Chronic high glucose

Chronic high glucose downregulates mitochondrial calpain 10and contributes to renal cell death and diabetes-induced renal injury

Marisa D.Covington 1and Rick G.Schnellmann 1,2

1

Center for Cell Death,Injury and Regeneration,Department of Pharmaceutical and Biomedical Sciences,Medical University of South Carolina,Charleston,South Carolina,USA and 2Ralph H.Johnson Veterans Administration Medical Center,Charleston,South Carolina,USA

Whereas most calpains are cytosolic proteases,calpain 10is resident in mitochondria and is important in mitochondrial homeostasis.Because calpain 10has been implicated in type 2diabetes,we studied its possible role in diabetes-induced renal dysfunction.We treated renal proximal tubular cells with high glucose (17mmol/l)and found decreased mitochondrial calpain 10mRNA and protein at 96h

compared with cells incubated with 0or 5mmol/l glucose or 17mmol/l D -mannitol.High glucose increased mitochondrial calpain 10substrates (NDUFB8and ATP synthase b ),

decreased basal and uncoupled respiration,and initiated cell apoptosis as indicated by cleaved caspase 3and nuclear condensation.Renal calpain 10protein and mRNA were specifically decreased in streptozotocin-induced diabetic rats with kidney dysfunction,and in diabetic ob/ob mice.In agreement with our in vitro data,the kidneys of

streptozotocin-induced diabetic rats had elevated calpain 10substrates and cleaved caspase 3.Finally,specific siRNA-induced knockdown of calpain 10in the proximal tubules of control rats resulted in decreased renal function as evidenced by increased serum creatinine,and increased caspase 3cleavage compared with rats receiving scrambled siRNA.Thus,the glucose-induced loss of calpain 10in vivo results in renal cell apoptosis and organ failure through accumulation of mitochondrial calpain 10substrates and mitochondrial dysfunction.Whether this is a major cause of the decreased renal function in diabetic nephropathy will require further studies.

Kidney International (2012)81,391–400;doi:10.1038/ki.2011.356;published online 19October 2011

KEYWORDS:acute kidney injury;apoptosis;diabetic nephropathy;hyperglycemia;mitochondria

The most prevalent cause of chronic renal failure and end-stage renal disease is diabetic nephropathy—previously described as a glomerulopathy associated with diffuse or nodular glomerulosclerosis—although fewer than one-third of diabetic patients have this type of glomerulopathy.1,2Proximal tubular functional and structural changes correlate better with diabetic nephropathy progression,and may be key to kidney dysfunction development in diabetes.3,4

Disruption of mitochondrial function is important in various diseases including diabetes,and diabetes induced in rats by streptozotocin (STZ)or alloxan treatment results in impaired mitochondrial respiration and disruption of energy production and mitochondrial protein synthesis in the kidney.5–8Ultimately,changes in mitochondrial viability lead to cell death via apoptosis or necrosis,but the exact mechanism of mitochondrial dysfunction in diabetic nephropathy is unclear.

Calpain 10is a ubiquitously expressed ‘atypical’calpain.9,10Calpain 10gained much attention following a genome-wide linkage scan for susceptibility genes associated with dia-betes.11,12Since calpain 10was identi?ed as a type 2diabetes susceptibility gene in 2000,there have been numerous reports supporting and refuting this proposed association.11,13Never-theless,calpain 10has been implicated in both insulin-stimulated glucose uptake 14,15and insulin secretion 16,17in islet cells.

