Glomerular Endothelial Mitochondrial Dysfunction Is Essential and Characteristic of Diabetic Kidney Disease Susceptibility - PubMed (original) (raw)

. 2017 Mar;66(3):763-778.

doi: 10.2337/db16-0695. Epub 2016 Nov 29.

Gabriella Casalena 1, Shaolin Shi 1, Liping Yu 1, Kerstin Ebefors 2, Yezhou Sun 1, Weijia Zhang 1, Vivette D'Agati 3, Detlef Schlondorff 1, Börje Haraldsson 2, Erwin Böttinger 1 4, Ilse Daehn 5 4

Affiliations

Glomerular Endothelial Mitochondrial Dysfunction Is Essential and Characteristic of Diabetic Kidney Disease Susceptibility

Haiying Qi et al. Diabetes. 2017 Mar.

Abstract

The molecular signaling mechanisms between glomerular cell types during initiation/progression of diabetic kidney disease (DKD) remain poorly understood. We compared the early transcriptome profile between DKD-resistant C57BL/6J and DKD-susceptible DBA/2J (D2) glomeruli and demonstrated a significant downregulation of essential mitochondrial genes in glomeruli from diabetic D2 mice, but not in C57BL/6J, with comparable hyperglycemia. Diabetic D2 mice manifested increased mitochondrial DNA lesions (8-oxoguanine) exclusively localized to glomerular endothelial cells after 3 weeks of diabetes, and these accumulated over time in addition to increased urine secretion of 8-oxo-deoxyguanosine. Detailed assessment of glomerular capillaries from diabetic D2 mice demonstrated early signs of endothelial injury and loss of fenestrae. Glomerular endothelial mitochondrial dysfunction was associated with increased glomerular endothelin-1 receptor type A (Ednra) expression and increased circulating endothelin-1 (Edn1). Selective Ednra blockade or mitochondrial-targeted reactive oxygen species scavenging prevented mitochondrial oxidative stress of endothelial cells and ameliorated diabetes-induced endothelial injury, podocyte loss, albuminuria, and glomerulosclerosis. In human DKD, increased urine 8-oxo-deoxyguanosine was associated with rapid DKD progression, and biopsies from patients with DKD showed increased mitochondrial DNA damage associated with glomerular endothelial EDNRA expression. Our studies show that DKD susceptibility was linked to mitochondrial dysfunction, mediated largely by Edn1-Ednra in glomerular endothelial cells representing an early event in DKD progression, and suggest that cross talk between glomerular endothelial injury and podocytes leads to defects and depletion, albuminuria, and glomerulosclerosis.

© 2017 by the American Diabetes Association.

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Figures

Figure 1

Figure 1

Diabetes-induced podocyte loss with progressive glomerular disease in D2 mice, not B6. ACR of control and STZ-treated B6 or D2 mice (A) and B6 Akita or D2 Akita mice (B) after 3, 6, and 12 weeks of diabetes (n = 6, ± SEM). Histopathology stain (PAS) of STZ-B6 (C) and STZ-D2 mice (D) after 12 weeks of diabetes (×40 magnification). Note the presence of sclerotic lesions (fibrosis and capillary loop destruction) in D. Picrosirius red staining for matrix deposits in 12-week diabetic B6 (E) and diabetic D2 (F) by brightfield (top) and polariscope (bottom). Arrows indicate large collagen fibers that showed yellow and orange birefringence under polarizing microscopy. G: Percentage glomerular methenamine SPA per glomerular section in control and 6 and 12 weeks of STZ-induced diabetes in B6 and D2 mice (SD). Podocyte number (WT-1–positive cells) per glomerular section in control and STZ-treated B6 or D2 mice (H) and B6 Akita or D2 Akita mice (I) after 3, 6, and 12 weeks of diabetes (mean ± SEM; >50 glomerular profiles/mouse, >5 mice per time point). **P < 0.01; ***P < 0.01.

