Mitochondrial dynamics and diabetic kidney disease: Missing pieces for the puzzle of therapeutic approaches

Abstract Diabetic kidney disease (DKD) is a common microvascular complication among diabetic patients. Once the DKD has developed, most of the patients inevitably progress to the end‐stage renal disease (ESRD). Although many new therapeutic strategies have attempted to demolish the root of the pathogenesis of DKD, the residual risks of ESRD still remained. Alteration of mitochondrial dynamics towards mitochondrial fission concurrent with the mitochondrial dysfunction is the characteristic that is usually seen in various diseases, including DKD. It has been proposed that those perturbation and their cooperative networks could be responsible for the residual risk of ESRD in DKD patients. In this review, the collective evidence of alteration in mitochondrial dynamics and their associations with the mitochondrial function from in vitro, in vivo and clinical reports of DKD are comprehensively summarized and discussed. In addition, both basic and clinical reports regarding the pharmacological interventions that showed an impact on the mitochondrial dynamics, and the correlation with the renal parameters in DKD is presented. Understanding these complex mechanisms in combination with the existing therapeutic modalities could bring a new opportunity to overcome the unresolvable problem of DKD.


| INTRODUC TI ON
Diabetic kidney disease (DKD) is the most common cause of chronic kidney disease (CKD), and the major aetiology of end-stage renal disease (ESRD), contributing to renal replacement therapy worldwide. 1 The presence of DKD among diabetic patients is associated with higher morbidity and mortality, comparing to other vascular complications. 2 In diabetes mellitus (DM), substantial comorbid diseases such as hypertension and dyslipidaemia, which are the common illness that often found in the same patient, can enhance the deterioration of DKD. 3 Nevertheless, recent data demonstrate that although the incidence of DKD is stabilized, this is due to improved treatment on those comorbidities, not DKD. 4 The pathogenesis of DKD consists of two main mechanisms including haemodynamic and metabolic pathways. 3 Molecular studies demonstrated that each pathway involved numerous complicated vital substrates that are responsible for the development of DKD.
Although many clinical studies using emerging therapies and possible beneficial strategies intended to reverse those pathological keys, none of those succeeded in stopping the disease progression. 4,5 It has been proposed that once the kidney involvement is observed, the progression to ESRD is inevitable overtime. 6 According to those facts, understanding the molecular mechanisms responsible for the development of DKD is crucial for developing strategies to combat with DKD and ESRD. Recently, the roles of mitochondria and mitochondrial dynamics have been extensively investigated and have been proposed as one of the jigsaw puzzles to complete the mechanistic picture in DKD. 7,8 Mitochondria are membrane-bound organelles which are known as the power generator of every cell. They incessantly alter their size, shape and number through the process of 'mitochondrial dynamics', which consists of mitochondrial fusion and fission, in response to cellular energy requirement and to maintain their homeostasis. 9 Mitochondrial fusion causes the mitochondria joining together for sharing the substrates between injured mitochondria to eliminate the damaged components and recover overall mitochondrial functions. 10 However, mitochondrial fission is activated if the cellular damage is occurred, and the broken fragments will be eliminated through the budding off vesicle which is sent to the recycling process, an event known as mitophagy. 11 Simultaneously, mitochondrial biogenesis is a process which promotes mitochondrial DNA duplication, and to synthesize revitalize organelles substituting the dysfunctional mitochondria. 12 Under physiological condition, the balance between fusion and fission must be maintained. 13 Growing evidence demonstrates that an alteration in mitochondrial dynamics is associated with the underlying pathogenesis of various diseases including malignancy, cardiovascular disease and neurodegenerative disease. [14][15][16][17] DKD has also been shown to involve in this mitochondrial networking abnormality. 18 Interestingly, these phenomena seem to occur preceding the development of both clinical and laboratory abnormalities in DKD. 7,19 Since impaired balance of mitochondrial dynamics could be responsible for DKD pathogenesis, the modulation on those processes to restore its homeostasis might be the therapeutic strategies to correct the unresolvable problem of DKD. Despite this proposed hypothesis, the current evidence from both basic and clinical reports is still limited regarding the precise mechanism of mitochondrial dynamics on DKD and the effective interventions targeting mitochondrial dynamics.
This review intends to summarize the evidence which is available from the in vitro, in vivo and clinical studies regarding the roles of mitochondrial dynamics on DKD. It also focuses on both mechanistic and interventional aspects, concurrently with the discussion of their potential application in the clinical practice in DKD patients. Lastly, this review aims to encourage more basic and clinical investigations on the roles of mitochondrial dynamics in DKD to complete the jigsaw puzzles on our understanding of DKD mechanisms and to pave ways for better therapeutic strategies in those patients.
The PubMed database was used for the search for literatures published in English language before October 2020. Diabetic kidney disease and mitochondrial dynamics were used as keywords. All the results from the search were reviewed, and the relevant articles were identified for further reviewed. The pertinent findings of each literature were extracted and summarized in this review.