Whereas calpains were traditionally thought to be cytosolic proteases,our laboratory identi?ed calpain 10as the resident mitochondrial calpain in isolated rabbit,mouse,and rat renal mitochondria with primary activity in the matrix.18,19In addition,calpain 10was shown to be a mediator of Ca 2t-induced mitochondrial dysfunction through cleavage of complex I subunits,NDUFV2and NDUFB8,of the electron transport chain.18Interestingly,overexpression of calpain 10in NIH-3T3cells resulted in mitochondrial swelling,autophagy,and cell death,18suggest-ing that calpain 10is important in mitochondrial homeo-stasis.In contrast,knockdown of calpain 10protein expression resulted in renal proximal tubular cell (RPTC)apoptosis,20illustrating the double-edged sword of calpain 10.

https://www.360docs.net/doc/6512369707.html, o r i g i n a l a r t i c l e

&2012International Society of Nephrology

Received 1June 2010;revised 1September 2011;accepted 7September 2011;published online 19October 2011

Correspondence:Rick G.Schnellmann,Center for Cell Death,Injury and Regeneration,Department of Pharmaceutical and Biomedical Sciences,Medical University of South Carolina,280Calhoun Street POB 250140,Charleston,South Carolina 29425,USA.E-mail:schnell@https://www.360docs.net/doc/6512369707.html,

We also observed a decrease in renal calpain10protein and

mRNA expression in aged humans,mice,and rats that was

attenuated with caloric restriction and correlated with

decreased kidney function in rats.20These studies provide

evidence that renal calpain10is important in age-related loss

of renal function.The goal of this study was to determine the

role of calpain10in renal dysfunction observed in diabetes

using two different animal models of diabetes:an RPTC

in vitro model and an in vivo rat kidney model after calpain10

knockdown.

RESULTS

Elevated glucose biphasically regulates mitochondrial calpain10in RPTCs

To investigate the effects of elevated glucose on calpain

protein expression over time,RPTCs were treated with

17mmol/l glucose for3–144h.Physiological glucose is B5mmol/l;17mmol/l is commonly used to simulate elevated glucose.21Cytosolic calpain1or2protein content

did not change at any time(Figure1a).Cytosolic calpain10

protein content did not change until120h(decreased46%).

In contrast,mitochondrial calpain10protein increased

after3,6,and12h of17mmol/l glucose exposure,returned

to control at24and48h,and then markedly decreased at

72,96,and120h(Figure1a).Calpains1and2are not

present in mitochondria of RPTCs.18Of note,control RPTCs

incubated in5mmol/l glucose,11mmol/l glucose,11mmol/l D-mannitol,or17mmol/l D-mannitol had no effect on calpain10protein expression at any time(Figure1b and Supplementary Figure S1online).These data reveal that glucose elevated threefold,but not twofold,regulates mito-chondrial calpain10protein expression in RPTCs biphasically, with increasing expression at early times(3–12h)and depleting mitochondrial calpain10at3days and beyond.

T o document that the gain/loss of mitochondrial calpain10 protein from elevated glucose exposure changed mitochondrial calpain10activity,RPTCs were treated as described above, mitochondrial and cytoplasmic fractions isolated,and calpain activity was examined using succinyl-Leu-Leu-Val-Tyr-7-ami-no-4-methylcoumarin.RPTCs treated with17mmol/l glucose underwent no cytoplasmic calpain activity changes at any point in time(data not shown).However,mitochondrial calpain activity increased at6and12h of17mmol/l glucose treatment and decreased at4days(Figure1c).The calpain inhibitor calpeptin inhibited mitochondrial calpain activity,providing evidence that the succinyl-leu-leu-val-tyr-7-amino-4-methyl-coumarin hydrolysis re?ects calpain activity(Figure1c). Mitochondrial calpain activity did not change with5mmol/l glucose or17mmol/l D-mannitol at any time(Figure1d).The data con?rm that elevated glucose regulates mitochondrial calpain10protein and activity in a biphasic manner. Calpain10mRNA expression in RPTCs is decreased specifically by chronic elevated glucose

Because chronic elevated glucose decreased calpain10 protein in RPTCs,we examined calpain10mRNA

expression

Con

Calpain 1

Calpain 10

Calpain 10

Calpain 10

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Calpain 10

(+) Calpeptin

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20

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612244872

Cytoplasm Cytoplasm

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0361296

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(–) Calpeptin250

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D-mannitol

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6h24h96h

96120

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Figure1|Calpain protein expression in glucose-treated renal proximal tubular cells(RPTCs).(a)RPTCs were incubated in5mmol/l (Con)or17mmol/l for various times,mitochondrial and cytoplasmic fractions isolated,and immunoblot analysis performed.b-Actin and heat shock protein60(HSP60)were used as loading controls.Results were reproduced in four different experiments.(b)RPTCs incubated were in17mmol/l D-mannitol over time and calpain10was measured in cytosol and mitochondria.(c)Mitochondrial calpain10activity 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,or5mmol/l glucose over time and mitochondrial calpain activity determined.Data are

means±s.e.m.,N X4.tSignificantly different from control;*Significantly different from time-matched sample(P p0.05).SLLVY-AMC, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin.

to determine if this decrease was transcriptionally regulated.Calpain 1mRNA expression did not change at any time in 17mmol/l glucose-treated RPTCs (Figure 2).However,cal-pain 10mRNA expression decreased,but only after 4days of elevated glucose exposure (Figure 2,data not shown),correlating with the decrease in calpain 10protein and activity.No change in calpain 10mRNA occurred in cells incubated with 17mmol/l D -mannitol.Thus,prolonged incubation with elevated glucose transcriptionally down-regulates calpain 10in RPTCs.

Mitochondrial function is decreased in RPTCs exposed to chronic elevated glucose

Because chronic elevated glucose caused loss of mito-chondrial calpain 10expression,RPTCs were treated with 17mmol/l glucose and RPTC respiration was measured at different times.There was no change in basal respiration until 4days of glucose treatment at which time it decreased 50%(Figure 3a).After basal respiration was determined,FCCP (carbonyl cyanide-p -tri?uoromethoxyphenylhydrazone)was added to obtain uncoupled QO 2,which is used here as a stress test to ascertain underlying defects in the electron transport chain when maximally stimulated.Uncoupled respiration remained constant until 4days of glucose treatment at which time it decreased 50%(Figure 3b).Basal

or uncoupled respiration did not change at any time in RPTCs treated with 17mmol/l D -mannitol (Figure 3a and b).Therefore,prolonged exposure to elevated glucose causes mitochondrial dysfunction in RPTCs.

Prolonged exposure to glucose elevates protein expression of NDUFB8and ATP synthase b

We previously reported that electron transport chain protein NDUFB8and adenosine triphosphate (ATP)synthase b are substrates for mitochondrial calpain 10.18Because 17mmol/l glucose decreased mitochondrial calpain 10expression,we hypothesized that mitochondrial calpain 10substrates may increase.With immunoblot,we noted no change in NDUFB8or ATP synthase b protein expression in 17mmol/l glucose-treated RPTCs at 6or 24h (Figure 4a).However,both proteins increased after 96h of glucose treatment.Accumula-tion of NDUFB8and ATP synthase b correlates with the loss of mitochondrial calpain 10at 96h.There was no change in NDUFB8or ATP synthase b in RPTCs treated with 17mmol/l D -mannitol (Figure 4b).In addition,mRNA of NDUFB8or ATP synthase b did not change,suggesting that increases in these proteins are not because of increased transcription under these conditions (Figure 4c and

d).

Glucose treatment

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Calpain 10

Calpain 1

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C a l p a i n 10 m R N A β-A c t i n (f o l d o f c o n t r o l )

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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 b -actin primers.Data are means ±s.e.m.,N X 4.*Significantly different from D -mannitol (P p 0.05).

40506040708017mmol/l glucose

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B a s a l r e s p i r a t i o n (n m o l O 2/m i n /m g )

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Basal respiration

4896

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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 10m mol/l carbonyl cyanide-p -trifluoromethoxyphenylhydra-zone (FCCP).Data are means ±s.e.m.(N ?4).*Significantly different from D -mannitol (P p 0.05).

