inactivation of p66shc decreases afferent arteriolar katp ... · 17/08/2018 · containing adaptor...

Inactivation of p66Shc Decreases Afferent Arteriolar KATP Channel Activity and

Decreases Renal Damage in Diabetic Dahl SS Rats

Bradley S. Miller1,3, Shoshana R. Blumenthal1, Alexey Shalygin4,5, Kevin D. Wright1,3,

Alexander Staruschenko3,4, John D. Imig2,3 and Andrey Sorokin1,3

Affiliation:

1Department of Medicine, Division of Nephrology, Medical College of Wisconsin, US

2Department of Pharmacology & Toxicology, Medical College of Wisconsin, US

3Cardiovascular Center, Medical College of Wisconsin, US

4Department of Physiology, Medical College of Wisconsin, US 5Institute of Cytology of the Russian Academy of Sciences, St. Petersburg, Russia Running title: p66Shc regulates vascular function in diabetic rats Corresponding author at: Cardiovascular Center, Medical College of Wisconsin, 8701 Watertown Plank Road, 53226, USA; e-mail address: [email protected] (Sorokin)

of 29 Diabetes

Diabetes Publish Ahead of Print, published online August 21, 2018

Abstract Increased expression of adaptor protein p66Shc has been associated with progression

of diabetic nephropathy. Afferent arteriolar dilation and glomerular hyperfiltration in

diabetes are due to increased KATP channel availability and activity. Hyperglycemia was

induced in Dahl SS (SS) rats in a model of diabetes via injection of streptozotocin (STZ).

Renal injury was evaluated in SS and genetically modified SS, either lacking p66Shc

(p66ShcKO), or expressing p66Shc mutant (p66Shc-S36A). Afferent arteriolar diameter

responses during STZ-induced hyperfiltration were determined using the juxtamedullary

nephron technique. Albuminuria and glomerular injury were mitigated in p66ShcKO and

p66Shc-S36A rats with STZ-induced diabetes. SS rats with STZ-induced diabetes had a

significant increase in the afferent arteriolar diameter, whereas p66ShcKO and p66Shc-

S36A rats did not. STZ SS rats, but not STZ p66ShcKO or p66Shc-S36A rats had an

increased vasodilator response to KATP channel activator pinacidil. Likewise, KATP

inhibitor glibenclamide resulted in a greater decrease in afferent arteriolar diameter in

STZ SS rats compared to STZ-treated SS p66ShcKO and p66Shc-S36A rats. Using

patch-clamp electrophysiology we demonstrated that p66Shc knockout decreases KATP

channels activity. These results indicate that inactivation of the adaptor protein p66Shc

decreases afferent arteriolar KATP channel activity and decreases renal damage in

diabetic SS rats.

of 29Diabetes

Introduction Patients with poorly-controlled diabetes mellitus sustain damage to the macro- and

microvasculature that is responsible for much of the morbidity and mortality associated

with the disease. The risk for myocardial infarction, stroke and amputation is 2-4 times

greater than in patients with normal blood glucose levels (1;2). Risk of blindness in

diabetics is 8-16 times greater than in non-diabetics (3), while the occurrence of end-

stage renal disease is elevated 10-fold (4).

The mechanism by which diabetes damages small and large blood vessels has been

investigated intensively. In the kidney, dysregulation of glomerular blood flow has been

implicated as one factor in the pathogenesis of diabetic glomerulosclerosis (5). In

animals, micropuncture studies found higher glomerular capillary pressures and flow in

diabetic animals (6). Hyperfiltration in type 1 diabetes is primarily driven by alterations in

afferent arteriolar resistance (7), which is likely regulated by ATP-senstitive potassium

channels (KATP) (8). At a molecular level, what causes these changes in microvascular

function are unresolved.

We recently found that p66Shc, one of three isoforms of SRC homology 2-Domain-

containing adaptor protein (Shc1), regulates renal vascular tone in a hypertensive model

of glomerular disease, the Dahl salt-sensitive (SS) rat (9). We observed increased

p66Shc expression in smooth muscle cells (SMC) of renal resistance vessels and

showed that the preglomerular afferent renal arterioles have defective autoregulatory

vasoconstrictive responses that were restored in rats deficient in p66Shc (p66ShcKO).

We have also previously shown that vasoactive peptide Endothelin-1 (ET-1) induces

Ser36 phosphorylation of p66Shc, which is required for mitochondrial translocation of

of 29 Diabetes

cytosolic p66Shc (10). Prooxidant properties of p66Shc are usually associated with its

mitochondrial location (11;12). In this study we extend observations done in

hypertensive SS rats to SS rats rendered diabetic with streptozotocin (STZ). Besides

p66ShcKO, we have also generated SS rats in which Ser36 in p66Shc is substituted

with Ala (p66Shc-S36A), the change which is expected to prevent p66Shc translocation

to mitochondria. p66ShcKO and p66Shc-S36A allow us to assess the role of p66Shc in

diabetic nephropathies. In particular, we examined the role of p66Shc in regulating

afferent arteriolar dilation via potential regulation of the KATP channel.