| NATUR AL HIS TORY OF DIAB E TIC K IDNE Y D IS E A S E AND ITS CORREL ATI ON WITH MITOCHONDRIAL DYNAMIC S
Diabetic kidney disease is one of the microvascular complications among diabetic patients. 20 Typical laboratory manifestations are the presence of impaired kidney function, concurrent with progressive increment of albuminuria, which are concordant with the duration of being diagnosed DM. 20 DKD consists of five stages including hyperfiltration, silence, incipient, overt nephropathy and ESRD. 20 The higher the stage of DKD, the worse of the glomerular filtration rate (GFR), the degree of albuminuria and the subsequent development of hypertension are to be found. 20 According to the natural history of DKD, hyperglycaemia is believed to be the central initiator of the disease. 21 Nonetheless, tight glycaemic control strategy was failed to demonstrate the effectiveness on the ESRD prevention, 22-26 and even worse with the possible harm in term of increasing mortality. 24 In addition, recent clinical studies reported the beneficial effect of sodium-glucose cotransporter 2 inhibitors (SGLT-2i), which is one of the glucose-lowering agent categories, on retarding DKD progression as a result beyond glucose-lowering effect. 27 All of these findings suggested that there are still undiscovering mechanisms for the pathogenesis of DKD.
Previous findings revealed that longstanding hyperglycaemic state generated intracellular oxidative stress accompanied with the disruption of mitochondrial dynamics by promoting mitochondrial fission 28 and suppressing mitochondrial fusion. 7 These alterations also impaired biogenesis and mitophagy, leading to increased mitochondrial fragmentation, enhanced mitochondria permeability transition pore (mPTP) opening, induced cytochrome C leakage into cytosol, and finally stimulated the cellular apoptosis. 7 Furthermore, mitochondrial fission was also found to be associated with the worsening of kidney parameters such as GFR and albuminuria in DKD. 29,30 Taken together, these findings accentuated the vital role of mitochondrial dynamics on the pathogenesis of DKD, and that it can be the potential modulated pathway that might lead to the novel targeted therapy for DKD.
In molecular studies, mitochondrial fusion is reflected through the presence of mitofusin 1 (MFN1), mitofusin 2 (MFN2) and optic atrophy protein 1 (OPA1). 31 MFN1 and MFN2 are located at the outer membrane of the mitochondria, while OPA1 is situated at the inner membrane. Interaction between MFN and OPA1 from each organelle proceeds the fusion process, which causes less mitochondrial fragmentation, and more elongated mitochondria as describe by increasing of aspect ratio (AR). 32,33 Mitochondrial fission is evaluated from the expression of dynamin-related protein 1 (DRP1), mitochondrial fission protein 1 (Fis1) and mitochondrial fission factor (MFF). 33 DRP1 is a main cytosolic fission effector that once activated, it will translocate to the outer membrane of mitochondria and adhere with MFF and Fis1 guided for forming a separation ring-like structure. 32 On the contrary to fusion, fission causes more mitochondrial fragmentation and mitochondrial length shortening as demonstrate by decreasing in AR. In biogenesis activity, it is detected from the expression of peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α). 34 For mitophagy, PTEN-induced putative kinase protein 1 (PINK1) and E3 ubiquitin-protein ligase parkin (Parkin) are the two important proteins for this process. 31 Both representatives are sequentially activated and tagged the injured mitochondria which had lost their membrane potential, in order to trigger the autophagosome formation. 31 After that, enzymes are released into the autophagosome to eliminate the content inside which subsequently either excreted or reutilized. 35 Interestingly, many new molecular markers related to mitochondrial dynamics and their co-operated networks in DKD have been identified in in vitro, in vivo and clinical studies.

