Protection of mitochondria prevents high-fat diet–induced glomerulopathy and proximal tubular injury
Authors: Hazel H. Szeto, Shaoyi Liu, Yi Soong, Nazia Alam, Glen T. Prusky, Surya V. Seshan
Journal: Kidney International (2016)

Obesity is a major risk factor for the development of chronic kidney disease, even independent of its association with hypertension, diabetes, and dyslipidemia. The primary pathologic finding of obesity-related kidney disease is glomerulopathy, with glomerular hypertrophy, mesangial matrix expansion, and focal segmental glomerulosclerosis. Proposed mechanisms leading to renal pathology include abnormal lipid metabolism, lipotoxicity, inhibition of AMP kinase, and endoplasmic reticulum stress. Here we report dramatic changes in mitochondrial structure in glomerular endothelial cells, podocytes, and proximal tubular epithelial cells after 28 weeks of a high-fat diet in C57BL/6 mice. Treatment with SS-31, a tetrapeptide that targets cardiolipin and protects mitochondrial cristae structure, during high-fat diet preserved normal mitochondrial structure in all kidney cells, restored renal AMP kinase activity, and prevented intracellular lipid accumulation, endoplasmic reticulum stress, and apoptosis. SS-31 had no effect on weight gain, insulin resistance or hyperglycemia. However, SS-31 prevented loss of glomerular endothelial cells and podocytes, mesangial expansion, glomerulosclerosis, macrophage infiltration, and upregulation of proinflammatory (TNF-α, MCP-1, NF-κB) and profibrotic (TGF-β) cytokines. Thus, mitochondria protection can overcome lipotoxicity in the kidney and represent a novel upstream target for therapeutic development. Obesity is a major risk factor for the development of chronic kidney disease, even independent of its association with hypertension, diabetes, and dyslipidemia. The primary pathologic finding of obesity-related kidney disease is glomerulopathy, with glomerular hypertrophy, mesangial matrix expansion, and focal segmental glomerulosclerosis. Proposed mechanisms leading to renal pathology include abnormal lipid metabolism, lipotoxicity, inhibition of AMP kinase, and endoplasmic reticulum stress. Here we report dramatic changes in mitochondrial structure in glomerular endothelial cells, podocytes, and proximal tubular epithelial cells after 28 weeks of a high-fat diet in C57BL/6 mice. Treatment with SS-31, a tetrapeptide that targets cardiolipin and protects mitochondrial cristae structure, during high-fat diet preserved normal mitochondrial structure in all kidney cells, restored renal AMP kinase activity, and prevented intracellular lipid accumulation, endoplasmic reticulum stress, and apoptosis. SS-31 had no effect on weight gain, insulin resistance or hyperglycemia. However, SS-31 prevented loss of glomerular endothelial cells and podocytes, mesangial expansion, glomerulosclerosis, macrophage infiltration, and upregulation of proinflammatory (TNF-α, MCP-1, NF-κB) and profibrotic (TGF-β) cytokines. Thus, mitochondria protection can overcome lipotoxicity in the kidney and represent a novel upstream target for therapeutic development. Obesity is a major risk factor for the development of chronic kidney disease, even independently of its association with hypertension, diabetes, and dyslipidemia.1Wang Y. Chen X. Song Y. et al.Association between obesity and kidney disease: a systematic review and meta-analysis.Kidney Int. 2008; 73: 19-33Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 2Chertow G.M. Hsu C.Y. Johansen K.L. The enlarging body of evidence: obesity and chronic kidney disease.J Am Soc Nephrol. 2006; 17: 1501-1502Crossref PubMed Scopus (68) Google Scholar, 3de Vries A.P. Ruggenenti P. Ruan X.Z. et al.Fatty kidney: emerging role of ectopic lipid in obesity-related renal disease.Lancet Diabetes Endocrinol. 2014; 2: 417-426Abstract Full Text Full Text PDF Scopus (268) Google Scholar The primary pathologic finding of obesity-related kidney disease is glomerulopathy, with glomerular hypertrophy, mesangial matrix expansion, and focal segmental glomerulosclerosis.3de Vries A.P. Ruggenenti P. Ruan X.Z. et al.Fatty kidney: emerging role of ectopic lipid in obesity-related renal disease.Lancet Diabetes Endocrinol. 2014; 2: 417-426Abstract Full Text Full Text PDF Scopus (268) Google Scholar, 4Chen H.M. Liu Z.H. Zeng C.H. et al.Podocyte lesions in patients with obesity-related glomerulopathy.Am J Kidney Dis. 2006; 48: 772-779Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar Increasing evidence shows that podocyte stress is the primary driving force in the development of proteinuria and glomerulosclerosis. Patients with obesity-related glomerulopathy have reduced podocyte density, widened foot processes, and proteinuria.4Chen H.M. Liu Z.H. Zeng C.H. et al.Podocyte lesions in patients with obesity-related glomerulopathy.Am J Kidney Dis. 2006; 48: 772-779Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar Similar glomerular findings have been reported in mice after just 2 to 4 months on a high-fat diet (HFD), even before there is any significant increase in blood glucose.5Deji N. Kume S. Araki S. et al.Structural and functional changes in the kidneys of high-fat diet-induced obese mice.Am J Physiol Renal Physiol. 2009; 296: F118-F126Crossref PubMed Scopus (196) Google Scholar, 6Shevalye H. Lupachyk S. Watcho P. et al.Prediabetic nephropathy as an early consequence of the high-calorie/high-fat diet: relation to oxidative stress.Endocrinology. 2012; 153: 1152-1161Crossref PubMed Scopus (35) Google Scholar, 7Sun Y.B. Qu X. Howard V. et al.Smad3 deficiency protects mice from obesity-induced podocyte injury that precedes insulin resistance.Kidney Int. 2015; 88: 286-298Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 8Ruggiero C. Ehrenshaft M. Cleland E. et al.High-fat diet induces an initial adaptation of mitochondrial bioenergetics in the kidney despite evident oxidative stress and mitochondrial ROS production.Am J Physiol Endocrinol Metab. 2011; 300: E1047-E1058Crossref PubMed Scopus (90) Google Scholar, 9Wicks S.E. Nguyen T.T. Breaux C. et al.Diet-induced obesity and kidney disease - in search of a susceptible mouse model.Biochimie. 2016; 124: 65-73Crossref Scopus (23) Google Scholar Another prominent feature of obesity-related kidney disease is the accumulation of lipid vacuoles in podocytes, mesangial cells, and proximal tubular (PT) epithelial cells, suggesting that abnormal lipid metabolism and lipotoxicity may be the major cause of renal dysfunction.3de Vries A.P. Ruggenenti P. Ruan X.Z. et al.Fatty kidney: emerging role of ectopic lipid in obesity-related renal disease.Lancet Diabetes Endocrinol. 2014; 2: 417-426Abstract Full Text Full Text PDF Scopus (268) Google Scholar, 10Herman-Edelstein M. Scherzer P. Tobar A. et al.Altered renal lipid metabolism and renal lipid accumulation in human diabetic nephropathy.J Lipid Res. 2014; 55: 561-572Crossref PubMed Scopus (311) Google Scholar, 11Martinez-Garcia C. Izquierdo-Lahuerta A. Vivas Y. et al.Renal lipotoxicity-associated inflammation and insulin resistance affects actin cytoskeleton organization in podocytes.PLoS One. 2015; 10: e0142291Google Scholar Intracellular lipid accumulation causes oxidative stress, endoplasmic reticulum (ER) stress, cytoskeletal changes, and activation of proinflammatory processes.10Herman-Edelstein M. Scherzer P. Tobar A. et al.Altered renal lipid metabolism and renal lipid accumulation in human diabetic nephropathy.J Lipid Res. 2014; 55: 561-572Crossref PubMed Scopus (311) Google Scholar, 11Martinez-Garcia C. Izquierdo-Lahuerta A. Vivas Y. et al.Renal lipotoxicity-associated inflammation and insulin resistance affects actin cytoskeleton organization in podocytes.PLoS One. 2015; 10: e0142291Google Scholar, 12van der Heijden R.A. Bijzet J. Meijers W.C. et al.Obesity-induced chronic inflammation in high fat diet challenged C57BL/6J mice is associated with acceleration of age-dependent renal amyloidosis.Sci Rep. 2015; 5: 16474Crossref Scopus (52) Google Scholar Mitochondrial fatty acid β-oxidation is the major source of adenosine triphosphate (ATP) for the kidney. Excess fatty acids that are not oxidized by mitochondria are esterified with glycerol and deposited as lipid droplets. Gene analysis of renal biopsy samples from diabetic nephropathy patients revealed significant upregulation of pathways involved in lipid uptake and decreased expression of enzymes supporting fatty acid β-oxidation.10Herman-Edelstein M. Scherzer P. Tobar A. et al.Altered renal lipid metabolism and renal lipid accumulation in human diabetic nephropathy.J Lipid Res. 2014; 55: 561-572Crossref PubMed Scopus (311) Google Scholar Mice fed HFD also showed significant downregulation of key enzymes in β-oxidation and upregulation of enzymes involved in lipogenesis.13Kume S. Uzu T. Araki S. et al.Role of altered renal lipid metabolism in the development of renal injury induced by a high-fat diet.J Am Soc Nephrol. 