Methods

Animals and induction of diabetes: All studies were performed on rats generated and

maintained on the SS/JrHsdMcwi (SS) genetic background. p66ShcKO and p66Shc-

S36A rats have been described previously (9). Male SS and p66Shc mutant rats (9-11

weeks old) were given a single injection of STZ (50mg/kg i.p.), without insulin treatment,

as described previously (13). One week after injection, diabetes was confirmed by

nonfasted blood glucose measurements using a blood glucose test strip meter (Contour,

Bayer HealthCare). Only animals in which hyperglycemia exceeded 300 mg/dL

throughout the 12-week induction period were used for analysis. All procedures were

approved by the Institutional Animal Care and Use Committee at the Medical College of

Wisconsin.

Measurement of urinary albumin excretion: Rats were individually housed in metabolic

caging for overnight urine collection every 3 weeks post STZ injection. Water and food

were provided ad libitum. Urine was collected after 17 to 24 hours and volume quantified

before being stored at –80°C until assayed. Urinary albumin was quantified with Albumin

of 29Diabetes

Blue 580 dye (Molecular Probes) using a fluorescent plate reader (FL600, Bio-Tek) and

normalized to a 24 hour collection period.

Analysis of glomerular injury: Following STZ-treatment, kidneys were processed for PAS

staining and scored for glomerular injury as previously described (9). The semi-

quantitative glomerular score of each animal was determined in a blinded manner by 2

investigators and derived as the mean of 50 glomeruli per rat.

Mesangial index quantification: Percent area of the glomerulus that is PAS stained and

expressed as the mesangial index was determined as described by the Animal Models

of Diabetic Complications Consortium (AMDCC, http://www.amdcc.org), with the

following modifications. 20 random glomeruli per animal were imaged and analyzed at

40X using the Hamamatsu Slide Scanner. Images were split into channels to separate

PAS pixel data from nuclei by batch process using a custom matrix derived from a

control slide using the Colour Deconvolution plugin found in the Fiji distribution of

ImageJ software (14). Glomerular tuft area (including capillary lumen) was defined by

manual selection, followed by manual segmentation of the PAS channel to account for

stain variability specific to the mesangial matrix using the global RenyiEntropy threshold

method.

Afferent arteriolar diameter response: Afferent arteriolar diameter responses were

determined six weeks following induction of STZ diabetes as described previously (9).

Wild type SS rats, SS p66ShcKO and SS p66Shc-S63A with and without STZ-induced

diabetes were analyzed for afferent arteriolar contraction response to KATP channel

activator, pinacidil (30-300 µM), or channel blocker, glibenclamide (30-300 µM) (15;16).

of 29 Diabetes

Patch-clamp electrophysiology: Patch-clamp electrophysiology was used to assess the

activity of KATP channels in isolated renal vascular SMC. SMC were isolated and cultured

in high glucose (4.5 g/L, 25mM) DMEM supplemented with 10% fetal bovine serum, as

previously described (9). Passages 15-17 were used for analysis. Single-channel data

were acquired and subsequently analyzed with Axopatch 200B amplifier (Molecular

Devices) interfaced via a Digidata 1440A to a PC running the pClamp 10.2 suite of

software (Molecular Devices). Signals were filtered with an eight-pole, low pass Bessel

filter LPF-8 (Warner Inst., Hamden, CT) at 1 kHz. The single-channel activity of KATP

channels in vascular SMCs was determined in cell-attached patches made under

voltage-clamp conditions. The pipette was pulled with a horizontal puller (Sutter P-97;

Sutter Inst., Novato, CA). The resistance of the pipette in the corresponding bath

medium was 7–12 MΩ. Bath and pipette solutions were (in mM) 150 NaCl, 5 KCl, 1

CaCl2, 10 HEPES, 2 MgCl2, 5 Glucose (pH=7.35) and 140KCl, 10 EDTA, 10 HEPES (pH

7.35), respectively. The channel events were analyzed by Clampfit 10.2 software (single-

channel search in analyze function). NPo, the product of the number of the channels,

was used to measure the channel activity within a patch. The channel open probabilities

(NPo) were determined by the following equation: NPo = (I)/i, where (I) is the mean

channel current and i is the unitary current amplitude. The (I) was estimated from the

time integrals of the currents above the baseline, and i was determined from the current

traces and all-point amplitude histograms.

