[Effects and mechanisms of total flavones of Abelmoschus manihot in attenuating diabetic tubulopathy by targeting endoplasmic reticulum stress-induced cell apoptosis].

Renal tubular injury in patients with diabetic kidney disease(DKD) may be accompanied by glomerular and microvascular diseases. It plays a critical role in the progression of renal damage in DKD, and is now known as diabetic tubulopathy(DT). To explore the multi-targeted therapeutic effects and pharmacological mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese medicine for treating kidney disease, in attenuating DT, the authors randomly divided all rats into four groups: a normal control group(normal group), a DT model group(model group), a DT model+TFA-treated group(TFA group) and a DT model+rosiglitazone(ROS)-treated group(ROS group). The DT rat model was established based on the DKD rat model by means of integrated measures. After successful modeling, the rats in the four groups were continuously given double-distilled water, TFA suspension, and ROS suspension, respectively by gavage every day. After 6 weeks of treatment, all rats were sacrificed, and the samples of their urine, blood, and kidneys were collected. The effects of TFA and ROS on various indicators related to urine and blood biochemistry, renal tubular injury, renal tubular epithelial cell apoptosis and endoplasmic reticulum stress(ERS), as well as the activation of the protein kinase R-like endoplasmic reticulum kinase(PERK)-eukaryotic translation initiation factor 2α(eIF2α)-activating transcription factor 4(ATF4)-C/EBP homologous protein(CHOP) signaling pathway in the kidney of the DT model rats were investigated. The results indicated that hypertrophy of renal tubular epithelial cells, renal tubular hyperplasia and occlusion, as well as interstitial extracellular matrix and collagen deposition occurred in the DT model rats. Moreover, significant changes were found in the expression degree and the protein expression level of renal tubular injury markers. In addition, there was an abnormal increase in tubular urine proteins. After TFA or ROS treatment, urine protein, the characteristics of renal tubular injury, renal tubular epithelial cell apoptosis and ERS, as well as the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney of the DT model rats were improved to varying degrees. Therein, TFA was superior to ROS in affecting the pathological changes in renal tubule/interstitium. In short, with the DT model rats, this study demonstrated that TFA could attenuate DT by multiple targets through inhibiting renal tubular ERS-induced cell apoptosis in vivo, and its effect and mechanism were related to suppressing the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney. These findings provided preliminary pharmacological evidence for the application of TFA in the clinical treatment of DT.

Beneficial Effect of Chloroquine and Amodiaquine on Type 1 Diabetic Tubulopathy by Attenuating Mitochondrial Nox4 and Endoplasmic Reticulum Stress.

BACKGROUND: Oxidative stress induced by chronic hyperglycemia is recognized as a significant mechanistic contributor to the development of diabetic kidney disease (DKD). Nonphagocytic nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) is a major source of reactive oxygen species (ROS) in many cell types and in the kidney tissue of diabetic animals. We designed this study to explore the therapeutic potential of chloroquine (CQ) and amodiaquine (AQ) for inhibiting mitochondrial Nox4 and diabetic tubular injury. METHODS: Human renal proximal tubular epithelial cells (hRPTCs) were cultured in high-glucose media (30 mM D-glucose), and diabetes was induced with streptozotocin (STZ, 50 mg/kg i.p. for 5 days) in male C57BL/6J mice. CQ and AQ were administered to the mice via intraperitoneal injection for 14 weeks. RESULTS: CQ and AQ inhibited mitochondrial Nox4 and increased mitochondrial mass in hRPTCs under high-glucose conditions. Reduced mitochondrial ROS production after treatment with the drugs resulted in decreased endoplasmic reticulum (ER) stress, suppressed inflammatory protein expression and reduced cell apoptosis in hRPTCs under high-glucose conditions. Notably, CQ and AQ treatment diminished Nox4 activation and ER stress in the kidneys of STZ-induced diabetic mice. In addition, we observed attenuated inflammatory protein expression and albuminuria in STZ-induced diabetic mice after CQ and AQ treatment. CONCLUSION: We substantiated the protective actions of CQ and AQ in diabetic tubulopathy associated with reduced mitochondrial Nox4 activation and ER stress alleviation. Further studies exploring the roles of mitochondrial Nox4 in the pathogenesis of DKD could suggest new therapeutic targets for patients with DKD.

