions [2,7]. Novel findings of MDVs generation during the past decade have amplified our understanding of non-mitophagy pathway for mitochondrial preservation and cell survival. Many lines of MDVs research have shown greater MDVs level in mild stress conditions than mitophagy, which is the canonical machinery for removing the damaged mitochondria [1,[8][9][10][11]. Recently, selective cargos of mitochondrial oxidized molecules, such as mitochondrial DNA (mtDNA), peptides, proteins and lipids, to be degraded by lysosomes have been shown [3,12,13]. Moreover, immune regulation by MDVs has been emphasized in several reports of inflammation-associated diseases [3,12,14]. As such, MDVs have gained a wide interest in many mitochondria-associated disorders/diseases, such as cancers [15,16], aging [17][18][19], cardiovascular diseases [20,21], and neurodegenerative disorders [22][23][24].
It is well known that kidney stone disease (KSD) is associated with oxidative stress and mitochondrial abnormalities in renal tissue [25][26][27][28][29]. Cellular mechanisms of mitochondrial dysfunction associated with kidney stone formation have been proposed [25]. For example, renal tubular inflammation and peroxidation of lipids and proteins in cell membranes induced by mitochondrial abnormalities can increase crystal deposition in the kidney Page 2 of 20 Chaiyarit and Thongboonkerd Journal of Translational Medicine (2023) 21:294 [25]. Components of dead cells and fragmented organelles, including mitochondria, also serve as the sources for stone nidus (core component) formation [25]. Additionally, the damaged mitochondria can promote renal interstitial inflammation that further enhances development and formation of the Randall's plaque, which is one of the common pathologies serving as the nidus for calcium oxalate (CaOx) kidney stone [25]. Therefore, preserving mitochondrial functions has been proposed as one of the preventive strategies against KSD [25].
In addition to the whole mitochondria and their fragments, several lines of recent evidence have implicated the involvement of intracellular and extracellular MDVs in kidney stone formation. This review therefore summarizes the current knowledge on roles of MDVs, particularly in KSD.

Overview of MDVs
The evolutionary origin of mitochondria is from archaebacteria that ordinarily transport vesicles in order to communicate with other living microorganisms, escape from host immune systems, and eliminate self-damaged materials [30,31]. Thus, MDVs formation has been proposed as the ancient homeostatic process in living cells at mitochondrial level under physiologic and mild stress conditions [21,32]. Although removal of the damaged mitochondria or mitochondrial contents by autophagy in the MQC system for cell homeostasis has been extensively studied [5,6], several mechanisms of mitochondrial reinforcement and repair remain unclear. Hence, recent concepts of micromitophagy [33,34], MDVs formation [1,8,10,22], and mitophagy-independent machinery [35][36][37] have been emerged to explain mitochondrial stability [8], prevention of cell death [37] and tissue repair [38,39].
MDVs are known as the key component of the first-line secure process in the MQC system, and their possible roles entirely differ from mitochondrial dynamics (fission/fusion) and mitophagy [1,4,5,10]. Additionally, the number of MDVs is increased by mild stress or early stage of mitochondrial dysfunction [21]. Two main types of MDVs have been recognized in the MQC system, including steady-state MDVs [32,60] and stress-induced MDVs [8,39], both of which can be characterized by their specific markers. Translocase of outer mitochondrial membrane 20 (TOMM20), an OMM protein, is mostly found in steady-state MDVs (TOMM + -MDVs) [32], whereas pyruvate dehydrogenase (PDH) is predominantly found in oxidative stress-triggered MDVs (PDH + -MDVs) [61]. Unveiling the MDVs formation and their functional roles would make the image of mitochondria-related intracellular and intercellular communications much clearer.

Biogenesis of MDVs
Previously, mitochondrial membrane blebbing and mitophagy-related machinery had been proposed as the possible mechanisms for MDVs formation [7]. However, later evidence has clearly shown that MDVs are independent of mitochondrial dynamics and mitophagy [5,40]. One of the newly proposed mechanisms for MDVs biogenesis is via PINK1 (phosphatase and tensin homolog-induced kinase 1)/Parkin (an E3 ubiquitin protein ligase containing ubiquitin-like domain at N-terminus)-dependent, but DRP1 (dynamin related protein 1)-independent process [7,52,61,62]. In mild stress condition or slight mitochondrial damage, mitochondrial membrane curvature is initiated followed by PINK1 accumulation [8,10,40]. Parkin is then recruited at OMM, and the MDVs are scissored and released by an unclear mechanism [7,8,10,40,52]. The involvement of DRP1 in MDVs generation has been excluded as MDVs can be formed even when DRP1 is knocked down [40].