Statistical analysis and data presentation: Pairwise comparisons between groups over

time were performed using the least square means method for unbalanced, two-factor

ANOVA for repeated measures with Holm-Sidak post-hoc tests (SigmaPlot 11.2). Data

of 29Diabetes

are reported as mean ± SEM. Box whisker plot data represent minimum, first quartile,

median (box divider), mean (X), third quartile, and maximum.

Results

SS rats on high salt diet develop hyperlipidemia and insulin resistance (17). IV glucose

tolerance tests established no difference between measured plasma glucose and insulin

levels of SS and Sprague Dawley rats, indicating that SS rats do not have metabolic

syndrome (Supplemental Figure 1). Upon induction of diabetes with STZ, we observed

an increase in renal p66Shc expression (Figure 1), a result in agreement with human

cases of DN (18), suggesting p66Shc involvement in progression of renal DN.

Using our genetically modified SS rats, we observed that p66ShcKO did not prevent

development of diabetes induced by injection of STZ, but prevented onset of renal

damage (Figure 2). Compared to controls, hyperglycemia (Figure 2A), increased

urination (Figure 2B) and decreased change in body weight (Figure 2C) were observed

to the same levels in STZ-treated SS, p66ShcKO and p66Shc-S36A rats, indicating that

loss or modification of p66Shc does not interfere with the establishment of diabetes.

Furthermore, there were no significant differences in end-stage renal hypertrophy as

measured by kidney weight to body weight ratio (Figure 2D) between STZ-treated

groups. In contrast, both STZ-treated p66ShcKO and p66Shc-S36A exhibited reduced

albuminuria at 6 weeks and 12 weeks post-induction, respectively, compared to STZ-

treated SS rats (Figure 2E). Glomeruli of all non-diabetic control rats displayed mild to

moderate levels of mesangial expansion with limited sclerosis (Figure 3A), features

common to Dahl SS rats on low-salt diet. A semi-quantitative assessment of PAS

staining determined that STZ-treated SS rats had increased glomerular injury, whereas

of 29 Diabetes

no significant glomerular injury was found in diabetic p66ShcKO and p66Shc-S36A

(Figure 3B). A significant increase in the percent of glomeruli undergoing sclerosis in

STZ-treated SS rats (38.1 ± 6.3%, n=9, versus SS control 16.7 ± 3.4%, n=6) was absent

in either p66ShcKO STZ (11.9 ± 1.6%, n=8, versus control 11.3 ± 2.0%, n=6) or p66Shc-

S36A STZ (16.0 ± 3.1%, n=8, versus control 7.0 ± 1.6%, n=6). There was no significant

difference in the percentage of glomeruli with mesangial matrix expansion among strains

or STZ treatment as determined by semi-quantitative scoring or by digital image analysis

of mesangial index (data not shown). This data suggests that mitochondrial action of

p66Shc plays a role in the renal damage observed in rats with STZ-induced DN.

As we have found that p66Shc knockout restores microvascular responses in isolated

perfused afferent arterioles from SS rats with hypertension induced nephropathy (9), we

tested the role of p66Shc in renal vascular dysfunction with STZ-induced DN. Afferent

arteriolar diameters of control rats at 100 mmHg averaged 22.9 ± 0.9 µm (n=8) in SS

rats, 21.1 ± 0.8 µm (n=7) and 21.1 ± 0.8 µm (n=6) in control p66Shc-S36A and

p66ShcKO, respectively. STZ-induced SS rats had significant increase in the afferent

arteriolar diameter (24.7 ± 1.3 µm, p<0.05; n=6) whereas STZ-induced p66ShcKO rats

and STZ-induced p66Shc-S36A did not (22.1 ± 1.2 µm; n=6) and (21.6 ± 1.2 µm; n=7)

correspondingly (Figure 4A). Afferent arteriolar dilation and glomerular hyperfiltration in

diabetes is due to increased KATP channel availability and activity (19). Afferent arteriolar

dilator responses to pharmacological vasodilator pinacidil, which targets KATP channels,

were not different between controls of SS rats, p66ShcKO, or p66Shc-S36A rats.

However, STZ-induced SS, but not p66ShcKO, or p66Shc-S36A rats had increased

vasodilator response to pinacidil (Figure 4B) suggesting that p66Shc increases afferent

arteriolar KATP channel activity in diabetes. Likewise, the KATP inhibitor glibenclamide

resulted in greater decrease in afferent arteriolar diameter in STZ-induced SS rats (23 ±

of 29Diabetes

4%, n =6) compared to STZ p66ShcKO rats (13 ± 2%, n =6) (Figure 4C), suggesting

that diabetic SS rats have increased KATP channel activity. Nitroprusside, whose

vasodilating effects are based on release of nitric oxide, had similar vasodilator

responses in the SS, p66Shc-S36A and p66ShcKO groups (Supplemental Figure 2),

indicating, that ability of vessels to dilate is not affected by targeted editing of Shc1 gene.

