Empagliozin Alleviates Diabetic Renal Tubular Lipid Accumulation and NLRP3 Inammasome Activation Through AGEs-RAGE Pathway

: 45 Background: Advanced glycation end products (AGEs) are pathogenic factors of renal 46 tubular lipid accumulation and play a negative role in diabetic kidney disease (DKD). 47 Glucose cotransporter (SGLT) 2 inhibition offers strong renoprotection in the 48 progression of DKD. The aim of the current study was to investigate the effects of 49 empagliflozin (EMPA, a potent and selective SGLT2 inhibitor) on AGEs-induced renal 50 tubular lipid accumulation in both diabetic mice fed with a high-AGEs diet and 51 AGEs-treated cultured human renal proximal tubular epithelial (HK-2) cells. 52 Methods: In vivo , EMPA was used to treat db/db mice fed a high-AGEs diet or an 53 AIN-76 basal diet. In an in vitro study, HK-2 cells were treated with AGEs-bovine 54 serum albumin (BSA) and/or EMPA. Sterol regulatory element binding protein 55 (SREBP) cleavage-activating protein (SCAP) translocation was detected by confocal 56 microscopy. 57 Results: EMPA reduced tubular lipid droplets and intracellular cholesterol content, as 58 well as the expression of proteins involved in the synthesis and absorption of 59 cholesterol in the kidneys of basal diet-fed db/db mice, high-AGEs diet-fed db/db mice 60 and AGEs-BSA-treated HK-2 cells. AGEs-BSA loading promoted the formation of 61 SCAP-SREBP-2 complexes and enhanced the transport of the complexes to the Golgi, 62 but these effects were markedly inhibited by EMPA in HK-2 cells. EMPA reduced 63 renal inflammation both in basal diet-fed db/db mice and high-AGEs diet-fed db/db 64 mice, and suppressed NLRP3 inflammasome activation in AGEs-BSA-treated HK-2 65 cells. In addition, EMPA reduced the serum AGEs level in vivo and inhibited renal tubular endoplasmic reticulum (ER) stress and receptor of AGEs (RAGE) expression 67 both in vivo and in vitro . Conclusions: EMPA attenuated AGEs synthesis and inhibited the AGEs-RAGE signaling pathway, thereby suppressing ER stress and inhibiting abnormal cholesterol 70 metabolism and release of inflammatory cytokines, thus alleviating renal tubular lipid 71 accumulation and inflammation.

12 ‡ These authors contributed equally to this work. 13 14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  Diabetic kidney disease (DKD) is a chronic kidney disease caused by diabetes, 90 and almost 40% of diabetic patients with DKD [1]. DKD plays a major role in the 91 global burden of disease. Although the research on DKD has lasted for many years, the 92 comprehensive mechanism of DKD is still not fully understood. Metabolic changes 93 associated with diabetes lead to renal pathological changes and functional damage, 94 which are generally considered the leading causes of DKD. Among the many key 95 metabolic changes that lead to the onset of DKD, nonenzymatic glycosylation (NEG) 96 is a significant presence. NEG attaches reducing sugar to the free amino group of 97 protein through a series of events, which is irreversible. In this process, Schiff base and 98 Amadori products are formed, and advanced glycation end products (AGEs) are finally 99 produced [2]. In addition, the serum level of AGEs in patients with diabetes 100 complicated with renal insufficiency will be significantly increased in this process. 101 What's more, renal structure will suffer progressive damage caused by renal AGEs, 102 which will further lead to renal function damage in DKD patients [3].

104
Through the integration and analysis of many years of research, it can be found 105 that some previous description of the characteristic histological changes of DKD is not 106 comprehensive, and the focus is on diffuse and nodular glomerulosclerosis. However, 107 in the aspect of renal insufficiency of DKD, changes within the tubulointerstitium are 108 more important than glomerulopathy [4,5]. Some   in the kidneys of db/db mice fed with a high-AGEs diet (Fig. 6C); at the same time, 390 we also obtained consistent results in HK-2 cells, which further confirmed the 391 above phenomenon (Fig. 6D). However, EMPA treatment clearly reduced the 392 expression of RAGE both in the basal diet-fed db/db mice and high-AGEs diet-fed 393 db/db mice, and consistent results were confirmed in HK-2 cells (Fig. 6C and D).