By contrast, several investigations have shown that MDVs can be formed in PINK1-deficient cells [4,7,62,63]. Recent proteome study has documented a new molecular model of MDVs biogenesis in resting stage that depends on the microtubule-associated motor proteins, MIRO1 and MIRO2 (MIRO1/2), and DRP1dependent mechanism for cutting and releasing MDVs from parental mitochondria, whereas Parkin and PINK1 are not involved in this mediated pathway [32]. MDVs formation begins at steady-state by mitochondrial membrane protrusion after MIRO1/2 formation followed by recruitment of DRP1 by 49-and 51-kDa mitochondrial dynamics DRP1 receptor protein (MiD49 and MiD51, respectively) or mitochondrial fission factor (MFF) [32].
To complete MDVs construction, DRP1 then catalyzes the cutting of thin membrane tube to release MDVs that can be delivered to their specific targets. However, further elucidations for precise mechanism are needed as this group of the investigators have previously demonstrated that DRP1 silencing does not affect MDVs formation [21,52,61,64] (in contrast to their own recent findings). They have described that the contradictory results were due to dissimilar gene knockout technique in each work. DRP1 was > 95% silencing in the prior study by simple molecular technique but was completely deleted by a more effective method, namely clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system, in a recent work [32]. Thus, MDVs can be formed in an incomplete DRP1-knockdown condition. Nevertheless, they have also suggested that the steady-state MDVs formation does not require Parkin and PINK1, which may be needed for generation and regulation of MDVs formation during oxidative stress and inflammatory conditions [48,65]. Moreover, the dynamicity of MDVs formation may be also affected by techniques of detection, isolation, and diverse states of diseases or study models. Therefore, future studies on MDVs should clearly provide sufficient details of methodology and conditioning used in each study for clarification. And more extensive investigations are required for further elucidations of the precise mechanism(s) of MDVs biogenesis.
Packaging of MDVs is a complex mechanism associated with their diverse functions and destinations. Therefore, MDVs subtypes may be classified based on their specific contents and targets (Fig. 1). For example, MDVs containing mitochondria-anchored protein ligase (MAPL) are transported to peroxisomes [64,91]. Similarly, MDVs containing peroxisomal biogenesis factor 3 (Pex3) and peroxisomal biogenesis factor 14 (Pex14) play crucial roles in peroxisomal biogenesis [92,93]. Although MDVs containing Pex3/Pex14 or MAPL share the same targets, each of them functions differently.
Additionally, MDVs can be classified based on the cellular status, including steady-state MDVs and stressinduced MDVs, which are the two distinct subtypes of MDVs widely investigated in several disease models [8,32,39,60]. The steady-state MDVs are typically demonstrated as TOMM + /PDH − MDVs, whereas TOMM − / PDH + MDVs (stress-induced MDVs) are predominantly found during oxidative stress [32,61]. The biogenesis of the steady-state MDVs is PINK1/Parkin-independent, in contrast to that of the stress-induced MDVs as discussed above [52,61]. After biogenesis, both TOMM + /PDH − and TOMM − /PDH + MDVs carry the damaged mitochondrial components and transfer them to lysosomes for degradation to maintain mitochondrial structure and functions.
Another subtype of extracellular nanovesicles that correlate with MDVs has been recently isolated by high-resolution density gradient separation and termed as "mitovesicles" [68,70]. Their size is approximately 6 nm and differs from other subtypes of MDVs or EVs. Mitovesicles are small double-membrane EVs that contain proteins involved in catabolic pathway, energy production and pro-fission process, but lack of proteins involved in biosynthesis, transport and pro-fusion process [19,68]. Mechanisms of mitovesicles formation and release to extracellular space are not specified at this stage, but has been postulated to fuse with MVB before  being secreted from the cells [68]. Moreover, mitovesicles serve as the functional vesicles based on the inside mitochondrial components [19,68].
Although several subtypes of MDVs have been reported, their molecular machineries and biogenesis remain not well understood. Hence, specific cargos, functions, targets and subtypes of MDVs still require further elucidations for clarification.