Figure 2

Figure 2

Decreased mitochondrial function and increased mtDNA damage in glomeruli of diabetic D2 mice. A: Mitochondrial respiratory reserve capacity was measured by OCR (OCR uncoupled respiration [FCCP] over basal respiration) of isolated glomeruli from B6 and D2 controls, STZ-B6, and STZ-D2 mice with diabetes for the days indicated (values are mean percentage reduction in OCR ± SEM; n > 6 mice/time point). B: Immunofluorescent detection of oxidative DNA marker 8-oxoG in glomeruli from B6 (top) and D2 mice (bottom) with 3, 6, 12, and 24 weeks of STZ-induced diabetes and untreated control mice 0 weeks. Arrow indicates 8-oxoG–positive staining in tubular cells. C: Urine 8-oxodG (nmol) relative to urine creatinine (Creat) in B6 and D2 control mice and 3-week diabetic STZ-B6 or STZ-D2 mice. D: Quantification of lesion frequencies in mtDNA and nuclear DNA (nucDNA) by quantitative PCR in isolated glomeruli of B6 and D2 mice after 1 and 21 days of hyperglycemia (n = 6; ±SEM) relative amplification normalized to nondamaged (0 days controls). E: Representative images of double immunofluorescence staining detecting endothelial cell marker CD31 (red, top left), 8-oxoG (green, top middle), and merge (top right) or podocyte marker synaptopodin (SYNPO; red, bottom left), 8-oxoG (green, bottom middle), and merge (bottom right) in glomeruli of 3-week diabetic STZ-D2 mouse. *P < 0.05, **P < 0.01 versus untreated control mice.

Figure 3

Figure 3

Diabetes induced mtStress specifically in glomerular endothelial cells. A: Double immunofluorescence staining detecting CD31 (red, top left), 8-oxoG (green, top middle), synaptopodin (SYNPO; red, bottom left), 8-oxoG (green, bottom middle), and merge (top and bottom right) in kidney biopsy from a subject diagnosed with DKD (patient groups in

Supplementary Table 2

). B: Urine 8-oxodG (nmol) relative to urine creatinine in human control subjects and patients with diabetes with nonprogressive, intermediate progressive, and rapid progressive DKD (Table 1). One-way ANOVA with Tukey posttest was used to compare control patients to patients with progressive, intermediate, and nonprogressive disease. *P < 0.05, **P < 0.01 vs. control subjects. CKD, chronic kidney disease.

Figure 4

Figure 4

MitoTEMPO prevented diabetes-mediated mtStress in glomerular endothelial cells, podocyte loss, and albuminuria. A: Immunofluorescence detecting CD31-positive glomerular endothelial cell and mtDNA 8-oxoG in kidneys of STZ-D2 mice with 3-week diabetes. STZ treatment showing prominent glomerular 8-oxoG staining (A, top) and STZ cotreated with mitoTEMPO (1 mg/kg/day) showing reduced staining (A, bottom). B: CD31 and 8-oxoG in kidneys of Akita D2 mice after 3 weeks of diabetes (top) or diabetes with mitoTEMPO (1 mg/kg/day) (bottom). Urine 8-oxodG/creatinine of control D2 mice, STZ-D2, or STZ-D2 cotreated with mitoTEMPO (C) and 3-week diabetic Akita-D2 mice or Akita-D2 cotreated with mitoTEMPO (D) (n = 5; ±SEM). Podocyte number (percent of WT-1–positive cells/glomeruli) of control D2 mice, STZ-D2, or STZ-D2 cotreated with mitoTEMPO (E) and Akita-D2 mice or Akita-D2 cotreated with mitoTEMPO (n = 6; ±SEM) (F). ACR of control D2 mice, STZ-D2, or STZ-D2 plus mitoTEMPO (G) or Akita-D2 mice or Akita-D2 plus mitoTEMPO (H) (n = 5; ±SEM). In E and F, the percentage of WT-1–positive cell counts/glomeruli is mean ± SEM of at least 250 glomeruli from 5 to 6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control mice; +P < 0.05, ++P < 0.01 vs. diabetic mice.

Figure 5

Figure 5

Diabetes-mediated glomerular endothelial cell injury is ameliorated by mitoTEMPO. Scanning electron micrographs of control D2 (A), 3-week diabetic STZ-D2 (B), and STZ-D2 mice (C) cotreated with mitoTEMPO (1 mg/kg/day). D: EMI (see

research design and methods

) of control, 3-week STZ-D2, and STZ-D2 plus mitoTEMPO mice. Mean ± SEM of at least 90 capillary vessels of glomeruli from 5 mice per group. ***P < 0.001 vs. control mice; ++P < 0.01 vs. diabetic mice.