| MITOCHONDRIAL DYNAMIC S ALTER ATI ON S IN D IAB E TI C K IDNE Y D IS E A S E: E VIDEN CE FROM IN VITRO A N D I N VIVO S TUD IE S
In vitro study of the kidney cells under diabetic milieu demonstrated that the renal mitochondrial dynamics were altered as indicated by increased mitochondrial fission, while the mitochondrial fusion was attenuated. 13,28,[36][37][38][39][40] Furthermore, the mitochondrial biogenesis was suppressed, 36,39 and the mitophagy was impaired. 13,40 All of these changes resulted in mitochondrial dysfunction as F I G U R E 1 Regulatory signalling of mitochondrial dynamics and their cooperative networks under diabetic milieu. Excess fission was mediated through the upstream signalling that directly stimulated DRP1 phosphorylation and DRP1 facilitated proteins. These included Rap1b -ERK1/2 -C/EBPβ cascade, ROCK1, NR4A1 -P53 and DUSP1 -JNK pathway. In addition, fission could indirectly activate through the excess of ROS generation and apoptosis as a result of decreased DsbA-L and increased of MIOX. The downregulation of mitochondrial fusion was regulated through the MIOX, which intervened the PINK1 facilitated Parkin and MFN2 interactions, and the direct inhibitory effect of high glucose state. Mitochondrial biogenesis was mainly suppressed via the decreased of PGC-1α in consequence of direct high glucose mediated, and the alteration of Rap1b -ERK1/2 -C/EBPβ pathway. Autophagy (mitophagy) was impaired through the perturbation of NR4A1-P53 and MIOX -PINK1 -Parkin signalling. Notably, alteration of each process was conversely deteriorated one another. C/EBPβ, CCAAT-enhancer binding protein; Cyt-C, cytochrome C; DsbA-L, Disulfide-bond A oxidoreductase-like protein; DUSP1, Dual-specificity protein phosphatase 1; ERK1/2, extracellular signal-related kinase 1/2; JNK, c-Jun N-terminal kinase; MFF, mitochondrial fission factor; MFN 1, mitofusin 1; MFN 2, mitofusin 2; MIOX, myo-inositol oxygenase; mPTP, mitochondrial permeability transition pore; PDrp1, phosphorylated dynamin-related protein 1; NR4A1, nuclear receptor subfamily 4 group A member 1; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator 1-alpha; Pink1, PTEN-induced putative kinase 1; Rap1b, Ras-proximate 1b; ROCK1, Rho-associated coiled coil-containing protein kinase 1; ROS, reactive oxygen species; ΔΨm, mitochondrial membrane potential

Apoptosis
Autophagy Biogenesis High glucose-induced mitochondrial fission through an activation of ROCK1.
High glucose-stimulated MIOX which subsequently activated mitochondrial fission, and impaired autophagy through ROS activation, and Pink 1 inhibition, respectively.

13
(Continues) demonstrated by the shortening of mitochondrial length, mitochondrial depolarization, increased oxidative stress generation and decreased adenosine triphosphate (ATP) production. 13,28,[36][37][38][39][40] The regulatory pathways that mediated the mitochondrial fission under high glucose exposure composed of many proteins ( Figure 1), including Rho-associated coiled coil-containing protein kinase 1 (ROCK1), 28 Ras-proximate 1b (Rap1b), 36 Disulphide-bond A oxidoreductase-like protein (DsbA-L), 37 Dual-specificity protein phosphatase 1 (DUSP1), 38 PGC-1α, 39 myo-inositol oxygenase (MIOX) 13 and the nuclear receptor subfamily 4 group A member 1 (NR4A1). 40 Some of these proteins activated the fission process through increasing transcription or phosphorylation of the fission substrates, such as DRP1 and MFF, while the others promoted mitochondrial fission via activation of ROS. 13,37 Unlike mitochondrial fission, the underlying signal that was responsible for the decreasing of mitochondrial fusion among diabetic milieu was not apparently defined. Most in vitro studies reported only the reduction of fusion activity without demonstrated the regulatory pathway. 36,38,40 However, there is one study proposed that fusion was attenuated through the increasing of MIOX, which then suppressed the PINK1 and led to the reduction of Parkin-MFN2 interactions. 13 For mitochondrial biogenesis and mitophagy, each process was demonstrated to be suppressed via the cascade that involved in reducing the expression of PGC-1α, and PINK1-Parkin, respectively. 13,36,39,40 A summary of findings from those in vitro reports is shown in Table 1.
Currently, whether the perturbation of mitochondrial dynamics occurred prior to the renal structure and the laboratory chemistry abnormalities are still debated. 7 The mapping-time course of mitochondrial dynamics in the kidney among diabetic rat models revealed that at the early phase, mitochondrial fusion and biogenesis were increased as the compensatory mechanism to the excessive mitochondrial fission, which was augmented by the hyperglycaemia. 7 Unfortunately, mitochondrial fission was predominant as demonstrated by an increasing of mitochondrial fragmentation, reducing of the AR, antioxidant and the mitochondrial bioenergetics. For the natural history of DKD, serum cystatin C was initially decreased, which related to the hyperfiltration stage, whereas the surrogate markers of renal injury and renal dysfunction were unremarkably changed. 7 With the persistence of hyperglycaemia, mitochondrial dynamics were shifting towards fission, while fusion was decreased.
At this time, there were evidence of glomerulosclerosis, progressive