Roles of MDVs in physiology and pathophysiology
Under physiologic state with mild stress, MDVs serve as a part of the crucial process in the MQC system to preserve mitochondrial functions [2,21,91]. MDVs formation has been proposed as the first-line mitochondrial safety to remove damaged mitochondrial components prior to detrimental derangement of the entire mitochondria and cell death activation [1,5,8,38,40,46]. In addition, the increase of MDVs is the finest compensatory mechanism of the MQC system, when mitophagy does not work to eliminate the impaired mitochondria [1,37,63]. Thereafter, biogenesis of mitochondrial proteins and lipids is activated to restore the mitochondrial functions [5,7,94]. MDVs are therefore considered as a novel potential therapeutic target for maintaining the MQC system and preventing mitochondrial dysfunction in normal and disease conditions. MDVs also get involved in communications between mitochondria and other intracellular organelles. They not only transport the damaged compartments to endolysosomes for degradation but also transfer proteins and lipids to peroxisomal activation and biogenesis [92,93]. Moreover, mitochondrial components such as BCL-2 (B-cell lymphoma 2) protein [5,39,40,68,95] and mtDNA from healthy mitochondria [21,96,97] can be sent to unhealthy mitochondria to recover their structure and functions, resulting in prevention of cell death [42,98,99].
Under pathophysiologic conditions, MDVs are the important regulator for immune response and inflammation [65,100,101]. During injury, mtDNA is recognized as one of the damage-associated molecular patterns (DAMPs) that can trigger pro-inflammatory response after binding to intracellular Toll-like receptors or nucleotide-binding oligomerization domain-containing protein (NOD)-like receptors [102,103]. Additionally, mitochondrial reactive oxygen species (mtROS) has been shown to regulate proinflammatory signaling by increasing nuclear factor kappa B (NF-κB) gene expression and involving in hypoxia-inducible factor 1α (HIF1α)induced inflammasome formation through NLRP3 (NLR family pyrin domain containing 3) [95,104,105]. Previous studies have also found that mitochondrial DAMPs can be released into the circulation, are recognized by pattern recognition receptors (PRRs), and promote tissue and organ injury [3,103,[106][107][108]. Moreover, mitochondrial DAMPs can mediate neutrophil migration and degranulation, thereby further enhancing cellular injury and tissue inflammation [3,103]. Many lines of evidence have shown that MDVs inhibit such inflammatory response and down-stream cascades by transferring the damaged mitochondrial components to be degraded by lysosomes and then fuse with MVB [109,110]. Mitochondrial DAMPs in MVB are then released out as extracellular MDVs, which can inhibit pro-inflammatory activation. Moreover, MDVs-mediated antigen presentation is crucial for regulating the immune system [40,48,65,111]. MDVs formation in the presence of Rab7 (a small GTPase that monitors vesicular transport to late endosomes and lysosomes), Rab9 and SNX9 (sorting nexin 9) can mediate antigen presentation after breaking down inside lysosomes by proteasome to load these mitochondrial antigens onto MHC class I molecules within endoplasmic reticulum and then transfer them to the cell surface [40,48,65,111]. Therefore, MDVs are the important regulator for development, activation, differentiation and survival of diverse immune cells, including T-lymphocytes and macrophages [40,48,101,112,113].
Mechanistically, oxidative stress-induced stimuli can activate ROS overproduction and induce mitochondrial damage [135][136][137]. The damaged mitochondria fail to keep membrane potential properties and, hence, release calcium ion, mtDNA, mtROS, mitochondrial matrix proteins, OMM and IMM into the cytoplasm [4,5,21,35]. These mitochondrial components further induce cell death, inflammatory response and renal tubulointerstitial tissue injury [134,138]. Such tubular cell injury has been reported to induce CaOx crystal adhesion onto the cells, leading to crystal retention inside the renal tissue that is one of the important mechanisms for kidney stone formation [134,[139][140][141][142]. Additionally, the adhered crystals can further grow and aggregate with the surrounding crystals, resulting in stone nidus formation [127,129,134,141].
Additionally, the damaged mitochondria and other cellular and organellar fragments can directly bind to CaOx crystals and serve as the stone nidus for crystal nucleation, growth and aggregation, which further enhance kidney stone formation [134,143,144]. Moreover, the damaged mitochondria can trigger inflammatory cascade at renal interstitial area [117,145] by recruiting numerous inflammatory cells into this area, leading to accumulation of various proinflammatory cytokines and tissue inflammation [116,129]. Together with supersaturation of calcium phosphate, which is common in the renal interstitium, Randall's plaque starts to form [126,146,147]. After erosion into the urinary space, where CaOx is frequently supersaturated, this plaque then serves as the nidus for CaOx stone to grow [25].