Empagliflozin attenuates diabetic tubulopathy by improving mitochondrial fragmentation and autophagy.

We examined the effects of empagliflozin, a selective inhibitor of Na+-glucose cotransporter 2, on mitochondrial quality control and autophagy in renal tubular cells in a diabetic environment in vivo and in vitro. Human renal proximal tubular cells (hRPTCs) were incubated under high-glucose conditions. Diabetes was induced with streptozotocin in male C57BL/6J mice. Improvements in mitochondrial biogenesis and balanced fusion-fission protein expression were noted in hRPTCs after treatment with empagliflozin in high-glucose media. Empagliflozin also increased autophagic activities in renal tubular cells in the high-glucose environment, which was accompanied with mammalian target of rapamycin inhibition. Moreover, reduced mitochondrial reactive oxygen species production and decreased apoptotic and fibrotic protein expression were observed in hRPTCs after treatment with empagliflozin, even in the hyperglycemic circumstance. Importantly, empagliflozin restored AMP-activated protein kinase-α phosphorylation and normalized levels of AMP-to-ATP ratios in hRPTCs subjected to a high-glucose environment, which suggests the way that empagliflozin is involved in mitochondrial quality control. Empagliflozin effectively suppressed Na+-glucose cotransporter 2 expression and ameliorated renal morphological changes in the kidneys of streptozotocin-induced diabetic mice. Electron microscopy analysis showed that mitochondrial fragmentation was decreased and 8-hydroxy-2'-deoxyguanosine content was low in renal tubular cells of empagliflozin treatment groups compared with those of the diabetic control group. We suggest one mechanism related to the renoprotective actions of empagliflozin, which reverse mitochondrial dynamics and autophagy.

Role of HSP60 (HSPD1) in diabetes-induced renal tubular dysfunction: regulation of intracellular protein aggregation, ATP production, and oxidative stress.

Because underlying mechanisms of diabetic nephropathy/tubulopathy remained poorly understood, we aimed to define a key protein involving in hyperglycemia-induced renal tubular dysfunction. All altered renal proteins identified from previous large-scale proteome studies were subjected to global protein network analysis, which revealed heat shock protein 60 (HSP60, also known as HSPD1) as the central node of protein-protein interactions. Functional validation was performed using small interfering RNA (siRNA) to knock down HSP60 (siHSP60). At 48 h after exposure to high glucose (HG) (25 mM), Madin-Darby canine kidney (MDCK) renal tubular cells transfected with controlled siRNA (siControl) had significantly increased level of HSP60 compared to normal glucose (NG) (5.5 mM), whereas siHSP60-transfected cells showed a dramatically decreased HSP60 level. siHSP60 modestly increased intracellular protein aggregates in both NG and HG conditions. Luciferin-luciferase assay showed that HG modestly increased intracellular ATP, and siHSP60 further enhanced such an increase. OxyBlot assay showed significantly increased level of oxidized proteins in HG-treated siControl-transfected cells, whereas siHSP60 caused marked increase of oxidized proteins under the NG condition. However, the siHSP60-induced accumulation of oxidized proteins was abolished by HG. In summary, our data demonstrated that HSP60 plays roles in regulation of intracellular protein aggregation, ATP production, and oxidative stress in renal tubular cells. Its involvement in HG-induced tubular cell dysfunction was most likely via regulation of intracellular ATP production.-Aluksanasuwan, S., Sueksakit, K., Fong-ngern, K., Thongboonkerd, V. Role of HSP60 (HSPD1) in diabetes-induced renal tubular dysfunction: regulation of intracellular protein aggregation, ATP production, and oxidative stress.

PTEN-induced kinase 1 exerts protective effects in diabetic kidney disease by attenuating mitochondrial dysfunction and necroptosis.