Chronic elevated glucose causes RPTC apoptosis

We previously showed that ‘knockdown’of mitochondrial calpain 10caused RPTC apoptosis.20To investigate the effects of chronic elevated glucose on RPTC apoptosis,we examined nuclear condensation and caspase 3activation.Examination of nuclear morphology using 4,6-diamidino-2-phenylindole staining revealed normal nuclei (495%)in controls and at 6h of 17mmol/l glucose treatment (Figure 5a and b).However,at 96h of glucose treatment,RPTCs contained B 30%condensed https://www.360docs.net/doc/6512369707.html,ing immunoblot analysis,we observed cleaved procaspase 3in RPTCs treated with 17mmol/l glucose for 96h (Figure 5c).No cleaved procas-pase 3was observed in control and RPTCs treated with glucose for 6h (Figure 5c).Thus,loss of calpain 10by chronic elevated glucose causes RPTC apoptosis.

Renal calpain 10protein expression is depleted in STZ-induced diabetic rats and diabetic ob/ob mice

Our RPTC data revealed that chronic elevated glucose decreased calpain 10expression,and hence we next measured calpain expression in two in vivo rodent diabetes models.At 10weeks after induction of STZ-induced diabetes in the rat,serum glucose was 408±30mg/dl and serum creatinine was 2.8±0.5mg/dl.In contrast,control,vehicle-treated animals had serum glucose of 128±17mg/dl and serum creatinine of 0.5±0.1mg/dl.Thus,this diabetic rat model has increased serum glucose and decreased kidney function at https://www.360docs.net/doc/6512369707.html,pared with controls,renal calpain 1or 2protein expression did not change in STZ-treated rats (Figure 6a).In contrast,calpain 10protein expression decreased in STZ-induced rats,suggesting that the effect of diabetes was unique

to calpain https://www.360docs.net/doc/6512369707.html,pared with controls,calpain 10mRNA was also decreased,suggesting that calpain 10is transcriptionally downregulated in STZ-induced diabetic kidneys (Figure 6b).Calpain 1mRNA did not change,correlating with the lack of change in calpain 1protein expression (Figure 6b).

We also measured renal calpain 10in another in vivo model of diabetes,the ob/ob mouse.We obtained blood and kidney samples from both control and ob/ob 8-week-old male mice.The average blood glucose in the ob/ob mice was 400±31mg/dl and 204±29mg/dl in lean mice.There was a decrease in renal calpain 10protein expression in the diabetic ob/ob mice compared with the control but no change in calpain 1protein expression (Figure 6c).These results reveal that calpain 10is speci?cally decreased in two in vivo models of diabetes.Importantly,in the STZ model,we observed a decrease in renal calpain 10protein and mRNA expression that correlates with decreased renal function.In addition,renal calpain 10is transcriptionally downregulated in STZ-induced diabetic rats.

NDUFB8and ATP synthase b protein expression and apoptosis increase in STZ-induced diabetic kidneys

Because we observed increases in the calpain 10substrates NDUFB8and ATP synthase b in RPTCs treated chronically with elevated glucose,we examined the expression of these proteins in STZ-induced diabetic https://www.360docs.net/doc/6512369707.html,pared with controls,there was an increase in both NDUFB8and ATP synthase b protein expression in the STZ-induced diabetic kidney,correlating with our in vitro data (Figure 7a and b).In addition,cleaved procaspase 3and increased terminal deoxynucleotidyl transferase dUTP nick end

labeling

17mmol/l glucose-treated RPTCs Con a

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NDUFB8NDUFB8NDUFB8 mRNA

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17mmol/l glucose 17mmol/l mannitol

Figure 4|NDUFB8and adenosine triphosphate (ATP)synthase b 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 NDUFB8and ATP synthase b .b -Actin was used as a loading control.mRNA was isolated and reverse transcriptase-PCR (RT-PCR)was performed using (c )NDUFB8and (d )ATP synthase b primers.Data are means ±s.e.m.(N ?4).

Calpain 1Calpain 1

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t r o l Ob/ob mice Figure 6|Calpain expression in kidneys of diabetic models.Sprague–Dawley rats,8weeks 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 10weeks 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 2was performed.(b )Reverse transcriptase-PCR (RT-PCR)analysis for calpains 10and 1was performed.(c )Immunoblot analysis was performed on ob/ob mice kidney samples.Results were reproduced in at least four different animals.