In Figure 4A differences in afferent arteriole diameters between SS and SS with STZ-

induced DN is due to increased KATP activity (P<0.05). The difference of afferent

arterioles diameter between untreated SS and p66ShcKO rats is due to increased

vascular tone of afferent arterioles in the absence of p66Shc (P<0.05). Finally, the

differences in the diameter of afferent arterioles between SS treated with STZ and

p66ShcKO treated with STZ is likely to be the consequence of increased myogenic tone

and decreased KATP influence (P<0.001).

To assess the possible functional implications of the inactivation of p66Shc in control

KATP channels, we assessed endogenous channels activity in renal SMC isolated from

wild type (SS), p66ShcKO and p66Shc-S36A mutant rats. KATP channels were

measured in cell-attached configuration to evaluate the basal level of activity.

Representative current trace recordings are displayed in Figure 5B. Channel activity

(Figure 5B, each point representing individual experiments) was observed in 6 out of 12,

2 out of 15, and 3 out of 8 experiments for SS, p66ShcKO and p66Shc-S36A SMC,

respectively. Figure 5C summarizes NPo for those experiments where KATP channels

were active. As shown in this figure, inactivation of p66Shc results in downregulation of

frequency of observed channels and activity of KATP channels. The two recordings from

SMC isolated from p66ShcKO rats, where channel activity was detected, demonstrated

of 29 Diabetes

very low NPo. To a lesser extent, KATP activity was also attenuated in p66Shc-S36A

SMC.

DISCUSSION

p66Shc plays a significant role in the progression of a number of renal pathologies (20).

We have developed Zinc Finger nuclease (ZFN)-mediated targeted knockouts of p66Shc

in SS rats and used this rat strain to evaluate the role of p66Shc in the development of

DN. All modifications of Shc1 gene were made on the genetic background of SS rats.

The SS rat is a well-established model for the study of salt-sensitive hypertension (21)

but only recently has been used to investigate DN (13;22). Unlike Sprague-Dawley rats,

which are resistant to STZ-induced type 1 diabetes, SS rats are susceptible to renal

injury induced by STZ. SS rats develop progressive proteinuria, hyperfiltration, and also

display renal histological lesions similar to those seen in DN patients (13;22). Our data

clearly indicates that loss of p66Shc does not prevent development of STZ-induced

diabetes, but mitigates subsequent renal damage which ensues following onset of

diabetes. One mechanism by which p66Shc mitigates renal damage could be a

restoration of vascular function of renal microvessels.

KATP channels are present in vascular SMC and by modulating vascular responses to

diverse stimuli contribute to physiological regulation of vascular tone (23). Our analysis

of renal microvascular reactivity using in vitro juxtamedullary nephron segments

revealed that increased afferent arteriolar KATP channel activity contributes to dilation of

the afferent arteriole, an increase in arteriolar diameter and hyperfiltration injury in SS

rats with STZ-induced diabetes. In diabetes mellitus elevated KATP function and

decreased calcium influx may contribute to pre-glomerular vasodilation and

of 29Diabetes

hyperfiltration (16;24). p66Shc expressed in renal vascular SMC inhibits TPRC-

dependent calcium influx (9) and, as shown by current study, promotes KATP channel

function. Conversely, inactivation of the adaptor protein p66Shc decreases afferent

arteriolar KATP channel activity, presumably restores TPRC function and decreases renal

damage in diabetic Dahl SS rats.

Increased oxidative stress within the kidney contributes to progression of DN

(11;18;25;26). p66Shc is capable of acting as a regulator of sensitivity to oxidative stress

and since it was reported to be overexpressed in proximal tubule cells and podocytes in

STZ-induced diabetic mice and other genetic models of diabetes (27;28). We analyzed

the progression of STZ-induced DN in p66Shc-S36A rats in which p66Shc is expressed,

but devoid of its mitochondrial action. Supporting the role of p66Shc as an inducer of

mitochondrial ROS in DN, STZ-induced p66Shc-S36A rats displayed decreased

albuminuria and glomerular damage. Interestingly, both restoration of vascular function

and decrease of albuminuria in diabetic p66Shc-S36A rats was less than in p66ShcKO,

implying that non-mitochondrial action of p66Shc, presumably related to KATP activity, is

also important. Importantly, it was recently reported that Sirt1-regulated lysine

acetylation of p66Shc contributes to vascular oxidative stress and diabetic vascular

pathophysiology (29).

In summary, our data suggest the crucial role of p66Shc in renal and microvascular

damage associated with DN.