Potential roles of MDVs in KSD
Several recent studies have continuously shown significant roles of urinary EVs (uEVs) in KSD [148][149][150][151][152]. uEVs are involved in inflammatory response and elimination of CaOx crystals, and may also serve as the composition of the stone matrix [150,152]. Also, recent clinical studies have identified specific subtypes of uEVs as the potential biomarkers in the urine of kidney stone patients compared with healthy subjects [149,150,153]. Furthermore, pattern of uEVs subtypes in females with KSD (but not those derived from non-stone females) is similar to that in males with or without KSD [154]. Although MDVs have not yet been examined directly in KSD, numerous mitochondrial proteins have been identified in these uEVs. According to recent proteome and lipidome studies of MDVs [22,46,68], a large number of mitochondrial proteins and lipids have been identified in both MDVs and EVs [155][156][157][158]. We have also compared all of the proteins identified in EVs based on Vesiclepedia database (http:// www. micro vesic les. org/) with those identified in mitochondria based on The Human Protein Atlas (https:// www. prote inatl as. org/). Interestingly, 244 proteins are commonly found in both EVs and mitochondria (Table 1). These findings are consistent with the data observed in recent proteome studies of EVs [46,73]. Therefore, MDVs are expected to play similar roles as of uEVs in KSD.
Remarkably, both EVs and mitochondria are acknowledged to be the crucial players in kidney stone formation [25,130,131,148,[159][160][161]. As such, using anti-oxidants and/or other means of preservation of mitochondrial functions are expected to be one of the ideal strategies for KSD prevention [129,[162][163][164][165][166]. Although mitochondrial dynamics and mitophagy have been investigated and proposed as the main processes in the MQC system in many diseases [167][168][169][170], their roles in KSD remain underinvestigated [171]. Interestingly, MDVs have been demonstrated as the novel key player in the MQC system that is the main mechanism for mitochondrial homeostasis and mitochondrial stress response in several diseases, including kidney disorders [5,171]. Recent studies of MDVs have demonstrated that MDVs can reduce inflammatory response and preserve healthy mitochondria in mild stress, leading to reduction of tissue injury [169][170][171][172].
The beneficial roles of MDVs are mediated via the MQC system to place a limit on mitochondrial dysfunction under the normal and mild stress conditions [8,10,40]. Also, they are the substitutable machineries to replace the other impaired processes in the MQC system such as mitochondrial dynamics and mitophagy [1,8,10,40,46]. Thus, the damaged mitochondrial components induced by oxidative stress, including oxidized mtDNA, proteins and lipids, are eradicated from the unhealthy mitochondria by MDVs to restore the healthy mitochondria inside the cells [3,7,22,100,173]. These processes can further reduce oxidative stress and prevent cell death. Besides, MDVs can remove the excessive mtROS and other proinflammatory molecules that tend to trigger proinflammatory signaling and cytokine production [21,39,100]. Therefore, MDVs formation is considered as the rapid and foremost protective response to prevent mitochondrial dysfunction, cell death and tissue inflammation/injury under the oxidative stress condition.
MDVs carry not only the damaged mitochondrial components but also the healthy mitochondrial compartments that can be transferred and released to the unhealthy mitochondria for maintaining cellular functions and survival. Previous studies have demonstrated that MDVs can transport functional mtDNA, mitochondrial matrix, IMM, OMM and fragmented mitochondria to other malfunctioned mitochondria inside the same cell or outside (adjacent cells) [41,[174][175][176][177][178][179]. Recently, the in vitro synthesis of MDVs has been developed and applied for reduction of cell apoptosis [2,40,73]. In the study of myocardial ischemic/hypoxic injury, administration of exogenous (synthetic) MDVs has been demonstrated to serve as the new and effective therapeutic 28S ribosomal protein S14, mitochondrial (MRP-S14) (S14mt) (Mitochondrial small ribosomal subunit protein uS14m)       strategy [39,57]. Most of previous studies have suggested that both intracellular and extracellular MDVs have the protective roles against mitochondrial damage, oxidative stress and tissue/organ injury. Although the clear evidence for the beneficial roles of MDVs in KSD prevention is not currently available, we propose that MDVs would also play such protective role to cope with mitochondrial dysfunction and oxidative stress that are common in KSD (Fig. 2). Therefore, MDVs may serve as the novel therapeutic target to prevent KSD related to mitochondrial dysfunction and oxidative stress as described above.
Recently, uEVs serve as the important source for biomarker discovery in several kidney and non-kidney diseases [180][181][182]. In KSD, a recent proteome study of urinary exosomes has demonstrated greater levels of proteins in S100A family (S100A8, S100A9 and S100A12) in urinary exosomes derived from stone patients compared with those from healthy individuals [183]. Therefore, these exosomal S100A proteins may serve as the biomarkers for KSD. As MDVs share similar proteome and lipidome profiles with EVs, another important role of MDVs in diagnostics and prognostics of KSD should be more extensively investigated.