2007; 18: 2715-2723Crossref PubMed Scopus (189) Google Scholar Importantly, there was a significant correlation between lipid metabolism gene expression and glomerular filtration rate in diabetic nephropathy.10Herman-Edelstein M. Scherzer P. Tobar A. et al.Altered renal lipid metabolism and renal lipid accumulation in human diabetic nephropathy.J Lipid Res. 2014; 55: 561-572Crossref PubMed Scopus (311) Google Scholar AMP kinase (AMPK) plays a major role in lipid metabolism. Activation of AMPK reduces fatty acid synthesis and increases uptake and oxidation of fatty acids by mitochondria. HFD decreases AMPK activity in multiple tissues, including the kidney.13Kume S. Uzu T. Araki S. et al.Role of altered renal lipid metabolism in the development of renal injury induced by a high-fat diet.J Am Soc Nephrol. 2007; 18: 2715-2723Crossref PubMed Scopus (189) Google Scholar, 14Lindholm C.R. Ertel R.L. Bauwens J.D. et al.A high-fat diet decreases AMPK activity in multiple tissues in the absence of hyperglycemia or systemic inflammation in rats.J Physiol Biochem. 2013; 69: 165-175Crossref PubMed Scopus (97) Google Scholar, 15Decleves A.E. Mathew A.V. Cunard R. et al.AMPK mediates the initiation of kidney disease induced by a high-fat diet.J Am Soc Nephrol. 2011; 22: 1846-1855Crossref PubMed Scopus (176) Google Scholar Treatment with the AMPK activator (5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside) prevented glomerulopathy and tubulointerstitial fibrosis.15Decleves A.E. Mathew A.V. Cunard R. et al.AMPK mediates the initiation of kidney disease induced by a high-fat diet.J Am Soc Nephrol. 2011; 22: 1846-1855Crossref PubMed Scopus (176) Google Scholar, 16Decleves A.E. Zolkipli Z. Satriano J. et al.Regulation of lipid accumulation by AMP-activated kinase in high fat diet-induced kidney injury.Kidney Int. 2014; 85: 611-623Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar Chronic 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside treatment also reduced lipid accumulation in tubular cells.16Decleves A.E. Zolkipli Z. Satriano J. et al.Regulation of lipid accumulation by AMP-activated kinase in high fat diet-induced kidney injury.Kidney Int. 2014; 85: 611-623Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar There is evidence to suggest that obesity and HFD may inhibit mitochondrial fatty acid β-oxidation in the kidney. There are numerous reports of abnormal mitochondrial structures associated with obesity. PT mitochondria of ob/ob mice are small and rounded with loss of cristae membranes and matrix density.17Stacchiotti A. Favero G. Giugno L. et al.Mitochondrial and metabolic dysfunction in renal convoluted tubules of obese mice: protective role of melatonin.PLoS One. 2014; 9: e111141Crossref PubMed Scopus (46) Google Scholar Mitochondrial fragmentation, decreased ATP content, and increased H2O2 emission in kidney mitochondria have also been reported in mice after HFD, in ob/ob mice, and in a rat model of type 1 diabetes.8Ruggiero C. Ehrenshaft M. Cleland E. et al.High-fat diet induces an initial adaptation of mitochondrial bioenergetics in the kidney despite evident oxidative stress and mitochondrial ROS production.Am J Physiol Endocrinol Metab. 2011; 300: E1047-E1058Crossref PubMed Scopus (90) Google Scholar, 18Munusamy S. do Carmo J.M. Hosler J.P. et al.Obesity-induced changes in kidney mitochondria and endoplasmic reticulum in the presence or absence of leptin.Am J Physiol Renal Physiol. 2015; 309: F731-F743Crossref Scopus (20) Google Scholar, 19Coughlan M.T. Nguyen T.V. Penfold S.A. et al.Mapping time-course mitochondrial adaptations in the kidney in experimental diabetes.Clin Sci (Lond). 