Acknowledgements

of 29 Diabetes

Authors thank Dr. J. Lazar (HudsonAlpha Institute for Biotechnology, AL) for carrying out

IV glucose tolerance tests. We also thank Dr Palygin (Medical College of Wisconsin, WI)

for discussion of results.

Funding: This work was supported by NIH grants R01 DK098159 (to AS), R01

DK103616 (to JDI), R35 HL135749 (to ASt) and a grant from the Wisconsin Breast

Cancer Showhouse, Inc. and the Medical College of Wisconsin Cancer Center (to AS).

Authors contribution: AS conceptualized the study. BM, SB, KW, ASh and JDI

conducted the investigation. ASt designed the experiments dealing with direct

measurement of KATP channel activity. All authors analyzed and interpreted the data.

BM, SB, KW, ASt and AS drafted the article. AS supervised the study. All authors

approved the final version and agreed to be accountable for all aspects of the work.

Conflict of Interest Statement: No potential conficts of interest relevant to this article

were reported. Dr. Andrey Sorokin is the guarantor of this work and, as such, had full

access to all the data in the study and takes responsibility for the integrity of the data

and the accuracy of the data analysis.

of 29Diabetes

Reference List

1. Al-Delaimy,WK, Merchant,AT, Rimm,EB, Willett,WC, Stampfer,MJ, Hu,FB:

Effect of type 2 diabetes and its duration on the risk of peripheral arterial

disease among men. Am J Med 116:236-240, 2004

2. Sarwar,N, Gao,P, Seshasai,SR, Gobin,R, Kaptoge,S, Di,AE, Ingelsson,E,

Lawlor,DA, Selvin,E, Stampfer,M, Stehouwer,CD, Lewington,S, Pennells,L,

Thompson,A, Sattar,N, White,IR, Ray,KK, Danesh,J: Diabetes mellitus, fasting

blood glucose concentration, and risk of vascular disease: a collaborative

meta-analysis of 102 prospective studies. Lancet 375:2215-2222, 2010

3. Camacho,P, Pitale,S, Abraira,C: Beneficial and detrimental effects of intensive

glycaemic control, with emphasis on type 2 diabetes mellitus. Drugs Aging

17:463-476, 2000

4. Prischl,FC, Auinger,M, Saemann,M, Mayer,G, Rosenkranz,AR, Wallner,M,

Kramar,R: Diabetes-related end-stage renal disease in Austria 1965-2013.

