1. Chronic high glucose downregulates mitochondrial calpain 10 and contributes to renal cell death and diabetes-induced renal injury 
Marisa D. Covington, Rick G. Schnellmann 
Kidney International 
Volume 81, Issue 4, Pages (February 2012)
DOI: /ki
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2. Figure 1
Calpain protein expression in glucose-treated renal proximal tubular cells (RPTCs). (a) RPTCs were incubated in 5mmol/l (Con) or 17mmol/l for various times, mitochondrial and cytoplasmic fractions isolated, and immunoblot analysis performed. β-Actin and heat shock protein 60 (HSP60) were used as loading controls.
Results were reproduced in four different experiments. (b) RPTCs incubated were in 17mmol/l D-mannitol over time and calpain 10 was measured in cytosol and mitochondria. (c) Mitochondrial calpain 10 activity was measured in RPTC mitochondria over time. Calpeptin, a calpain inhibitor, was used to verify calpain activity. (d) RPTCs were incubated with 17mmol/l D-mannitol, 17mmol/l glucose, or 5mmol/l glucose over time and mitochondrial calpain activity determined. Data are means±s.e.m., N≥4. +Significantly different from control; *Significantly different from time-matched sample (P≤0.05). SLLVY-AMC, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin.
Kidney International  , DOI: ( /ki )
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3. Figure 2
Calpain mRNA expression in glucose-treated renal proximal tubular cells (RPTCs). RPTCs were incubated in 17mmol/l glucose or 17mmol/l D-mannitol over time. Cells were collected, mRNA was isolated, and reverse transcriptase-PCR (RT-PCR) was performed using calpain 10, calpain 1, and β-actin primers. Data are means±s.e.m., N≥4. *Significantly different from D-mannitol (P≤0.05).
Kidney International  , DOI: ( /ki )
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4. Figure 3
Mitochondrial respiration in glucose-treated renal proximal tubular cells (RPTCs). RPTCs were incubated in 17mmol/l glucose or 17mmol/l D-mannitol over time. Cells were collected and (a) basal and (b) uncoupled respiration was measured. Uncoupled respiration was measured in the presence of 10μmol/l carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP). Data are means±s.e.m. (N=4). *Significantly different from D-mannitol (P≤0.05).
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5. Figure 4
NDUFB8 and adenosine triphosphate (ATP) synthase β protein expression in glucose-treated renal proximal tubular cells (RPTCs). RPTCs were incubated in (a) 17mmol/l glucose or (b) 17mmol/l D-mannitol over time. Cell lysates were subjected to immunoblot analysis for NDUFB8 and ATP synthase β. β-Actin was used as a loading control. mRNA was isolated and reverse transcriptase-PCR (RT-PCR) was performed using (c) NDUFB8 and (d) ATP synthase β primers. Data are means±s.e.m. (N=4).
Kidney International  , DOI: ( /ki )
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6. Figure 5
Apoptotic cell death in glucose-treated renal proximal tubular cells (RPTCs). RPTCs were incubated in 17mmol/l over time. Apoptosis was measured by (a, b) nuclear condensation and (c) procaspase 3 cleavage. (a) Nuclei were identified by 4,6-diamidino-2-phenylindole (DAPI) staining and visualized using a Nikon TE300 Eclipse Fluorescence microscope (Nikon, Melville, NY) with excitation and emission filters of 350 and 486nm, respectively. (b) Condensed nuclei from 10 non-overlapping fields were counted for each treatment group. Data are means±s.e.m., N=4. *Significantly different from control and 6h (P<0.05). (c) Cell lysates were subjected to immunoblot analysis for procaspase 3 and cleaved caspase 3 (17kDa). β-Actin was used as a loading control.
Kidney International  , DOI: ( /ki )
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7. Figure 6
Calpain expression in kidneys of diabetic models. Sprague–Dawley rats, 8 weeks of age (200–250g), were starved for 16h and injected once into the tail vein with streptozotocin (STZ; 55mg/kg) in sodium citrate buffer. At 10 weeks after induction of diabetes, rats were killed and blood and kidneys were harvested. Renal cell lysates and mRNA were isolated. (a) Immunoblot analysis for calpains 10, 1, and 2 was performed. (b) Reverse transcriptase-PCR (RT-PCR) analysis for calpains 10 and 1 was performed. (c) Immunoblot analysis was performed on ob/ob mice kidney samples.
Results were reproduced in at least four different animals.
Kidney International  , DOI: ( /ki )
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8. Figure 7
NDUFB8, adenosine triphosphate (ATP) synthase β, and caspase 3 protein expression in streptozotocin (STZ)-induced diabetic kidneys. STZ-induced diabetic kidneys were isolated and the tissue was homogenized. Immunoblot analysis for β-actin, NDUFB8, ATP synthase β, procaspase 3, and cleaved caspase 3 (17kDa) was (a) performed and (b) quantified (open bars are control animals and black bars are STZ-treated animals). (c) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells were determined. (d) NDUFB8 and ATP synthase β mRNA was examined using reverse transcriptase-PCR (RT-PCR).
Results were reproduced in at least four different animals. Data are means±s.e.m., N≥4. *Significantly different from control (P<0.05).
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

9. Figure 8
The effect of small interfering RNA (siRNA) on calpain protein in rat kidney. Eight-week-old rats were treated with 20nmol of siRNA directed against calpain 10 (Cal 10) or a scrambled siRNA (Neg) by tail vein injection. (a) Kidney cortex was isolated and calpain 10 protein was measured by immunoblot analysis. Calpain 10 and 1 mRNA was measured by reverse transcriptase-PCR (RT-PCR). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. (b) Blood was collected from rats and serum creatinine was determined. (c) Kidney cortex was isolated and procaspase 3 and cleaved caspase 3 (17kDa) protein was measured by immunoblot analysis. GAPDH served as a loading control. (d) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells were determined.
Results were reproduced in at least four different animals. Data are means±s.e.m., N≥4. *Significantly different from control (P<0.05).
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions