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.

P0979BENEFICIAL EFFECT OF CHLOROQUINE AND AMODIAQUINE ON DIABETIC TUBULOPATHY BY ATTENUATING MITOCHONDRIAL NOX4 AND ENDOPLASMIC RETICULUM STRESS

Background and Aims 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 and amodiaquine for inhibiting mitochondrial Nox4 and diabetic tubular injury. Method 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 C57/BL6J mice. Chloroquine and amodiaquine were administered to the mice via intraperitoneal injection for 14 weeks. Results Chloroquine and amodiaquine 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, chloroquine and amodiaquine 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 chloroquine and amodiaquine treatment. Conclusion We substantiated the protective actions of chloroquine and amodiaquine 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. Figure None

Clinical Utility of Urinaryβ2-Microglobulin in Detection of Early Nephropathy in African Diabetes Mellitus Patients

Background. Studies have indicated that diabetic tubulopathy may occur earlier than glomerulopathy, therefore providing a potential avenue for earlier diagnosis of diabetic nephropathy. Urinary beta-2-microglobulin (β2m) was investigated in this study as a potential biomarker in the detection of early nephropathy in type 2 diabetics.Methods. One hundred and two diabetic subjects and 103 controls that met the inclusion criteria had data (sociodemographic, medical history, physical examination, and laboratory) collected. Urinaryβ2m levels and urinary albumin concentration (UAC) were determined.Results. Elevated urinaryβ2m was more frequent among the diabetics (52%, 95% CI: 42.1–61.8%) than among the controls (32%, 95% CI: 22.9–41.2%). The frequency of microalbuminuria was higher in the diabetics (35.3%, 95% CI: 25.9–44.7%) than in the controls (15.5%, 95% CI: 8.4–22.6%). There was a positive correlation between urinaryβ2m and UAC (rho = 0.38,p<0.001). Multivariate analysis showed BMI (OR: 1.23, 95% CI: 1.05–1.45), eGFR (OR: 0.97, 95% CI: 0.94–0.99), and presence of microalbuminuria (OR: 3.94, 95% CI: 1.32–11.77) as independent predictors of elevated urinary beta-2-microglobulin among the diabetics.Conclusion. Urinaryβ2m may be useful, either as a single test or as a component of a panel of tests, in the early detection of diabetic nephropathy.

PGC1αActivators Mitigate Diabetic Tubulopathy by Improving Mitochondrial Dynamics and Quality Control

Purpose.In this study, we investigated the effect of PGC1αactivators on mitochondrial fusion, fission, and autophagic quality control in renal tubular cells in a diabetic environment in vivo and in vitro. We also examined whether the upregulation of PGC1αattenuates diabetic tubulopathy by normalizing mitochondrial homeostasis.Methods. HKC8 cells were subjected to high-glucose conditions (30 mM D-glucose). Diabetes was induced with streptozotocin (STZ, 50 mg/kg i.p. for 5 days) in male C57/BL6J mice. AICAR or metformin was used as a PGC1αactivator.Results.Treatment with the PGC1αactivators AICAR and metformin improved functional mitochondrial mass in HKC8 cells in high-glucose conditions. Moreover, in renal proximal tubular cells, increased PGC1αactivity correlated with the reversal of changes in Drp1, Mfn1, and LC3-II protein expression in a high-glucose environment. Normalized mitochondrial life cycles resulted in low ROS production and reduced apoptosis. AICAR and metformin treatment effectively mitigated albuminuria and renal histopathology and decreased the expression of TGFβ1 andαSMA in the kidneys of diabetic mice.Conclusions. Our results demonstrate that increases in PGC1αactivity improve diabetic tubulopathy by modulating mitochondrial dynamics and autophagy.