2016; 130: 711-720Crossref Scopus (83) Google Scholar Here we report structural changes indicative of lipid accumulation in PT cells and dramatic mitochondrial damage in tubular cells, podocytes, and glomerular endothelial cells after 28 weeks of HFD in C57BL/6 mice. Protection of mitochondrial structures by SS-31 (d-Arg-2′,6′-dimethylTyr-Lys-Phe-NH2), a peptide that targets cardiolipin and protects mitochondrial cristae structure,20Birk A.V. Liu S. Soong Y. et al.The mitochondrial-targeted compound SS-31 re-energizes ischemic mitochondria by interacting with cardiolipin.J Am Soc Nephrol. 2013; 24: 1250-1261Crossref PubMed Scopus (297) Google Scholar, 21Birk A.V. Chao W.M. Bracken C. et al.Targeting mitochondrial cardiolipin and the cytochrome c/cardiolipin complex to promote electron transport and optimize mitochondrial ATP synthesis.Br J Pharmacol. 2014; 171: 2017-2028Crossref PubMed Scopus (190) Google Scholar, 22Szeto H.H. Birk A.V. Serendipity and the discovery of novel compounds that restore mitochondrial plasticity.Clin Pharmacol Ther. 2014; 96: 672-683Crossref PubMed Scopus (116) Google Scholar preserved normal mitochondrial structure in all kidney cells, restored AMPK activity, and prevented intracellular lipid accumulation and ER stress during HFD. SS-31 also prevented the loss of glomerular endothelial cells and podocytes, mesangial expansion, glomerulosclerosis, macrophage infiltration, and upregulation of proinflammatory and profibrotic cytokines. Interestingly, SS-31 had no effect on weight gain, insulin resistance, or hyperglycemia. These findings suggest that mitochondria protection can overcome lipotoxicity of the kidney and support the mitochondrion as a novel target for development of renal therapeutics. C57BL/6 mice were maintained on a normal diet (ND) or HFD for 28 weeks. This specific diet has been reported to cause a significant increase in body weight, body fat, blood glucose, and insulin, after 16 weeks in C57BL/6 mice.23Kang L. Dai C. Lustig M.E. et al.Heterozygous SOD2 deletion impairs glucose-stimulated insulin secretion, but not insulin action, in high-fat-fed mice.Diabetes. 2014; 63: 3699-3710Crossref PubMed Scopus (36) Google Scholar HFD mice received a 5-day regimen of low-dose streptozotocin (STZ) 4 weeks after the start of HFD to minimize hyperinsulinemia. HFD mice were then treated with either saline solution or SS-31 daily (1 mg/kg s.c.) for 8 to 28 weeks. The body weight of mice fed HFD was only slightly increased after 8 weeks of HFD relative to ND, but was dramatically increased after 28 weeks (Figure 1a). Nonfasting blood glucose was slightly increased after 8 weeks (Figure 1b), indicating that the dose of STZ did not cause a significant loss of pancreatic β cells. Oral glucose tolerance tests administered after 8 and 28 weeks of HFD revealed abnormal glucose clearance (Figure 1c and d), suggesting the development of insulin resistance before the onset of hyperglycemia. Plasma insulin has been reported to be significantly elevated in C57BL/6 mice after just 4 weeks of HFD.13Kume S. Uzu T. Araki S. et al.Role of altered renal lipid metabolism in the development of renal injury induced by a high-fat diet.J Am Soc Nephrol. 2007; 18: 2715-2723Crossref PubMed Scopus (189) Google Scholar Treatment with SS-31 had no effect on body weight, glucose clearance, or blood glucose after 8 and 28 weeks of HFD (Figure 1a–d). Pathology in the early stages of obesity-related renal disease in humans is primarily related to glomerular changes.24Thakur V. Morse S. Reisin E. Functional and structural renal changes in the early stages of obesity.Contrib Nephrol. 2006; 151: 135-150Crossref PubMed Scopus (11) Google Scholar Periodic acid–Schiff staining of mouse kidneys after 28 weeks of HFD revealed capillary collapse and mesangial expansion (Figure 2a). Preliminary studies showed that HFD alone, but not STZ alone, caused these glomerular changes (Supplementary Figure S1). Collagen IV deposition was detected by immunohistochemical staining (Figure 2b). Trichrome staining showed glomerulosclerosis (Figure 2c). Mesangial expansion is confirmed by transmission electron microscopy (TEM) (Figure 2d). There was no change in glomerular size with HFD (Figure 2e), but there was a significant increase in periodic acid–Schiff–positive matrix and collagen IV deposition (Figure 2f and g). Transforming growth factor-β (TGF-β) plays a key role in renal fibrosis, and we detected a significant