Con 4030352520151050

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Glucose (17mmol/l)*

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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 3cleavage.(a )Nuclei were identified by 4,6-diamidino-2-phenylindole (DAPI)staining and visualized using a Nikon TE300Eclipse Fluorescence microscope (Nikon,Melville,NY)with excitation and emission filters of 350and 486nm,respectively.(b )Condensed nuclei from 10non-overlapping fields were counted for each treatment group.Data are means ±s.e.m.,N ?4.*Significantly different from control and 6h (P o 0.05).(c )Cell lysates were subjected to immunoblot analysis for procaspase 3and cleaved caspase 3(17kDa).b -Actin was used as a loading control.

(TUNEL)staining were observed in the STZ-induced diabetic kidney,providing evidence that STZ-induced diabetes is causing renal apoptosis (Figure 7a–c).There was no change in mRNA expression of NDUFB8and ATP synthase b (Figure 7d),correlating with our in vitro data and providing evidence that the increase in NDUFB8and ATP synthase b is not due to increased synthesis.

In vivo siRNA knockdown of renal calpain 10

To determine whether decreased calpain 10is suf?cient to induce renal dysfunction,rats were treated with calpain 10small interfering RNA (siRNA)to decrease renal calpain 10.This effective approach has been used by many investigators because siRNA accumulates in the renal proximal tubule and decreases its target protein.22–26Rats were treated with siRNA (20nmol)directed against calpain 10or scrambled siRNA by tail vein injection.Tissues were isolated,homogenized,and immunoblot analysis performed.A single intravenous dose of a siRNA directed against calpain 10,but not that of a negative siRNA (scrambled),decreased calpain 10protein and mRNA expression in rat kidney cortex (Figure 8a).The siRNA directed against calpain 10had no effect on calpain 1in kidney cortex or calpain 10in the liver (Supplementary Figure S2online).Therefore,we have an in vivo model of renal-speci?c calpain 10knockdown to examine the functional consequences thereof.

To examine the effects of calpain 10loss in rat kidneys,we measured serum creatinine,cleavage of caspase 3,and TUNEL staining.In rats treated with the calpain 10siRNA,there was an increase in serum creatinine to 1.7±0.2mg/dl

at 7days (Figure 8b).The serum creatinine for negative siRNA was 0.7±0.1mg/dl and did not change over time.These data suggest that the loss of renal calpain 10causes renal dysfunction.In addition,immunoblots of caspase 3showed activation of caspase 3after 5and 7days of calpain 10siRNA but not in negative siRNA-treated rat kidneys (Figure 8c).There was also an increase in TUNEL-positive cells in the calpain 10siRNA-treated kidney (Figure 8d).We suggest that the loss of calpain 10in vivo results in renal apoptosis and renal dysfunction.

DISCUSSION

Our data reveal that diabetic glucose speci?cally decreases mitochondrial calpain 10expression in RPTCs and that renal calpain 10is decreased in diabetic rats and mice.Further-more,the loss of renal calpain 10resulted in apoptosis in vitro and in vivo ,and decreased renal function in the rat.We suggest that the loss of calpain 10is key in diabetes-induced renal dysfunction and,ultimately,diabetic nephrop-athy.These data complement our previous work in which we reported that mitochondrial calpain 10is required for cell viability,and calpain 10speci?cally decreases in aging rat,mouse,and human kidney tissues when renal function decreases.20We provide evidence that calpain 10is required for renal function and contributes to two common renal diseases.Finally,calpain 10is ubiquitously expressed and our ?ndings have broader implications in other tissues and diseases.