TA B L E 1 (Continued)
increasing of urinary albumin excretion and urinary kidney injury molecule-1 (KIM-1). 7 All of these findings from in vivo studies indicated that alterations of renal mitochondrial dynamics occurred prior to the development of abnormal histology and laboratory parameters related to DKD. The association between an alteration of mitochondrial dynamics and other cooperative networks with the laboratory parameters of diabetic kidney disease is illustrated in Figure 2.
In vivo studies also provided additional association of mitochondrial dynamics with the laboratory parameters related to DKD.
Disturbance of renal mitochondrial dynamics contributed to the abnormal kidney histology and renal parameters. 7,28,32,36,37,41,42 Notably, genetically modification on mitochondrial associated proteins which involved in the regulatory signalling pathway could alleviate the deteriorating effect of DKD. 28,32,36,37,41 All of these findings suggested that early modulation on renal mitochondrial dynamics to restore their balance before the structural and functional changes are observed could be the solution not only preventing the disease progression but also eliminating the risk of ESRD.
A summary of the findings from in vivo reports is shown in Table 2.

| MITOCHONDRIAL DYNAMIC S ALTER ATI ON S IN D IAB E TI C K IDNE Y D IS E A S E: REP ORTS FROM CLINIC AL S TUD IE S
Currently, available clinical report on this topic is still scarce. The perturbation of mitochondrial dynamics among DKD patients compared with the non-diabetic kidney disease group were consistent with the data reported from both in vitro and in vivo studies. 36,42,43 Interestingly, there is evidence demonstrating the correlation between the mitochondrial dynamics and several clinical renal parameters. 36,42,43 A positive correlation between mitochondrial AR and the degree of proteinuria, 43 Table 3.

Interpretation Ref Apoptosis
Autophagy Biogenesis NR4A1 -p53 signalling was activated under diabetic milieu, which accentuated mitochondrial fission and suppressed mitophagy.

| Mitochondrial dynamic modulators
Since DRP1 and other co-operated fission proteins are the main effector components in mitochondrial fission process that are upregulated in diabetic milieu, the mitochondrial dynamic modulators are investigated mainly to attenuate these substrates activities. 32 Table 4.

| Antidiabetic drug: Sodium-glucose cotransporter 2 inhibitors
Sodium-glucose cotransporter 2 inhibitors (SGLT-2i) is a new group of pharmacological intervention that has been shown to slow the DKD progression. [45][46][47] Although SGLT-2i is categorized to be one of the glucose-lowering agents, their beneficial effects are proven to be beyond the glycaemic control. 48 Not only the result of recovering tubulo-glomerular feedback, 49 lowering blood pressure 50 and weight reduction 51 that can explain the underlying reno-protective effect but also the normalization of mitochondrial dysregulation that play a crucial role. 52 Nevertheless, the mechanistic evaluation on those mitochondrial dynamics and functions was not apparently elucidated.
Administration of empagliflozin, one of the SGLT-2i, to human renal proximal tubular cells (hRPTCs) under a diabetic milieu could increase mitochondrial fusion and reduce mitochondrial fission. 53 Furthermore, it also improved mitochondrial function, autophagy and biogenesis, concurrently with an attenuation of apoptosis and the tubular injury.
These favourable outcomes were comparable to the SGLT2-silencing hRPTCs under high glucose environment. 53 Taken together, this study emphasized the potential additional reno-protective benefit of SGLT-2i on DKD and supported that mitochondrial abnormality was one of the underlying pathogenesis, leading to the kidney progression and ESRD among diabetes patients. A summary of the effects of the antidiabetic drug from the in vitro reports is shown in Table 4.