Nephrol Dial Transplant 30:1920-1927, 2015

5. Carlstrom,M, Wilcox,CS, Arendshorst,WJ: Renal autoregulation in health and

disease. Physiol Rev 95:405-511, 2015

of 29 Diabetes

6. Zatz,R, Dunn,BR, Meyer,TW, Anderson,S, Rennke,HG, Brenner,BM: Prevention

of diabetic glomerulopathy by pharmacological amelioration of glomerular

capillary hypertension. J Clin Invest 77:1925-1930, 1986

7. Yang,GK, Maahs,DM, Perkins,BA, Cherney,DZ: Renal hyperfiltration and

systemic blood pressure in patients with uncomplicated type 1 diabetes

mellitus. PLoS ONE 8:e68908, 2013

8. Carmines,PK: The renal vascular response to diabetes. Curr Opin Nephrol

Hypertens 19:85-90, 2010

9. Miller,B, Palygin,O, Rufanova,VA, Chong,A, Lazar,J, Jacob,HJ, Mattson,D,

Roman,RJ, Williams,JM, Cowley,AW, Jr., Geurts,AM, Staruschenko,A, Imig,JD,

Sorokin,A: p66Shc regulates renal vascular tone in hypertension-induced

nephropathy. J Clin Invest 126:2533-2546, 2016

10. Foschi,M, Franchi,F, Han,J, La Villa,G, Sorokin,A: Endothelin-1 induces serine

phosphorylation of the adaptor protein p66Shc and its association with 14-3-

3 protein in glomerular mesangial cells. J Biol Chem 276:26640-26647, 2001

11. Francia,P, Cosentino,F, Schiavoni,M, Huang,Y, Perna,E, Camici,GG, Luscher,TF,

Volpe,M: p66(Shc) protein, oxidative stress, and cardiovascular

complications of diabetes: the missing link. J Mol Med 87:885-891, 2009

of 29Diabetes

12. Giorgio,M, Migliaccio,E, Orsini,F, Paolucci,D, Moroni,M, Contursi,C, Pelliccia,G,

Luzi,L, Minucci,S, Marcaccio,M, Pinton,P, Rizzuto,R, Bernardi,P, Paolucci,F,

Pelicci,PG: Electron transfer between cytochrome c and p66Shc generates

reactive oxygen species that trigger mitochondrial apoptosis. Cell 122:221-

233, 2005

13. Slaughter,TN, Paige,A, Spires,D, Kojima,N, Kyle,PB, Garrett,MR, Roman,RJ,

Williams,JM: Characterization of the development of renal injury in Type-1

diabetic Dahl salt-sensitive rats. Am J Physiol Regul Integr Comp Physiol

305:R727-R734, 2013

14. Schindelin,J, Arganda-Carreras,I, Frise,E, Kaynig,V, Longair,M, Pietzsch,T,

Preibisch,S, Rueden,C, Saalfeld,S, Schmid,B, Tinevez,JY, White,DJ,

Hartenstein,V, Eliceiri,K, Tomancak,P, Cardona,A: Fiji: an open-source

platform for biological-image analysis. Nat Methods 9:676-682, 2012

15. Carmines,PK, Fujiwara,K: Altered electromechanical coupling in the renal

microvasculature during the early stage of diabetes mellitus. Clin Exp

Pharmacol Physiol 29:143-148, 2002

16. Troncoso Brindeiro,CM, Fallet,RW, Lane,PH, Carmines,PK: Potassium channel

contributions to afferent arteriolar tone in normal and diabetic rat kidney.

Am J Physiol Renal Physiol 295:F171-F178, 2008

of 29 Diabetes

17. Schulz,A, Kreutz,R: Mapping genetic determinants of kidney damage in rat

models. Hypertens Res 35:675-694, 2012

18. Xu,X, Zhu,X, Ma,M, Han,Y, Hu,C, Yuan,S, Yang,Y, Xiao,L, Liu,F, Kanwar,YS,

Sun,L: p66Shc: A novel biomarker of tubular oxidative injury in patients with

diabetic nephropathy. Sci Rep 6:29302, 2016

19. Vallon,V, Albinus,M, Blach,D: Effect of KATP channel blocker U37883A on

renal function in experimental diabetes mellitus in rats. J Pharmacol Exp Ther

286:1215-1221, 1998

20. Wright,KD, Staruschenko,A, Sorokin,A: Role of adaptor protein p66Shc in

renal pathologies. Am J Physiol Renal Physiol 314:F143-F153, 2018

21. Kotchen,TA, Cowley,AW, Jr., Frohlich,ED: Salt in health and disease--a

delicate balance. N Engl J Med 368:1229-1237, 2013

22. Ilatovskaya,DV, Levchenko,V, Lowing,A, Shuyskiy,LS, Palygin,O,

Staruschenko,A: Podocyte injury in diabetic nephropathy: implications of

angiotensin II-dependent activation of TRPC channels. Sci Rep 5:17637, 2015

23. Brayden,JE: Functional roles of KATP channels in vascular smooth muscle.

Clin Exp Pharmacol Physiol 29:312-316, 2002

of 29Diabetes

24. Salomonsson,M, Brasen,JC, Sorensen,CM: Role of renal vascular potassium

channels in physiology and pathophysiology. Acta Physiol (Oxf) 221:14-31,

2017

25. Menini,S, Amadio,L, Oddi,G, Ricci,C, Pesce,C, Pugliese,F, Giorgio,M,

Migliaccio,E, Pelicci,P, Iacobini,C, Pugliese,G: Deletion of p66Shc longevity

gene protects against experimental diabetic glomerulopathy by preventing

diabetes-induced oxidative stress. Diabetes 55:1642-1650, 2006

26. Yang,SK, Xiao,L, Li,J, Liu,F, Sun,L: Oxidative stress, a common molecular

pathway for kidney disease: role of the redox enzyme p66Shc. Ren Fail

36:313-320, 2014

27. Bock,F, Shahzad,K, Wang,H, Stoyanov,S, Wolter,J, Dong,W, Pelicci,PG,

Kashif,M, Ranjan,S, Schmidt,S, Ritzel,R, Schwenger,V, Reymann,KG, Esmon,CT,

Madhusudhan,T, Nawroth,PP, Isermann,B: Activated protein C ameliorates

diabetic nephropathy by epigenetically inhibiting the redox enzyme p66Shc.