increase in TGF-β expression in renal cortical tissue after HFD (Figure 2h). SS-31 treatment completely prevented the upregulation of TGF-β collagen deposition and glomerulosclerosis (Figure 2a–h) despite having no effect on body weight, blood glucose, or glucose clearance (Figure 1).Figure 2SS-31 prevents glomerular mesangial expansion and collagen IV deposition in glomeruli of mice fed a high-fat diet (HFD). (a) Representative images of periodic acid–Schiff (PAS) stain of glomeruli from normal diet (ND), HFD, and HFD+SS-31 mice. (b) Representative images of collagen IV immunostaining of glomeruli from ND, HFD, and HFD+SS-31 mice. (c) Representative images of trichrome stain of glomeruli from ND, HFD, and HFD+SS-31 mice. (d) Representative transmission electron microscopic images of glomeruli from ND, HFD, and HFD+SS-31 mice showing podocytes (P), endothelial cells (E), capillary loops (CL), and mesangium (M). (e) Glomerular size in ND, HFD, and HFD+SS-31 mice. (f,g) Densitometric analyses of PAS and collagen IV staining show significant mesangial expansion and collagen deposition in HFD mice that was significantly reduced by SS-31 treatment (4 in each group). (h) Representative Western blot and densitometric analysis of transforming growth factor-β (TGF-β expression in renal cortical tissue from ND, HFD, and HFD+SS-31 mice [4 in each group]). All data are shown as mean ± SEM. Statistical analyses were performed by 1-way analysis of variance followed by Tukey's multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.View Large Image Figure ViewerDownload (PPT) TEM indicated compression of glomerular capillaries in HFD mice (Figure 2d). A significant decrease in CD31, the endothelial marker, confirmed the loss of glomerular capillaries (Figure 3a). Treatment with SS-31 significantly preserved CD31 expression (Figure 3b). In addition to the loss of glomerular endothelial cells, HFD led to endothelial injury, as indicated by a dramatic increase in the expression of von Willebrand factor (vWF) (Figure 3a). vWF is a glycoprotein that mediates platelet adhesion to the subendothelium at sites of vascular injury and is not normally expressed in human glomeruli.25Pusztaszeri M.P. Seelentag W. Bosman F.T. Immunohistochemical expression of endothelial markers CD31, CD34, von Willebrand factor, and Fli-1 in normal human tissues.J Histochem Cytochem. 2006; 54: 385-395Crossref PubMed Scopus (585) Google Scholar vWF was barely detected in the glomeruli from ND mice, but expression was increased 10-fold after 28 weeks of HFD and was significantly prevented by SS-31 treatment (Figure 3c). Vascular endothelial growth factor is constitutively expressed in normal kidneys but is upregulated in experimental animals and humans with diabetes.26Schrijvers B.F. Flyvbjerg A. De Vriese A.S. The role of vascular endothelial growth factor (VEGF) in renal pathophysiology.Kidney Int. 2004; 65: 2003-2017Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar Vascular endothelial growth factor expression was increased almost 4-fold after 28 weeks of HFD and was significantly reduced by SS-31 treatment (Figure 3d). Obesity-related glomerulopathy is associated with decreased podocyte density and an increase in foot-process width.4Chen H.M. Liu Z.H. Zeng C.H. et al.Podocyte lesions in patients with obesity-related glomerulopathy.Am J Kidney Dis. 2006; 48: 772-779Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 5Deji N. Kume S. Araki S. et al.Structural and functional changes in the kidneys of high-fat diet-induced obese mice.Am J Physiol Renal Physiol. 2009; 296: F118-F126Crossref PubMed Scopus (196) Google Scholar Wilms tumor 1 and the cyclin-dependent kinase inhibitor p57 are widely used as markers for podocytes within the glomerulus.27Pichaiwong W. Hudkins K.L. Wietecha T. et al.Reversibility of structural and functional damage in a model of advanced diabetic nephropathy.J Am Soc Nephrol. 