Diabetes and Inflammasomes in the Bladder

J Todd Purves MD, PhD Duke University Medical Center

Division of Urology

Disclosures: None

Introduction

• Diabetic Bladder Dysfunction (DBD) is the most common complication seen in diabetic patients.

• DBD is a progressive complication

– Early DBD = irritative voiding symptoms (diminished sensation, frequency, urge incontinence)

– Chronic DBD = decompensated bladder (insensate bladder, poor compliance, overflow incontinence)

Endocrine referral-DONE!!(?) • Normalize blood sugars; stop diuresis • 58% of patients achieve ADA goals1

• DCCT found strict glycemic control decreased retinopathy, nephropathy. DBD not so much! 2,3,4

• What else can we do?

1 Selvin E et al. Ann Intern Med 2014; 160(8): 517-25 2 Genuth S Endocr Pract 2006; 12 Suppl 1:34-41 3 Sarma AV et al. Urology 2009; 73(6): 1203-9 4 Van den Eden SK et al Diabetes Care 2009; 32(4): 664-70

Bladder Damage from Diabetes

• Neuropathy • Smooth muscle dysfunction • Urothelial (barrier) dysfunction

DBD as manifestation of Peripheral Neuropathy

• PN correlates with OAB in diabetic women Variable OAB syndrome Without OAB syndrome P-value

No. of patients 26 (32.5) 54 (67.5) Polyneuropathy 0.003* With 22 (27.5) 27 (33.8) Without 4 (5.0) 27 (33.8) Retinopathy 0.763 With 5 (6.3) 9 (11.3) Without 21 (26.3) 45 (56.3) Nephropathy 0.572 With 7 (8.8) 11 (13.8) Without 19 (23.8) 43 (53.8)

Tanik, et al. Int Neurourol J 2016 Sep; 20(3):232-9

DBD as manifestation of Peripheral Neuropathy

• PN correlates with OAB in diabetic men

Tanik, et al. Int Neurourol J 2016 Sep; 20(3):232-9

Variable OAB syndrome Without OAB syndrome P-value

No. of patients 14 (35.0) 26 (65.0) Polyneuropathy 0.022* With 11 (27.5) 10 (25.0) Without 3 (7.5) 16 (40.0) Retinopathy 0.115 With 3 (7.5) 1 (2.5) Without 11 (27.5) 25 (62.5) Nephropathy 0.089 With 8 (20.0) 7 (17.5) Without 6 (15.0) 19 (47.5)

Rodent Studies • Decrease in myelinated nerve density

Time point Group Muscle NF200-IR area, mm2

Muscle NF200-IR/muscle area

1 week Control 0.110 ± 0.013 0.017 ± 0.001 Diabetic 0.112 ± 0.005 0.013 ± 0.001 Diuretic 0.114 ± 0.023 0.011 ± 0.002

9 week Control 0.129 ± 0.007 0.019 ± 0.001

Diabetic 0.112 ± 0.013 0.012 ± 0.001*

Diuretic 0.128 ± 0.013 0.012 ± 0.001*

20 week Control 0.128 ± 0.001 0.021 ± 0.001

Diabetic 0.108 ± 0.008 0.010 ± 0.001*

Diuretic 0.129 ± 0.004 0.011 ± 0.001*

Liu, et al. BJUI 2011Jun 113(4):1988-93.

Rodent Studies • Afferent nerve conduction velocity is decreased

Aizawa, N et al. J Urol 2013 Apr 189(4): 1580-1587

Velo

city

(m/s

ec)

DM and Smooth Muscle Dysfunction • Smooth muscle contractility changes according

to compensated versus de-compensated state

Daneshgari, et al. J Urol Jul 176(1): 380-6

DM effects on urothelial barrier

Hanna-Mitchell et al. Am J Physiol Integr Comp Physiol 2013 Jan 304(2): R84-93

Etiology of Diabetic Bladder Dysfunction

• Osmotic diuresis (polyuria)

• Hyperglycemia

Polyuria and hyperglycemia • Diuresis causes bladder hypertrophy

Xiao N, et al. J Urol 2013 Mar 189(3): 1130-6

Diabetes +

Diversion

Polyuria and hyperglycemia • Diabetes/hyperglycemia, but not diuresis alone,

leads to oxidative stress

Xiao N, et al. J Urol 2013 Mar 189(3): 1130-6

What is the molecular mechanism?

• Brownlee’s Unified Theory

• Inflammation

Brownlee’s Unified Theory of Diabetic Complications

Inflammation

Diabetic metabolites uric acid lipids

ROS mitochondrial dysfunction

K+ cellular efflux

NLRP3 Activation INFLAMMATION

Canonical Pathway -Activation The NLRP3 Inflammasome

Activating DAMPs and PAMPS

ROS

Pyroptosis

IL-1β

IL-18

Gasdermin D pore K+ efflux

K+

Active Caspase-1

NLRP3 Inflammasome

Pro-IL-1β Pro-IL-18

Gasdermin D

NEK7 NEK7

Ca2+

IP3

ER

Ca2+

Inouye et al. Curr Urol (2017) in press

NLRP3 mediates DM complications

• Retinopathy • Nephropathy • Cardiomyopathy • Neuropathy • Endothelial dysfunction

Inflammasomes in the bladder? • NLRP3 found in human bladder (Tschopp 2007) • Rat urothelium (Hughes 2014)

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And in the mouse!!