Proc Natl Acad Sci U S A 110:648-653, 2013

28. Sun,L, Xiao,L, Nie,J, Liu,FY, Ling,GH, Zhu,XJ, Tang,WB, Chen,WC, Xia,YC,

Zhan,M, Ma,MM, Peng,YM, Liu,H, Liu,YH, Kanwar,YS: p66Shc mediates high-

glucose and angiotensin II-induced oxidative stress renal tubular injury via

mitochondrial-dependent apoptotic pathway. Am J Physiol Renal Physiol

299:F1014-F1025, 2010

of 29 Diabetes

29. Kumar,S, Kim,YR, Vikram,A, Naqvi,A, Li,Q, Kassan,M, Kumar,V,

Bachschmid,MM, Jacobs,JS, Kumar,A, Irani,K: Sirtuin1-regulated lysine

acetylation of p66Shc governs diabetes-induced vascular oxidative stress and

endothelial dysfunction. Proc Natl Acad Sci U S A 114:1714-1719, 2017

Figure legends Figure 1. Renal p66Shc expression is increased in STZ-treated rats. Western Blot

(WB) analysis reveals increase of p66Shc expression in STZ-treated rats, when

compared with control (citrate-treated rats). Renal cortex tissues were subjected to

immunoprecipitation (IP) and immunoblotting (IB) with anti-Shc antibodies (which

recognize all three Shc isoforms). Every lane corresponds to a separate animal. Asterisk

denotes ~50kDa heavy chain of antibody used for IP.

Figure 2. Knockout of p66Shc inhibits progression of DN. Type 1 diabetes was

induced in control and genetically modified male rats by injection of STZ and evaluated

for 12 weeks after treatment. (A) Hyperglycemia and (B) polyuria indicative of diabetes

is induced after one week and maintained throughout protocol; *p<0.001 compared to

controls. (C) Induction of diabetes significantly reduced weight gain in all rats; * p<0.001

compared to week 1 controls. STZ SS rats gained weight by week 9; ** p<0.01 versus

STZ SS week 1, # p<0.05 versus STZ p66ShcKO week 12. (D) Kidney weight/body

weight ratio increased in STZ-treated rats but did not differ between genotype; *

p<0.001. (E) Albuminuria significantly increased in STZ-induced SS by week 6; p<0.001,

whereas p66ShcKO and p66Shc-S36A rats had significantly reduced albuminuria when

compared to STZ SS at 6 and 12 weeks, respectively; * p<0.01, # p<0.05.

of 29Diabetes

Figure 3. p66Shc knockout reduces diabetes-induced glomerular injury. (A)

Representative images of PAS-stained kidneys comparing glomeruli of STZ-treated to

control animals. Scale bar indicates 50 µm at 40x magnification. Glomeruli of STZ-treated

SS exhibit increased mesangial expansion and sclerosis. Similar lesions in p66ShcKO

and p66Shc-S36A were significantly reduced. (B) Quantification of glomerular injury. At

least 6 rats per group and 50 glomeruli per rat were scored.* p<0.001 vs SS, # p<0.001

vs SS STZ, using two-factor ANOVA with Bonferroni correction, alpha=0.01.

Figure 4. p66Shc knockout restored renal microvascular responses in diabetic rats.

(A) Comparison of afferent arterioles diameters in SS, p66ShcKO and p66Shc-S36A rats

before and after induction of STZ-induced DN. # p<0.05 compared to SS. * p<0.01

compared to STZ SS. (B) Effect of pinacidil. Pinacidil resulted in a greater increase in

afferent arteriolar diameter in SS rats with STZ-induced DN, compared to p66ShcKO with

STZ-induced diabetes and p66Shc-S36A with STZ-induced diabetes. Untreated SS rats,

p66ShcKO rats and p66Shc-S36A rats are also shown. (C) Effect of Glibenclamide.

Glibenclamide resulted in a greater decrease in afferent arteriolar diameter in SS rats

with STZ-induced DN, compared to p66ShcKO with STZ-induced diabetes and p66Shc-

S36A with STZ-induced diabetes. Untreated SS rats, p66ShcKO rats and p66Shc-S36A

rats are also shown. For panels B and C, * p<0.01 compared to SS, and † p<0.05

compared to STZ SS.

Figure 5. Knock out of p66Shc inhibits KATP channels. (A) Representative cell-attached

patch clamp recording of vSMC isolated from wild type (WT), p66Shc, and p66Shc-S36A

mutant rats. Membrane potential (Vp) = 0 mV. Scales are the same for all three

of 29 Diabetes

recordings. Also shown is activity at expanded scale. Closed (c) state is upwards. B.

Scattered plots demonstrating individual channel activity (NPo) for KATP channels

recorded in cell-attached patches from SS, p66ShcKO and p66Shc-S36A SMC cells at 0

mV holding potential. C. Mean ± SE of NPo for experiments with observed channels

activity (shown in B).

Supplemental material

Supplemental Figure 1. Glucose tolerance test indicates that SS rats do not have

metabolic syndrome. Glucose (1 g/kg) was introduced IV at time 0 and levels of

plasma insulin (upper panel) and glucose (lower panel) were measured at indicated time

points in Sprague Dawley (blue, n=5) and SS (red, n=5) rat strains.