2013; 24: 1088-1102Crossref PubMed Scopus (130) Google Scholar Podocyte number, as determined by nuclear staining of Wilms tumor 1, was significantly reduced with HFD but abolished by SS-31 treatment (Figure 4a and b ). The reduction in Wilms tumor 1–positive nuclei corresponded to a reduction in the expression of p57 as determined by Western blot (Figure 4c). TEM shows that surviving podocytes in HFD mice have irregular shapes with flattened foot processes and some areas of effacement, which were prevented by SS-31 treatment (Figure 4d). HFD feeding is known to cause macrophage infiltration in the kidney and increase plasma tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein 1 (MCP-1).12van der Heijden R.A. Bijzet J. Meijers W.C. et al.Obesity-induced chronic inflammation in high fat diet challenged C57BL/6J mice is associated with acceleration of age-dependent renal amyloidosis.Sci Rep. 2015; 5: 16474Crossref Scopus (52) Google Scholar We found a significant increase in the number of CD68+ macrophages per glomerulus after 28 weeks of HFD (Figure 5a and b ). Glomerular macrophage infiltration was associated with a significant increase in proinflammatory markers in renal cortical tissue, including MCP-1 (Figure 5c) and TNF-α (Figure 5d). The nuclear factor-κB is the primary regulator of the inflammatory response, and HFD significantly increased p65–nuclear factor-κB expression (Figure 5e). Treatment with SS-31 significantly reduced the number of macrophages by 50% (Figure 5b) and abolished the increase in all the proinflammatory markers (Figure 5c–e). Tubular dysfunction occurs later than glomerular injury in obesity-related kidney injury or diabetic nephropathy.28Reidy K. Kang H.M. Hostetter T. et al.Molecular mechanisms of diabetic kidney disease.J Clin Invest. 2014; 124: 2333-2340Crossref PubMed Scopus (505) Google Scholar After 28 weeks of HFD, periodic acid–Schiff stain of the outer medulla showed PT cells to be relatively normal with a preserved brush border (Figure 6a). Closer examination, however, revealed vacuolization in most PT cells (Figure 6b). Such vacuolization was previously described as lipid droplets in C57BL/6 mice after 14 weeks of HFD.29Mount P. Davies M. Choy S.W. et al.Obesity-related chronic kidney disease-the role of lipid metabolism.Metabolites. 2015; 5: 720-732Crossref Scopus (45) Google Scholar PT cells were further examined by TEM (Figure 6c–j). Mice fed ND have a normal brush border and many elongated mitochondria (Figure 6c). Although the brush border appears normal in mice fed HFD, numerous lipid droplets fill the cytoplasm (Figure 6d–h). Some of the droplets are very large (Figure 6d), but most are microscopic, with or without a limiting membrane (Figure 6e–h). Some lipid droplets are surrounded by crescent-shaped, double-walled membranes, consistent with macrolipophagy (Figure 6f–h).30Dong H. Czaja M.J. Regulation of lipid droplets by autophagy.Trends Endocrinol Metab. 2011; 22: 234-240Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar Others are autophagosomes containing only lipids (Figure 6g) or myelin figures (Figure 6h). The peripheral chromatin condensation seen in Figure 6d is suggestive of early apoptotic activity caused by HFD. PT cells from HFD mice treated with SS-31 do not show accumulation of lipid droplets or autophagic activity (Figure 6i and j). Terminal deoxynucleotidyltransferase–mediated dUTP nick end-labeling (TUNEL) stain was used to quantitatively assess the incidence of apoptosis in the kidney. TUNEL-positive cells were rarely found in glomeruli of ND mice (Figure 7a). In HFD mice, there was a significant increase in TUNEL-positive cells in the glomerular tuft (Figure 7c) and in parietal epithelial cells (Figure 7d). A significant increase in TUNEL-positive cells was also found in the outer medulla of HFD mice (Figure 7e). Treatment with SS-31 significantly reduced apoptosis in all cell types (Figure 7a–e). Despite a significant increase in tubular apoptosis, there was no significant change in peritubular capillary density, macrophage infiltration, or interstitial fibrosis in HFD mice (Supplementary Figure S2). The AMPK pathway is a key regulator of intracellular lipid metabolism in the kidney and other tissues.13Kume S. Uzu T. Araki S. et al.Role of altered renal lipid metabolism in the development of renal injury induced by a high-fat diet.J Am Soc Nephrol. 2007; 18: 2715-2723Crossref PubMed Scopus (189) Google Scholar, 15Decleves A.E. Mathew A.V. Cunard R. et al.AMPK mediates the initiation of kidney disease induced by a high-fat diet.J Am Soc Nephrol. 