Examining the effect of elevated glucose on calpain expression over time,we observed an acute increase

in

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kDa kDa kDa 350STZ-induced diabetic rats

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300200150100ATP synthase βNDUFB8

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ATP synthase βNDUFB8

Con Control STZ

STZ 1.210.40.60.8m R N A /β-A c t i n

(f o l d o f c o n t r o l )

0.20

250500

P r o t e i n e x p r e s s i o n % c o n t r o l

ATP synthase β

Caspase 3Cleaved caspase 3

Figure 7|NDUFB8,adenosine triphosphate (ATP)synthase b ,and caspase 3protein expression in streptozotocin (STZ)-induced diabetic kidneys.STZ-induced diabetic kidneys were isolated and the tissue was homogenized.Immunoblot analysis for b -actin,NDUFB8,ATP synthase b ,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 )NDUFB8and ATP synthase b 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 X 4.*Significantly different from control (P o 0.05).

calpain 10protein and activity between 3and 12h after 17mmol/l glucose exposure that returned to control at 24h.Increased calpain 10was not due to increased transcription and we propose that increased calpain 10is due to decreased degradation.Finally,the rapid increase in calpain 10by glucose suggests that renal mitochondrial calpain 10is important in renal metabolism and is regulated by nutrients.

It is noteworthy that the acute and chronic effects of glucose act speci?cally on mitochondrial calpain 10;two calpain family members (calpains 1and 2)did not change over time.In contrast,the decrease in calpain 10protein and activity in RPTCs after 3–5days of 17mmol/l glucose treatment was associated with a decrease in calpain 10mRNA,suggesting glucose induced a decrease in calpain 10transcription.A mechanism for decreased calpain 10transcription by glucose has not been reported.

The decrease in mitochondrial calpain 10protein occurred before a decrease in cytoplasmic calpain 10.We reported similar results when calpain 10was ‘knocked down’with short hairpin RNA 20and suggest that mitochondrial calpain 10turnover is greater than cytosolic calpain 10.

The decrease in RPTC calpain activity observed during chronic glucose exposure also has been reported in other cellular models.Diaz-Villasenor et al .27reported a decrease in calpain activity in lymphocytes from diabetic patients.The increase in RPTC calpain activity by short-term glucose also has been reported to occur in cardiomyocytes,28but the calpain isoform that was being measured was not identi?ed.

Given that calpain 10is a protease,decreased calpain 10expression may result in the accumulation of its substrates.There was no change in NDUFB8and ATP synthase b after acute glucose treatment of RPTCs,but chronic glucose-induced RPTC mitochondrial calpain 10loss resulted in increased NDUFB8and ATP synthase b .We also explored this possibility in the rat STZ diabetic model and observed that renal NDUFB8and ATP synthase b increased in concert with the loss of renal calpain 10.Thus,we hypothesize that the initiating mitotoxic event induced by the loss of mitochondrial calpain 10is the accumulation of mitochondrial calpain 10substrates.Supporting our hypoth-esis,we observed that chronic glucose treatment decreased RPTC basal and uncoupled RPTC respiration,markers of mitochondrial dysfunction.We observed no changes in mitochondrial function at earlier times of glucose treatment (that is,12,24,and 48h).

We previously reported that knockdown of calpain 10with short hairpin RNA resulted in RPTC apoptosis.20We observed apoptosis at 96h after glucose treatment,when calpain 10was decreased,but not at 6h.We also explored this possibility in the rat STZ diabetic model and observed caspase 3cleavage in concert with the loss of renal calpain 10.Importantly,we show the loss of renal calpain 10by siRNA in vivo results in activation of caspase 3in the kidney cortex.These results combined with the results from our earlier study 20provide evidence that the accumulation of mitochondrial proteins and mitochondrial

dysfunction

2.5211.50.50

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Calpain 10 siRNA *

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S e r u m c r e a t i n i n e (m g /d l )

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5++++++––––––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 10protein was measured by immunoblot analysis.Calpain 10and 1mRNA 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 3and 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 X 4.*Significantly different from control (P o 0.05).

associated with the loss of renal calpain10lead to apoptosis in vitro and in vivo.