Mitochondrial dysfunction plays a pivotal role in diabetic kidney disease initiation and progression. PTEN-induced serine/threonine kinase 1 (PINK1) is a core organizer of mitochondrial quality control; however, its function in diabetic kidney disease remains controversial. Here, we aimed to investigate the pathophysiological roles of PINK1 in diabetic tubulopathy, focusing on its effects on mitochondrial homeostasis and tubular cell necroptosis, which is a specialized form of regulated cell death. PINK1-knockout mice showed more severe diabetes-induced tubular injury, interstitial fibrosis, and albuminuria. The expression of profibrotic cytokines significantly increased in the kidneys of diabetic Pink1-/- mice, which eventually culminated in aggravated interstitial fibrosis. Additionally, the knockdown of PINK1 in HKC-8 cells upregulated the fibrosis-associated proteins, and these effects were rescued by PINK1 overexpression. PINK1 deficiency was also associated with exaggerated hyperglycemia-induced mitochondrial dysfunction and defective mitophagic activity, whereas PINK1 overexpression ameliorated these negative effects and restored mitochondrial homeostasis. Mitochondrial reactive oxygen species triggered tubular cell necroptosis under hyperglycemic conditions, which was aggravated by PINK1 deficiency and improved by its overexpression. In conclusion, PINK1 plays a pivotal role in suppressing mitochondrial dysfunction and tubular cell necroptosis under high glucose conditions and exerts protective effects in diabetic kidney disease.

Non-steroidal mineralocorticoid receptor antagonist finerenone ameliorates mitochondrial dysfunction via PI3K/Akt/eNOS signaling pathway in diabetic tubulopathy.

Diabetic tubulopathy (DT) is a recently recognized key pathology of diabetic kidney disease (DKD). The mitochondria-centric view of DT is emerging as a vital pathological factor in different types of metabolic diseases, such as DKD. Finerenone (FIN), a novel non-steroidal mineralocorticoid receptor antagonist, attenuates kidney inflammation and fibrosis in DKD, but the precise pathomechanisms remain unclear. The role of mineralocorticoid receptor (MR) in perturbing mitochondrial function via the PI3K/Akt/eNOS signaling pathway, including mitochondrial dynamics and mitophagy, was investigated under a diabetic state and high glucose (HG) ambiance. To elucidate how the activation of MR provokes mitochondrial dysfunction in DT, human kidney proximal tubular epithelial (HK-2) cells were exposed to HG, and then mitochondrial dynamics, mitophagy, mitochondrial ROS (mitoROS), signaling molecules PI3K, Akt, Akt phosphorylation and eNOS were probed. The above molecules or proteins were also explored in the kidneys of diabetic and FIN-treated mice. FIN treatment reduced oxidative stress, mitochondrial fragmentation, and apoptosis while restoring the mitophagy via PI3K/Akt/eNOS signaling pathway in HK-2 cells exposed to HG ambiance and tubular cells of DM mice. These findings linked MR activation to mitochondrial dysfunction via PI3K/Akt/eNOS signaling pathway in DT and highlight a pivotal but previously undiscovered role of FIN in alleviating renal tubule injury for the treatment of DKD.

Inhibition of ferroptosis of renal tubular cells with total flavones of Abelmoschus manihot alleviates diabetic tubulopathy.

Ferroptosis-related renal tubular lesions play important roles in diabetic kidney disease (DKD) progression, and these pathophysiological responses are collectively described as diabetic tubulopathy (DT), which lacks an effective treatment. Total flavones from Abelmoschus manihot (TFA), a natural extract that extensively used in patients with chronic kidney disease, has been used for treatment of renal tubular injury in DKD; however, whether TFA alleviates DT and its potential mechanisms remain unclear. Hence, we investigated the effects of TFA, compared to dapagliflozin, in DT management both in vivo and in vitro, using a DKD rat model and the NRK-52 E cells. Following modeling, the DKD rats received TFA, dapagliflozin, or vehicle for 6 weeks. For the in vitro research, the NRK-52 E cells were exposed to advanced glycation end products (AGEs) plus ferrostatin-1 (Fer-1), dapagliflozin, or TFA. Changes in biochemical parameters and renal tubular injury were analyzed in vivo, while changes in ferroptosis of renal tubular cells and the ferroptosis-related proteins expression were analyzed both in vivo and in vitro. We found that TFA and dapagliflozin improved biochemical parameters, renal tubular injury, and ferroptosis in the DKD rats. Moreover, TFA and dapagliflozin inhibited ferroptosis by ameliorating iron deposition, lipid peroxidation capacity, and ferroptosis-related proteins expression in vitro, which was similar to the effects of Fer-1. Collectively, this study demonstrated that TFA treated DT in a manner similar to dapagliflozin by inhibiting ferroptosis of renal tubular cells via improving iron deposition and antioxidant capacity. Our findings provide new pharmacological evidence for TFA application in DT treatment.

