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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)
Page 1 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.
Page 2 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
Page 3 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
Page 4 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).
Page 5 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
Page 6 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
Page 7 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 ±
Page 8 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
Page 9 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
Page 10 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
Page 11 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.
Page 12 of 29Diabetes
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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.
Page 18 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
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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.
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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)
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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)
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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)
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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)
Page 24 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)
Page 25 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)
Page 26 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)
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338x190mm (96 x 96 DPI)
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338x190mm (96 x 96 DPI)
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