In an earlier study,25mmol/l glucose for6and24h produced necrotic cell death in LLC-PK cells.29However,we observed no RPTC death at6or24h.These results could be due to different glucose concentrations used but,more likely,the difference arises from the use of an immortalized cell line and our use of primary cultures cultured under aerobic conditions.30,31

STZ-induced diabetic rats had elevated glucose and increased serum creatinine after10weeks.As we observed in RPTCs,renal calpain10mRNA and protein were decreased and calpains1and2were not,suggesting that decreases in renal calpain10observed in diabetes is because of decreased calpain10transcription.We conducted a similar experiment in the diabetic ob/ob mouse and observed that with elevated glucose,renal function decreased,suggesting that calpain10decreases with elevated glucose in multiple animal models.

We developed an in vivo model of renal calpain10 knockdown using siRNA.This effective approach has been used by a number of investigators:siRNA accumulates in the renal proximal tubule and decreases its target protein.22–26 The loss of both protein and mRNA expression of calpain 10in the kidney cortex results in kidney dysfunction.Caspase 3activation increased,suggesting that the loss of calpain 10in vivo results in apoptosis.These data provide strong evidence that the loss of renal calpain10is suf?cient to induce kidney injury.

In summary,renal calpain10protein and mRNA decrease with chronic elevated glucose,both in vitro and in vivo,a decrease that correlates with increased calpain10substrates resulting in mitochondrial dysfunction and apoptosis.Loss of renal calpain10by siRNA in vivo results in renal dysfunction and apoptosis,suggesting a direct relationship between loss of calpain10protein expression and renal dysfunction.These data collectively indicate that the loss of calpain10in vivo results in renal apoptosis and renal dysfunction,underscoring that the loss of calpain10causes mitochondrial dysfunction leading to cell death in diabetic nephropathy.

MATERIALS AND METHODS

Renal proximal tubules

Isolation and culture of renal proximal tubules were performed as described previously.30,31RPTCs were isolated from female New Zealand White rabbits(1.5–2.5kg)using the iron oxide perfusion method and grown under improved conditions.The advantage of this model is that RPTCs cultured under these conditions exhibit greater differentiated function,an in vivo–like rate of oxidative metabolism,and limited glycolysis and are gluconeogenic.30,31 Mitochondrial isolation and fractionation

RPTC mitochondria were isolated via differential centrifugation as previously described.18Mitochondrial purity was determined by measuring the levels of glyceraldehyde3-phosphate dehydrogenase (GAPDH)and heat shock protein60(HSP60)in the cytosol and mitochondria(Supplementary Figure S3online).Immunoblot analysis

Rat and mouse kidney tissue,and RPTCs were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes.Membranes were incubated with primary antibodies to calpains1,2,and10,and HSP60(used to normalize mitochondrial fractions),or GAPDH.The primary antibodies used were m-calpain(domain IV)(Calbiochem,La Jolla,CA;1:1000), m-calpain(domains III and IV)(Abcam,Cambridge,MA;1:1000), calpain10(Abcam;1:1000),caspase3(StressGene,San Diego,CA; 1:1000),HSP60(Abcam;1:1000),ATP Synthase b(1:1000),NDUFB8 (Invitrogen,Carlsbad,CA;1:1000),and GAPDH(Fitzgerald Anti-bodies,Acton,MA;1:1000).We previously reported that ND6was a substrate for calpain10.18The anti-ND6antibody(Invitrogen) manufacturer recently informed us that the antibody actually identi?es the mitochondrial complex1protein NDUFB8.Thus, NDUFB8is a substrate of mitochondrial calpain10.Antibody incubation was followed by a horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody(Santa Cruz,Santa Cruz, CA;1:1000).Immunoreactive protein was visualized by enhanced chemiluminescence(Amersham,GE Healthcare,Piscataway,NJ)and imaged using an Alpha Innotech imaging station(Santa Clara,CA). STZ diabetic model

Male Sprague–Dawley rats,8weeks of age(200–250g),were starved for 16h and injected once with STZ(55mg/kg,tail vein)in sodium citrate buffer.32–34Afterward,rats were given drinking water supplemented with sucrose(15g/l)for48h to limit mortality as stores of insulin are released from damaged pancreatic islets.Aged matched males rats were controls.After24h,diabetes was con?rmed in STZ-treated rats by measuring tail vein plasma glucose.At10weeks after diabetes induction,rats were killed and blood and kidneys were harvested. Ob/ob mice