Mitochondrial dysfunction in diabetic tubulopathy.

Diabetic kidney disease (DKD) is a devastating microvascular complication associated with diabetes mellitus. Recently, the major focus of glomerular lesions of DKD has partly shifted to diabetic tubulopathy because of renal insufficiency and prognosis of patients is closely related to tubular atrophy and interstitial fibrosis. Indeed, the proximal tubule enriching in mitochondria for its high energy demand and dependence on aerobic metabolism has given us pause to focus primarily on the mitochondria-centric view of early diabetic tubulopathy. Multiple studies suggest that diabetes condition directly damages renal tubules, resulting in mitochondria dysfunction, including decreased bioenergetics, overproduction of mitochondrial reactive oxygen species (mtROSs), defective mitophagy and dynamics disturbances, which in turn trigger a series of metabolic abnormalities. However, the precise mechanism underlying mitochondrial dysfunction of renal tubules is still in its infancy. Understanding tubulointerstitial's pathobiology would facilitate the search for new biomarkers of DKD. In this Review, we summarize the current literature and postulate that the potential effects of mitochondrial dysfunction may accelerate initiation of early-stage diabetic tubulopathy, as well as their potential therapeutic strategies.

Correlation between albuminuria and interstitial injury marker reductions associated with SGLT2 inhibitor treatment in diabetic patients with renal dysfunction.

BACKGROUND: We investigated the effects of sodium-glucose cotransporter 2 inhibitor (SGLT2i) administration focusing on its involvement in tubulo-interstitial disorders in diabetic kidney. METHODS: Enrolled patients with diabetic kidney disease received a mean dose of 52.3 mg of an SGLT2i (ipragliflozin) daily. Blood and urine were sampled at 0, 1, and 12 months (M). RESULTS: Non-renal-dysfunction patients (NRD: baseline eGFR ≥ 60 mL/min/1.73 m2, n = 12) and renal-dysfunction patients (RD: baseline eGFR < 60 mL/min/1.73 m2, n = 9) were analyzed separately. The median urine albumin-to-Cr ratio (ACR) was significantly decreased at 1 M in both groups (NRD: 163.1 at 0 M vs 118.5 mg/g Cr at 1 M, RD: 325.2 at 0 M vs 136.0 mg/g Cr at 1 M). In the RD, but not the NRD group, reduction of urine monocyte chemotactic protein-1 (MCP-1) by SGLT2i showed a significant difference between high-responders (HR: - 25.7 ± 11.4%) and low-responders (LR: 59.2 ± 17.0%), defined by ACR reduction at 1 M. Univariate analysis showed a significant correlation between the reduction of ACR and MCP-1 (R = 0.683, p = 0.042) in RD. CONCLUSION: SGLT2i exerted an anti-albuminuric effect regardless of the presence/absence of renal dysfunction. However, the anti-albuminuric effect of SGLT2i in patients with renal dysfunction appears more closely associated with amelioration of tubulo-interstitial disorders compared to patients without renal dysfunction.

Cellular Senescence and Regulated Cell Death of Tubular Epithelial Cells in Diabetic Kidney Disease.

Cellular senescence is frequently evident at etiologic sites of chronic diseases and involves essentially irreversible arrest of cell proliferation, increased protein production, resistance to apoptosis, and altered metabolic activity. Regulated cell death plays a vital role in shaping fully functional organs during the developmental process, coordinating adaptive or non-adaptive responses, and coping with long-term harmful intracellular or extracellular homeostasis disturbances. In recent years, the concept of 'diabetic tubulopathy' has emerged. tubular epithelial cells are particularly susceptible to the derangements of diabetic state because of the virtue of the high energy requirements and reliance on aerobic metabolism render. Hyperglycemia, oxidative stress, persistent chronic inflammation, glucose toxicity, advanced glycation end-products (AGEs) accumulation, lipid metabolism disorders, and lipotoxicity contribute to the cellular senescence and different patterns of regulated cell death (apoptosis, autophagic cell death, necroptosis, pyroptosis, and ferroptosis) in tubular epithelial cells. We now explore the 'tubulocentric' view of diabetic kidney disease(DKD). And we summarize recent discoveries regarding the development and regulatory mechanisms of cellular senescence, apoptosis, autophagic cell death, necroptosis, pyroptosis, and ferroptosis in the pathogenesis of DKD. These findings provide new perspectives on the mechanisms of DKD and are useful for designing novel therapeutic approaches for the treatment of DKD.