Several antioxidants including Polydatin and D-glucaric acid have
been investigated under the diabetic milieu. A summary of those reports regarding the use of antioxidants in in vitro studies is shown in Table 4.
Polydatin (PD) is resveratrol glycoside, which is extracted from the radix of Polygonum cuspidatum. 54 Current evidence disclosed that PD has the anti-oxidative property. 55 Table 5.

Podocin-Specific cA-ROCK1 transgenic (overexpression) mice
Rap1b was downregulated in diabetic rats and was associated with increased mitochondrial fission, oxidative stress, cellular apoptosis and worsen renal parameters.

36
DsbA-L was reduced in diabetic mice, which contributed to ROS generation, increased mitochondrial fission, and reduced mitochondrial fusion.

Wild-type mice
Tamoxifen generated podocyte Drp1 null

db/db diabetic mice
Tamoxifen generated podocyte Drp1 null

Wild-type mice
Hemizygous Hq mutation in AIFm1 gene

STZ-induced diabetes mice
Hemizygous Hq mutation in AIFm1 gene

41
- Partial AIF knockout gene did not have the effect on mitochondrial dynamics and function among diabetes mice.

42
- The benefit of SGLT-2i in CKD from other aetiologies is still limited. However, data from the pre-specified analysis of DAPA-CKD trial which is the study aimed to evaluate the efficacy of dapagliflozin, one of the SGLT-2i, on the primary kidney outcome demonstrated that dapagliflozin also had benefit in participants with non-diabetes CKD, specifically, IgA nephropathy. 47 Nonetheless, the precise mechanisms related to these outcomes were not clearly defined, and therefore, whether this benefit is also possibly due to the improvement of the renal mitochondrial dynamics as demonstrated in DKD models are to be determined. Hence, future studies are needed to explore the precise mechanisms and to warrant its use in DKD and CKD from other aetiologies.

| Polydatin and D-glucaric
Oxidative stress is found to involved in the development of DKD through directly affect the kidney cells, and also worsen the alteration of mitochondrial dynamics. 19 In diabetic mice, both Polydatin Although the clinical studies of Bardoxolone methyl, an antioxidant inflammatory modulator, demonstrated that it could improve eGFR, 62 it was reported to be associated with the increased blood pressure and albuminuria, and the increased incidence of the cardiovascular events. 63 In addition, the beneficial effect on the development of ESRD was not determined. Future studies regarding the use of novel antioxidant therapy should be further investigated for their efficacies and safety profiles.

| Melatonin
Melatonin is a pleiotropic hormone which regulates circadian rhythm and preserves mitochondrial stability through antioxidant and anti-inflammatory properties. 64 Previous studies demonstrated the reno-protective effects of melatonin under diabetes and obesity-induced nephropathy. 65,66 These satisfactory

Histology Renal parameters Interpretation Ref Apoptosis
---Increased mitochondrial fission and decreased aspect ratio were found in diabetic kidney disease patients.

43
- Rap1b was decreased among diabetic patients and was negatively correlated with Urine β -NAG, and degree of interstitial fibrosis and tubular atrophy.

Mitochondrial dynamic modulators
Conditionally immortalized mouse podocytes

32
• Berberine ameliorated the effect of palmitic acid-induced alteration of mitochondrial dynamics by decreasing the transcription of Drp1 and its protein receptors expression.

Renal parameters Interpretation Ref Apoptosis
Autophagy Biogenesis

32
- Berberine reversed the effect of diabetes on mitochondrial dynamics by inhibiting the mitochondrial fission, ameliorating oxidative stress, and promoting mitochondrial biogenesis.

TA B L E 5 (Continued)
results were linked to the modulation on renal mitochondrial dynamics by increasing of MFN2 among obesity mouse model. 66 Melatonin could also increase the expression of mitochondrial fusion proteins, including MFN2 and OPA1, whereas it suppressed the mitochondrial fission process in diabetic fatty rat model. 67 In addition, it promoted the superoxide dismutase (SOD), an anti-oxidative enzyme, concurrent with decreased oxidative stress. 67 These findings were associated with the improvement of creatinine clearance and albuminuria in diabetic fatty rat model. 67 Although whether the beneficial effects of melatonin on DKD were directly through modulating the renal mitochon- BioRender.com.

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