NLRP3

Wild Type

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Diabetic

NLRP3 in urothelium

Hyperglycemia

Inflammasomes and sterile cystitis

• NLRP3 mediates inflammation in cyclophosphamide induced cystitis and BOO

• NLRP3 inhibitor (glyburide) prevents inflammationpreserves tissue and function

Hughes et al. Am J Physiol-Renal (2017) 313:F603-F610 Lutolf et al. Neurourol Urodyn (2017) in press Hughes et al. J. Urol (2016) 195:1598-1605 Hughes et al. Am J Physiol-renal (2014) 306:F299-308

Does NLRP3-induced inflammation lead to DBD?

Diabetic metabolites activate NLRP3

6

10

12

14

16

Glucose

0 3 6 9 12

Glucose (mM added)

8

**

** **

** **

Cas

p-1

Act.

(R

FUs

x 10

00)

0

15

MSU

0 0.3 0.6 0.9 1.2

MSU (µg/ml)

10

Cas

p-1

Act.

(%

max

ATP

res

pons

e)

5

** ** ** *

0

15

HMGB1

0 1 2 3 4

HMGB1 (µg/ml)

10

Cas

p-1

Act.

(%

max

ATP

res

pons

e)

5

** ** **

0

15

AGE

0 50 100 150 250

AGE (gly-BSA; µg/ml)

10

Cas

p-1

Act.

(%

max

ATP

res

pons

e)

5

** **

***

200

***

Akita Mouse Model

• Type 1 DM mouse • Heterozygous Ins2 mutation

Materials and Methods • Female Ins2 (Akita) mice were compared to age-matched controls for

the following end-points:

• Voiding Dysfunction: Cystometry • Active Caspase-1 Activity: FAM-FLICA assay

o Surrogate for activated NLRP3 inflammasome

• Inflammation: Evans Blue extravasation assay

DBD Occurs by Week 15 in Akita Mice

5

15

25

35

45

55

Bla

dder

Pre

ssur

e (c

m H

2O)

Wildtype Mouse Bladder Pressure Tracing

5

15

25

35

45

55

0 300 600 900 1200 1500

Bla

dder

Pre

ssur

e (c

m H

2O)

Time (s)

Diabetic Mouse Bladder Pressure Tracing

DBD Occurs by Week 15 in Akita Mice

0 20 40 60 80

100 120 140 160 180 200

Wildtype Diabetic

Volu

me

(ul)

Void Volume

0

2

4

6

8

10

12

Wildtype Diabetic

Freq

uenc

y (v

oids

/hr)

Frequency

*

**

Increased Caspase-1 Activity at 15 Weeks

14

16

18

20

22

Wildtype Diabetic

Mea

n Fl

uore

scen

ce In

tens

ity

(RFU

s)

Active Caspase-1 *

Increased Bladder Inflammation at Week 15

*

0

500

1000

1500

2000

2500

Wildtype Diabetic

Conc

entr

atio

n in

Bla

dder

(n

g/m

g)

Evans Blue Extravasation

DBD in Akita Mice

• Early DBD appears at 15 weeks in Akita mice • Early DBD associated with

– Inflammation – Activation of Caspase-1

So what happens without NLRP3?

Akita diabetic mouse with NLRP3 knocked out

Akita/NLRP3 KO

Blo

od g

lc (

mg/

dL)

wt KO wt KO

0 50

200

300 350

150

250

100

NLRP3:

Diabetic wild type

*** ***

Akita/NLRP3 KO - No change in hyperglycemia

Void Volume

µl

***

0

50

100

150

200 ###

### ###

Frequency

Mic

turit

ions

/hr ***

0

2.5

5

7.5

10 ***

* ### ###

###

µL

0

50

100

150

200

**

# ##

##

PVR

wt KO wt KO NLRP3:

Diabetic wild type wt KO wt KO NLRP3:

Diabetic wild type

wt KO wt KO NLRP3:

Diabetic wild type

Akita/NLRP3 KO - DBD is attenuated

# ne

rves

/µm

2 (x1

06)

wt KO wt KO 0

10

40

50

30

20

NLRP3:

Diabetic wild type

Akita/NLRP3 KO - Nerve density is preserved

NRP3 inflammation and DBD

High Glucose

DBD

IL-1β

DAMPs

NLRP3

Neuropathy Smooth muscle hypertrophy

Urothelia dysfunction

NLRP3 and Neuropathy

• Rat BOO model • BOO (pressure, stretch, hypoxia) activates NLRP3 • Decrease in bladder nerve density • IL1β causes neuronal apoptosis

Gly Ana

Nerve Density

Control Sham Veh 0

10

20

30

40

BOO

* * *

*

Ner

ve D

ensi

ty

(# n

erve

s x

10-6

)/µm

2 )

Gly Ana

Gly Ana

*

Control Sham Veh 0

50

100

150

200

BOO

Tot

al n

umbe

r of N

erve

s

# Nerves

*

Control Sham Veh 0

4

8

12

16

Bla

dder

Are

a (x

106 µ

m2 )

BOO

Bladder Area

IL-1𝑏𝑏 Apoptosis **

*

Apo

ptot

ic/T

otal

cel

ls (%

)

0 100 250 500 0

10

20

30

40

50

IL-1β (ng/ml)

Pelvic Ganglion Nerves

Apoptosis

Conclusion The NLRP3/IL-1β pathway mediates denervation during BOO

IL-1β

DAMPs (ATP)

NLR

Inflammasome

Casp-1

Adaptor

IL-1β

Pro-IL-1β

Pyroptosis

High pressure

Stretch Hypoxia/reperfusion

NLRP3 Mediates Denervation during BOO IL-1β Induces Apoptosis in Pelvic Neurons in vitro

Future Work • NLRP3 impact on:

– Neuropathy – Smooth muscle dysfunction – Urothelium/barrier function

• Natural course: DM vs. DM/NLRP3-/-

• NLRP3 inhibition as adjunct to Glycemic control

Acknowledgements