Inhibition of Renal Tubular Epithelial Mesenchymal Transition and Endoplasmic Reticulum Stress-Induced Apoptosis with Shenkang Injection Attenuates Diabetic Tubulopathy.

Background: The proximal renal tubule plays a critical role in diabetic kidney disease (DKD) progression. Early glomerular disease in DKD triggers a cascade of injuries resulting in renal tubulointerstitial disease. These pathophysiological responses are collectively described as diabetic tubulopathy (DT). Thus, therapeutic strategies targeting DT hold significant promise for early DKD treatment. Shenkang injection (SKI) has been widely used to treat renal tubulointerstitial fibrosis in patients with chronic kidney disease in China. However, it is still unknown whether SKI can alleviate DT. We designed a series of experiments to investigate the beneficial effects of SKI in DT and the mechanisms that are responsible for its effect on epithelial-to-mesenchymal transition (EMT) and endoplasmic reticulum (ER) stress-induced apoptosis in DT. Methods: The modified DKD rat models were induced by uni-nephrectomy, streptozotocin intraperitoneal injection, and a high-fat diet. Following the induction of renal injury, these animals received either SKI, rosiglitazone (ROS), or vehicle, for 42 days. For in vitro research, we exposed NRK-52E cells to high glucose (HG) and 4-phenylbutyric acid (4-PBA) with or without SKI or ROS. Changes in parameters related to renal tubular injury and EMT were analyzed in vivo. Changes in the proportion of apoptotic renal tubular cells and ER stress, and the signaling pathways involved in these changes, were analyzed both in vivo and in vitro. Results: SKI and ROS improved the general condition, the renal morphological appearance and the key biochemical parameters, and attenuated renal injury and EMT in the rat model of DKD. In addition, SKI and ROS alleviated apoptosis, inhibited ER stress, and suppressed PERK-eIF2α-ATF4-CHOP signaling pathway activation both in vivo and in vitro. Notably, our data showed that the regulatory in vitro effects of SKI on PERK-eIF2α-ATF4-CHOP signaling were similar to those of 4-PBA, a specific inhibitor of ER stress. Conclusion: This study confirmed that SKI can alleviate DT in a similar manner as ROS, and SKI achieves this effect by inhibiting EMT and ER stress-induced apoptosis. Our findings thereby provide novel information relating to the clinical value of SKI in the treatment of DT.

A more tubulocentric view of diabetic kidney disease.

Diabetic nephropathy (DN) is a common complication of Diabetes Mellitus (DM) Types 1 and 2, and prevention of end stage renal disease (ESRD) remains a major challenge. Despite its high prevalence, the pathogenesis of DN is still controversial. Initial glomerular disease manifested by hyperfiltration and loss of glomerular size and charge permselectivity may initiate a cascade of injuries, including tubulo-interstitial disease. Clinically, 'microalbuminuria' is still accepted as an early biomarker of glomerular damage, despite mounting evidence that its predictive value for DN is questionable, and findings that suggest the proximal tubule is an important link in the development of DN. The concept of 'diabetic tubulopathy' has emerged from recent studies, and its causative role in DN is supported by clinical and experimental evidence, as well as plausible pathogenetic mechanisms. This review explores the 'tubulocentric' view of DN. The recent finding that inhibition of proximal tubule (PT) glucose transport (via SGLT2) is nephro-protective in diabetic patients is discussed in relation to the tubule's potential role in DN. Studies with a tubulocentric view of DN have stimulated alternative clinical approaches to the early detection of diabetic kidney disease. There are tubular biomarkers considered as direct indicators of injury of the proximal tubule (PT), such as N-acetyl-ß-D-glucosaminidase, Neutrophil Gelatinase-Associated Lipocalin and Kidney Injury Molecule-1, and other functional PT biomarkers, such as Urine free Retinol-Binding Protein 4 and Cystatin C, which reflect impaired reabsorption of filtered proteins. The clinical application of these measurements to diabetic patients will be reviewed in the context of the need for better biomarkers for early DN.