Conclusions and perspectives
MDVs are one of the most significant players in the MQC system to preserve mitochondrial structure and functions in normal and mild oxidative stress conditions [1,4,5,10]. MDVs are also involved in various diseases, particularly cardiovascular diseases [20,21] and neurodegenerative disorders [22][23][24]. In the kidney, the abundance of mitochondria per cell and their functions are critical for maintaining renal tubular cell functions along the nephron. During oxidative stress, mitochondrial dysfunction and tubulointerstitial inflammation occur and induce kidney stone formation [25,116,184]. Therefore, mitochondria are the key player in KSD development.
The MQC system serves as the central machinery for mitochondrial homeostasis to prevent cell death and tissue injury [5,6,172]. MDVs, as the essential compartment of the MQC system [1][2][3][4], play the protective roles to rescue the malfunctioned mitochondria during mild stress to preserve their normal structure and functions ( Fig. 2).
At early stage of oxidative stress with mild mitochondrial damage, MDVs formation is the rapid and effective process for preserving mitochondrial functions. Under severe oxidative stress condition, mitochondrial dynamics (fission/fusion) and mitophagy are predominantly activated to rescue mitochondrial structure and functions [185][186][187][188]. Additionally, MDVs generation can be also triggered as the major MQC machinery to cope with unhealthy mitochondria when mitophagy is unsuccessful for eliminating the damaged mitochondria or mitochondrial fission/fusion fails to recover the mitochondrial structure and functions [1,8,10,40]. When the MQC system is overwhelmed by extremely severe oxidative stress, mitochondrial dysfunction occurs, leading to ROS overproduction, mitochondrial degradation, inflammation, cell death, and renal tubulointerstitial injury. All these detrimental derangements lead to CaOx crystal deposition, growth, aggregation, nidus formation, Randall's plaque development and, finally, kidney stone formation [25,129,146,147] (Fig. 2).
Nevertheless, the current knowledge on roles of MDVs under physiologic and pathophysiologic conditions remains incomplete. Several advanced methods/ techniques have been continuously developed to further clarify the MDVs biology and functions, such as MDVs formation mechanisms, subtypes, specific contents, targets, and diagnostic/therapeutic potential [68,75,78,189]. As MDVs seem to be more dynamic than we  Fig. 2 Roles of MDVs and MQC system in KSD. At early stage of oxidative stress with mild mitochondrial damage, MDVs (as a part of the MQC system) are formed to eliminate the malfunctioned mitochondrial components. Under severe oxidative stress condition, mitochondrial dynamics (fission/fusion) and mitophagy are predominantly activated to rescue mitochondrial structure and functions. When the MQC system is overwhelmed by extremely severe oxidative stress, mitochondrial dysfunction occurs, leading to ROS overproduction, mitochondrial degradation, inflammation, cell death, and renal tubulointerstitial injury. All these detrimental derangements lead to CaOx crystal deposition, growth, aggregation, nidus formation, Randall's plaque development and, finally, kidney stone formation initially anticipated, isolation and purification of MDVs also need further development to obtain the specific subtype(s) of the purified MDVs. Differential ultracentrifugation is the primary method for MDVs isolation but still requires further improvement for better yield and higher purity [68,75]. After isolation, characterizations can be done by morphological examination using high-resolution electron microscopy [19,68]. To validate MDVs subtypes, proteome and lipidome studies should be performed followed by immunodetection [19,68]. Recent evidence has demonstrated the therapeutic potential of MDVs in several diseases, including Parkinson's disease [190], Down syndrome [68], Alzheimer's disease [191], and myocardial ischemia [39,192]. Interestingly, the synthetic MDVs have been successfully generated in vitro [39,[193][194][195][196]. These synthetic (exogenous) MDVs can be produced by activating the isolated mitochondria by chemical reaction, energy regenerating system, or mild stress-inducing reagents [39,193]. This technique is therefore promising for further characterizations of MDVs and for developing MDVs-based therapeutic strategies in various diseases, including KSD.
In addition to the therapeutic/preventive potential, MDVs also have a promising role in diagnostics/prognostics of KSD. Future studies on biomarker discovery for KSD should focus on MDVs and their specific types. For example, at an initial phase of kidney stone development with mild stress condition or slight tissue injury, mtDNA and mtROS can be excreted through PDH + -MDVs and transferred to blood circulation and/or urine. Therefore, identification of urinary PDH + -MDVs containing mtDNA or mitochondrial proteins, together with evidence of supersaturation of crystalline compounds in the urine would yield an early biomarker for KSD.