396
This research is groundbreaking to some extent, mainly because this research has 397 created a new research topic, that is, the effect of EMPA on lipid accumulation and 398 inflammation induced by AGEs in diabetic renal tubules. Previously, our team found 399 that renal tubular injury is associated with lipid accumulation in high fat/sucrose diet 400 and streptozotocin-induced T2DM rats [14].    Values of intracellular cholesterol content are expressed as the means±S.E.M. of 5 independent experiments. **P < 0.01 versus the db/m group or the control cells (Ctr); # P < 0.05 and ## P < 0.01 versus db/db group or AGEs-BSA treated cells; P < 0.05 and P < 0.01 versus the AGEs+db/db group.

Figure 3
Effects of EMPA on HMGCoAR, LDLr, SREBP-2, nSREBP-2 and SCAP expression in the kidneys of diabetic mice and HK-2 cells. The protein expression of HMGCoAR, LDLr, SREBP-2, nSREBP-2 and SCAP in the kidneys of mice (A) and in HK-2 cells (B) was determined by Western blotting. ImageJ was used to quantify the relative levels of proteins. GAPDH or Lamin A was used as an internal control. The values are expressed as the mean ± S.E.M. of 5 independent experiments. *P < 0.05 and **P < 0.01 versus the db/m group or the control cells (Ctr); # P < 0.05 and ## P < 0.01, versus db/db group or AGEs-BSA treated cells; P < 0.01 versus the AGEs+db/db group.

Figure 4
Effect of EMPA on SCAP-SREBP-2 complexes in HK-2 cells. The translocation of GFP-SCAP from the ER to the Golgi was investigated using confocal microscopy after staining with anti-Golgi antibody. EMPA inhibited the translocation of GFP-SCAP from the ER to the Golgi in HK-2 cells (A). The interaction between SCAP and SREBP-2 proteins was investigated using Co-IP. AGEs-BSA increased the interactions of SCAP and SREBP-2 in HK-2 cells, whereas this could be inhibited by EMPA (B) Figure 5 Effects of EMPA on renal tubular in ammation in diabetic mice and HK-2 cells. HE (A) staining in the kidneys of mice was observed under a light microscope (×400). The protein expression of NLRP3 and IL-1β in the kidneys of mice (B) and in HK-2 cells (C) was determined by Western blotting. ImageJ was used to quantify the relative levels of proteins. GAPDH was used as an internal control. Values are expressed as the mean ± S.E.M. of 5independent experiments. NLRP3 in ammasome activation was detected by coimmunoprecipitation (D). **P < 0.01 versus the db/m group or the control cells (Ctr); # P < 0.05 and ## P < 0.01 versus db/db group or AGEs-BSA treated cells; P < 0.05 versus the AGEs+db/db group.

Figure 6
Effects of EMPA on ER stress and RAGE expression. The protein expression of GRP78, CHOP and RAGE in the kidneys of mice (A) and HK-2 cells (B) was determined by Western blotting. ImageJ was used to quantify the relative levels of proteins. GAPDH was used as an internal control. The values are expressed as the mean ± S.E.M. of 5 independent experiments. **P < 0.01 versus the db/m group or the control cells (Ctr); # P < 0.05 and ## P < 0.01 versus db/db group or AGEs-BSA treated cells; P < 0.05 and 715 P < 0.01 versus the AGEs+db/db group.

Figure 7
Role of EMPA in AGEs induced renal tubular lipid accumulation. AGEs-RAGE induces ER stress in the renal tubular epithelial cells, which stimulates the expression of SCAP and SREBP-2, causing increased formation of SCAP-SREBP-2 complexes and enhanced transport of the complexes to the Golgi, where SREBP-2 is hydrolyzed. The active fragments of SREBP-2 (nSREBP-2) enter the nucleus, thereby upregulating HMGCoAR and LDLr expression levels and ultimately, increase the cholesterol synthesis and uptake. Simultaneously, the NLRP3 in ammasome is activated and promotes the release of IL-1β, leading to renal tubulointerstitial in ammation. However, EMPA attenuates AGEs synthesis and inhibits the AGEs-