For the ob/ob mice study,8-week-old male ob/ob(leptinà/à)and lean controls(Jackson Labs,Bar Harbor,Maine)were used.Mice were killed and blood and kidneys were harvested.Kidneys were homogenized and immunoblot analyses were performed. Reverse transcription PCR

Rat kidney samples were homogenized with TRIZOL reagent (Invitrogen)according to the manufacturer’s protocol to extract total RNA.The mRNA was subjected to reverse transcription to DNA using Moloney murine leukemia virus(MMLV)reverse transcriptase (Invitrogen)in the presence of oligo dT primers.PCR was performed using primers for calpains1,2,and10,which has previously been reported.15Ampli?cation of a-GAPDH was used as internal control for normalizing PCR ef?ciency.Products were electrophoresed on 1.5%agarose gel and stained with ethidium bromide.

Calpain activity

Calpain activity was assayed spectrophotometrically using the calpain substrate succinyl-Leu-Leu-V al-Tyr-7-amino-4-methylcoumarin (Bachem,T orrance,CA)as previously described.18

TUNEL assay

The In Situ Cell Death Detection kit(Roche,Indianapolis,IN)was used.After deparaf?nization,the sections were treated with proteinase K(20g/ml)and kit protocol was followed.TUNEL-positive cells were counted in10non-overlapping?elds in each section under?100magni?cation.

Measurement of RPTC oxygen consumption(QO2)

QO2was monitored as previously described using a Clark-type oxygen electrode.18After the basal rate of QO2was determined, carbonyl cyanide-p-tri?uoromethoxyphenylhydrazone(?nal con-centration?10m mol/l)was injected to obtain uncoupled QO2. Assessment of nuclear morphology

Nuclear morphology was assessed as previously described.35Visua-lization of4,6-diamidino-2-phenylindole staining was performed using a?uorescence microscope and condensed nuclei in12cells from10high-powered?elds were counted for each treatment group. In vivo siRNA

In vivo siRNA was administered as previously described.26Male Sprague–Dawley rats,6–8weeks of age,were treated with calpain 10siRNA(50-CCAGGACAUUUGUGCCACACCUCAA-30)(Invitro-gen)or a negative control(scramble)(Invitrogen),with a phospho-rothioate backbone that decreases degradation and extends its effectiveness,in a sterile siRNA resuspension buffer(Invitrogen)to decrease renal calpain10.Rats were treated with20nmol of siRNA directed against calpain10or scrambled siRNA by tail vein injection.Tissues were isolated,homogenized,and immunoblot performed.Serum creatinine measurements were performed with the appropriate kit(Bioassay Systems,Hayward,CA).

Statistical analysis

RPTCs isolated from one rabbit represents one experiment(n?1). The appropriate analysis of variance was performed for each data set using SigmaStat statistical software(Ashburn,VA).Individual means were compared with Fisher’s protected least signi?cant difference test with P p0.05being considered statistically signi?cant. DISCLOSURE

All the authors declared no competing interests. ACKNOWLEDGMENTS

This research was supported by NIEHS grant ES-012239(to RGS)and NIGMS grant GM-084147(to RGS),and by the Biomedical Laboratory Research and Development Program of the Department of Veterans Affairs.MDC was supported by the National Institutes of Health Training grant T32HL007260.Animal facilities were funded by NIH grant C06RR-015455.

Disclaimer

The article content does not represent the views of the Department of Veterans Affairs of the US Government.

SUPPLEMENTARY MATERIAL

Figure S1.The effect of11mM glucose and11mM D-mannitol on cytosolic and mitochondrial calpain10.

Figure S2.The effect of siRNA on calpain protein in rat kidney and liver.

Figure S3.Relative purity of RPTC mitochondrial and cytosolic fractions.

Supplementary material is linked to the online version of the paper at https://www.360docs.net/doc/6512369707.html,/ki

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