Figure 6

Figure 6

Expression of Ednra in glomerular endothelial cells, circulating Edn1 exclusively in DKD-susceptible mice, and increased EDNRA expression in glomerular endothelial cells in human DKD. A: Double immunofluorescence detecting Ednra (green) and endothelial cell marker CD31 (red) in glomeruli of 3-week diabetic STZ-D2 and STZ-B6 mice. B: Serum levels of Edn1 from control and 3-week diabetic B6 and D2 mice (n = 5 to 6 mice/group; ±SEM). C: EDNRA (red) and 8-oxoG (green) in kidney biopsy from a human control subject (top) and a patient diagnosed with DKD (bottom). Colocalization indicated by arrows. *P < 0.05 vs. control mice.

Figure 7

Figure 7

Edn1 plus Ednra mediates glomerular endothelial mitochondrial dysfunction and progression of DKD. A: Immunofluorescence detecting mtDNA 8-oxoG in prominent CD31-positive glomerular endothelial cells of 3-week diabetic STZ-D2 (top) and STZ-D2 mice cotreated with BQ-123 (0.1 nmol/kg/day) (bottom). B: Urine 8-oxodG/creatinine levels of control mice, STZ-D2, or STZ-D2 cotreated with BQ-123. ACR (C) and podocyte number (WT-1–positive cells/glomeruli) (D) of control mice, STZ-D2, or STZ-D2 cotreated with BQ-123. E: PAS staining of kidneys from STZ-D2 (3 weeks diabetic; left) and STZ-D2 cotreated with BQ-123 (right). F: The sclerosis index score from STZ-D2 ± BQ-123 was performed as previously described (58), and glomeruli were graded as follows: 0 represents no lesion, 1+ represents sclerosis of <25% of the glomerulus, whereas 2+, 3+, and 4+ represent sclerosis of >25–50, >50–75, and >75% of the glomerulus, respectively. WT-1–positive cell counts/glomeruli per section in D are mean ± SEM of at least 180 glomeruli from 6 mice per group. *P < 0.05, ***P < 0.001 vs. control mice; +P < 0.05, ++P < 0.01 vs. diabetic mice.

Figure 8

Figure 8

hGluc–induced glomerular endothelial Ednra expression is linked to increased mitochondrial ROS. A: Percent increase of MitoSOX bright fluorescent mGEC control cells in control (5 μmol glucose plus 25 mmol

d

-mannitol) or after culture with hGluc (30 mmol) or hGluc plus Edn1 (200 ng/mL) for 24 h in the absence or presence of mitoTEMPO (1 μg/mL). B: Superresolution image of mitochondrial networks by MitoTracker Red in control or hGluc-treated mGECs ± mitoTEMPO. C: mRNA expression of ND1 and NDUFV1 in mGEC controls treated with hGluc or hGluc plus mitoTEMPO for 24 h. D: Mean fluorescence intensity (MFI) of Ednra mGEC surface expression measured by FACS in control RPMI and hGluc in the absence or presence of mitoTEMPO (1 μg/mL). E: Quantification by ELISA of Edn1 release into the culture media after 24 h by control or hGluc-treated mGEC. F: mRNA expression of Edn1 and Ednra in mGEC controls or treated with hGluc for 24 h as indicated. Bars represent mean ± SEM of three to five independent experiments. *P < 0.05 vs. controls; +P < 0.05 vs. hGluc.

References

    1. Collins AJ, Kasiske B, Herzog C, et al. Excerpts from the United States Renal Data System 2004 annual data report: atlas of end-stage renal disease in the United States. Am J Kidney Dis 2005;45:A5–A7 - PubMed
    1. Finne P, Reunanen A, Stenman S, Groop PH, Grönhagen-Riska C. Incidence of end-stage renal disease in patients with type 1 diabetes. JAMA 2005;294:1782–1787 - PubMed
    1. Qi Z, Fujita H, Jin J, et al. Characterization of susceptibility of inbred mouse strains to diabetic nephropathy. Diabetes 2005;54:2628–2637 - PubMed
    1. Breyer MD, Böttinger E, Brosius FC 3rd, et al.; AMDCC . Mouse models of diabetic nephropathy. J Am Soc Nephrol 2005;16:27–45 - PubMed
    1. Meyer TW, Bennett PH, Nelson RG. Podocyte number predicts long-term urinary albumin excretion in Pima Indians with type II diabetes and microalbuminuria. Diabetologia 1999;42:1341–1344 - PubMed

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