2011; 22: 1846-1855Crossref PubMed Scopus (176) Google Scholar, 31Long Y.C. Zierath J.R. AMP-activated protein kinase signaling in metabolic regulation.J Clin Invest. 2006; 116: 1776-1783Crossref PubMed Scopus (764) Google Scholar As reported previously,14Lindholm C.R. Ertel R.L. Bauwens J.D. et al.A high-fat diet decreases AMPK activity in multiple tissues in the absence of hyperglycemia or systemic inflammation in rats.J Physiol Biochem. 2013; 69: 165-175Crossref PubMed Scopus (97) Google Scholar, 15Decleves A.E. Mathew A.V. Cunard R. et al.AMPK mediates the initiation of kidney disease induced by a high-fat diet.J Am Soc Nephrol. 2011; 22: 1846-1855Crossref PubMed Scopus (176) Google Scholar, 16Decleves A.E. Zolkipli Z. Satriano J. et al.Regulation of lipid accumulation by AMP-activated kinase in high fat diet-induced kidney injury.Kidney Int. 2014; 85: 611-623Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar we found a significant decrease in the phospho-AMPK/AMPK ratio in renal cortical tissue of HFD mice, and this was ameliorated by SS-31 treatment (Figure 8a). In contrast, HFD had no effect on Akt activity (phospho-Akt/Akt ratio) (Figure 8b), suggesting no change in insulin sensitivity in the kidney with HFD with or without SS-31 treatment. Decreased AMPK activity and increased intracellular lipid content may reflect mitochondrial dysfunction caused by HFD.32Reznick R.M. Shulman G.I. The role of AMP-activated protein kinase in mitochondrial biogenesis.J Physiol. 2006; 574: 33-39Crossref PubMed Scopus (291) Google Scholar PT cells in ND mice contain numerous elongated mitochondria with densely stacked cristae membranes that are organized along membrane compartments (Figure 9a and b ). In contrast, PT mitochondria in HFD mice are small and disorganized (Figure 9c and d). Cristae membranes are few in most HFD mitochondria and are replaced by homogenized matrix (Figure 9e and f). Some mitochondria appear to have lipid deposited between the inner and outer mitochondrial membranes, often with rupture of the outer mitochondrial membrane (Figure 9g). Conversely, HFD mice treated with SS-31 had elongated mitochondria with normal cristae density (Figure 9h and i). The cell body of ND podocytes contains abundant rough ER and many mitochondria (Figure 10a–c ). The mitochondria are typically very small with double membranes and a dense matrix, even though individual cristae membranes are often difficult to visualize (Figure 10c). Podocytes from HFD mice show fewer organelles in the cytoplasm (Figure 10e). Some HFD podocytes contain large round vacuoles that are distended rough ER (Figure 10f). In addition, mitochondria are very small, and there are a loss of cristae membranes and reduced matrix density (Figure 10g). In contrast, podocytes from HFD mice treated with SS-31 show normal density of cellular organelles (Figure 10i and j), and mitochondria have normal matrix density and cristae membranes (Figure 10k). Few mitochondria are normally seen in endothelial cells (Figure 10d). Although they are very small, they can be identified by their double membrane and cristae structure. Endothelial mitochondria in HFD mice have intact double membranes but are rounded and have lost cristae membranes and matrix density (Figure 10h). Endothelial mitochondria in HFD mice treated with SS-31 maintained their elongated appearance, cristae structure, and matrix density (Figure 10-l). Human and experimental studies have revealed an association between lipid accumulation and structural and functional changes in podocytes, mesangial cells, and PT cells.3de Vries A.P. Ruggenenti P. Ruan X.Z. et al.Fatty kidney: emerging role of ectopic lipid in obesity-related renal disease.Lancet Diabetes Endocrinol. 2014; 2: 417-426Abstract Full Text Full Text PDF Scopus (268) Google Scholar However, the exact mechanism by which lipid accumulation produces glomerulopathy or tubulointerstitial fibrosis is not fully understood. Our results show that excess lipids in the kidney lead to mitochondrial damage in all renal cell types, with reduced size, loss of cristae membranes, and decreased matrix density. Because the protein complexes of the electron transport chain are located on the cristae membranes, the loss of cristae membranes contributes to a loss of ATP prod