Supplemental Figure 2. Comparison of afferent arterioles diameters in SS and

p66ShcKO rats. Nitroprusside vasodilator responses were similar in SS, p66ShcKO and

p66Shc-S36A.

of 29Diabetes

Figure 1. Renal p66Shc expression is increased in STZ-treated rats. Western Blot (WB) analysis reveals increase of p66Shc expression in STZ-treated rats, when compared with control (citrate-treated rats). Renal cortex tissues were subjected to immunoprecipitation (IP) and immunoblotting (IB) with anti-Shc antibodies

(which recognize all three Shc isoforms). Every lane corresponds to a separate animal. Asterisk denotes ~50kDa heavy chain of antibody used for IP.

23266x8356mm (1 x 1 DPI)

of 29 Diabetes

Figure 2. Knockout of p66Shc inhibits progression of DN. Type 1 diabetes was induced in control and genetically modified male rats by injection of STZ and evaluated for 12 weeks after treatment. (A)

Hyperglycemia and (B) polyuria indicative of diabetes is induced after one week and maintained throughout

protocol; *p<0.001 compared to controls. (C) Induction of diabetes significantly reduced weight gain in all rats; * p<0.001 compared to week 1 controls. STZ SS rats gained weight by week 9; ** p<0.01 versus STZ SS week 1, # p<0.05 versus STZ p66ShcKO week 12. (D) Kidney weight/body weight ratio increased in STZ-treated rats but did not differ between genotype; * p<0.001. (E) Albuminuria significantly increased in STZ-induced SS by week 6; p<0.001, whereas p66ShcKO and p66Shc-S36A rats had significantly reduced

albuminuria when compared to STZ SS at 6 and 12 weeks, respectively; * p<0.01, # p<0.05.

50393x37769mm (1 x 1 DPI)

of 29Diabetes

Figure 3. p66Shc knockout reduces diabetes-induced glomerular injury. (A) Representative images of PAS-stained kidneys comparing glomeruli of STZ-treated to control animals. Scale bar indicates 50 µm at 40x magnification. Glomeruli of STZ-treated SS exhibit increased mesangial expansion and sclerosis. Similar

lesions in p66ShcKO and p66Shc-S36A were significantly reduced. (B) Quantification of glomerular injury. At least 6 rats per group and 50 glomeruli per rat were scored.* p<0.001 vs SS, # p<0.001 vs SS STZ, using

two-factor ANOVA with Bonferroni correction, alpha=0.01.

33248x36195mm (1 x 1 DPI)

of 29 Diabetes

Figure 4. p66Shc knockout restored renal microvascular responses in diabetic rats. (A) Comparison of afferent arterioles diameters in SS, p66ShcKO and p66Shc-S36A rats before and after induction of STZ-

induced DN. # p<0.05 compared to SS. * p<0.01 compared to STZ SS.

15570x16840mm (1 x 1 DPI)

of 29Diabetes

Effect of pinacidil. Pinacidil resulted in a greater increase in afferent arteriolar diameter in SS rats with STZ-induced DN (white circles), compared to p66ShcKO with STZ-induced diabetes (black squares) and p66Shc-S36A with STZ-induced diabetes (black diamonds). Untreated SS rats (black circles), p66ShcKO rats (white

squares) and p66Shc-S36A rats (white diamonds) are also shown.

39166x17830mm (1 x 1 DPI)

of 29 Diabetes

Effect of Glibenclamide. Glibenclamide resulted in a greater decrease in afferent arteriolar diameter in SS rats with STZ-induced DN, compared to p66ShcKO with STZ-induced diabetes and p66Shc-S36A with STZ-induced diabetes. Untreated SS rats, p66ShcKO rats and p66Shc-S36A rats are also shown. For panels B

and C, * p<0.01 compared to SS, and † p<0.05 compared to STZ SS.

39039x17729mm (1 x 1 DPI)

of 29Diabetes

Figure 5. Knock out of p66Shc inhibits KATP channels. (A) Representative cell-attached patch clamp recording of vSMC isolated from wild type (WT), p66Shc, and p66Shc-S36A mutant rats. Membrane

potential (Vp) = 0 mV. Scales are the same for all three recordings. Also shown is activity at expanded

scale. Closed (c) state is upwards. B. Scattered plots demonstrating individual channel activity (NPo) for KATP channels recorded in cell-attached patches from SS, p66ShcKO and p66Shc-S36A SMC cells at 0 mV holding potential. C. Mean ± SE of NPo for experiments with observed channels activity (shown in B).

709x528mm (72 x 72 DPI)

of 29 Diabetes

338x190mm (96 x 96 DPI)

of 29Diabetes

338x190mm (96 x 96 DPI)

of 29 Diabetes

inactivation of p66shc decreases afferent arteriolar katp ... · 17/08/2018 · containing adaptor...

Documents