Lessons Learned from Endoscopy

Mayo Clinic O’Brien Urology Research Center

August 22, 2015Amy Krambeck, MD

Mayo Clinic Department of UrologyJohn C. Lieske, MD

Fang Zhao, MDMayo Clinic Division of Nephrology & Hypertension

Nicholas Chia, PhDDepartment of Surgery

New AimsSpecific Aim #1: Accurately map kidney stone location and precursor lesions after percutaneous nephrolithotomy in defined groups of patients

• Determine clinical and laboratory factors that predict the presence of plaque versus plug

• Correlate stone composition and stone precursor lesions with findings on dual energy CT scan using refined data analysis algorithms

Hypothesis: Renal stone precursor lesions will differ in each group of patients, and within the CaOx group. Hypercalciuria will predict plaque coverage while urinary pH and CGI will predict plugs. Refined dual-energy CT technology will allow accurate classification of stone localization and composition.

New AimsSpecific Aim #2: Define dual energy CT algorithms to predict stone fragilityHypothesis: Stone characteristics by DECT will allow prediction of stone susceptibility prior to intraoperative disruption.

Specific Aim #3: Determine the gene expression profile of papillary tip biopsies with minimal and maximal plaque and plugs Hypothesis: Patients with extensive amounts of plaque will have different gene expression profiles from those with plugs, and both will differ from controls

New Aims

Specific Aim #4: Characterize the microbiome of urine and kidney stones of patients with diverse types of stones.Hypothesis: The urinary and stone microbiome contributes to stone composition, growth upon plaque or plug, and will differ by stone type.

Mapping protocol• 78 consecutive patients

undergoing PCNL • Stones were removed

• Analyzed using µ-cT and IR• Each accessible calyx was

endoscopically mapped using a flexible digital nephroscope

• Biopsies were acquired• 24hr urine chemistries

• Supersaturations (EQUIL2)• Crystal inhibition

• CaOx seeded crystal growth assay

Kuo et al. J Urol, 170:2186, 2003

Randall’s Plaque Summary

• 98.7% patients mapped had some RP• 67.9% had plaque in all calyces

• Plaque SA covered a mean of 3.0% (±3.3%) • 3.5 % (±4.6%) in CaOx

• 1.7 % (±1.6%) in UA

• No correlation between urinary analytes and amount of plaque

• Volume p= 0.20• pH p= 0.07• Calcium p= 0.72

Linnes et al: Kidney Int. 2013 Oct;84(4):818-25

Ductal plugs are common in CaP stone formers

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Stone Type

Linnes et al: Kidney Int. 2013 Oct;84(4):818-25

Urine Correlations with Tubular Plugging

Plugging correlates with:•High pH (p=0.02)•Low Citrate (p=0.02)•High Apatite & Br SS

• (p=0.007 & 0.005)

Linnes et al: Kidney Int. 2013 Oct;84(4):818-25

Further Plaque Evaluation• 42 ICSF PCNL patients with urolithiasis

• 15 men and 27 women • Mean age 62 years old

• 10 non-stone forming controls undergoing PCNL for other indications

• 8 men and 2 women • Mean age 73 years old

Wang X, et al. Distinguishing Characteristics of Idiopathic Calcium Oxalate Kidney Stone Formers with Low Amounts of Randall's Plaque. Clin J Am Soc Nephrol. 2014

Results• 90% of controls and 75% stone formers had plaque

coverage less than 5% (p=0.67)

Upper 25%

Lower 75%

Controls ICSF

Clinical Characteristics

ICSF with high plaque

(n=10)

ICSF with low plaque

(n=32)P value

Age ( yr) 61±7 62±10 0.78

Male 6(60%) 8(25%) 0.059

BMI 27±2.5 31±7.6 0.11

BMI>30 1(10%) 16(50%) 0.03

History of stone events 10(100%) 21(66%) 0.04

Prior stone events ≥4 8(80%) 7(22%) 0.002

History of UTI 0(0%) 11(34%) 0.04

Hypertension 4(40%) 13(42%) 0.92

DM 1(10%) 10(31%) 0.25

Urine ChemistryICSF with high plaque

(n=9)

ICSF with low plaque

(n=27)P value

pH 6.3±0.4 6.2±0.6 0.71

Osmolality 444±128 473±167 0.73

Urine volume (l/24h) 2.5±0.6 1.9±0.8 0.038

Uric acid (mg/24h) 555±244 502±184 0.49

Sodium (mmol/24h) 170±97 134±45 0.14

Potassium (mmol /24h) 65±22 55±20 0.23

Calcium (mg/24h) 291±99 187±86 0.006

Phosphate (mg/24h ) 980±620 679±257 0.047

Magnesium (mg/24h) 112±32 91±38 0.17

Chloride (mmol/24h) 148±88 126±46 0.32

Creatinine ( mg/24h) 1369±593 1134±365 0.16

Sulfate (mmol /24h) 21±11 15±5 0.04

Stone Culture

• 19% of low plaque ICSF had a + stone culture• Diverse organisms: Corynebacterium (n=2), E. coli (n=1),

enterococcus (n=1), Garnerella vaginalis (n=1), and Pseudomonas (n=1)

• None of high plaque group (P=0.059)

• Plaque coverage trended lower among low plaque ICSF with + stone culture • 1.4% vs 4.0%, p=0.19

Stone morphology by micro CT

Likely to arise upon a Randall’s plaque

Unlikely to arise upon a Randall’s plaque

26% of low plaque ICSF had unlikely RP stonesNone of high plaque ICSF did (p=0.04)

Calcium Deposits by Yasue Staining

Punctate Punctate Dense

Low plaque ICSF High plaque ICSF

Low plaque ICSF tended to have lesser amounts of punctate and dense crystallization

Frac

tion

of I

CSF

, %

Conclusion Low Plaque vs. High Plaque

• Stone formers with low amount of plaque had distinguishing characteristics

• Fewer prior stone events • Lower urine calcium excretion• Obese • Past history of UTI and positive stone culture• Stones often lack a calcium phosphate core• Less punctate and dense crystallization

Gene Expression StudiesHypothesis

• ICSFs with low amount of plaque have a different gene expression profile than those with high amount of plaque

Methods• RNA samples from papillary tip biopsies

• 3 low plaque ICSFs • 2 high plaque ICSFs

• Next Generation Sequencing • Platform Type: Illumina HiSeq 2000• Application Type: Transcriptome

• Secondary Analysis • performed by Bio-Informatics Core• LogFC ≥ 2 or ≤ - 2 was used as cut-off for differential

expression analysis• Validation study

• Real-time RT-PCR• Control group: 8 samples• high plaque group: 6 new samples• low plaque group: 8 new samples

Results

61 differential expression (DE) genes between low and high plaque group

• 25 up-regulated DE genes in HP group• 36 down-regulated DE genes in HP group

Selected genes for validation study

Gene aka logFCUp-regulated in High Plaque group CHI3L1 Chitinase 3-Like 1 (Cartilage Glycoprotein-39) 4.096

ALOX15B Arachidonate 15-Lipoxygenase, Type B ( 15-LOX-2) 2.622

EPHA3 EPH Receptor A3 2.546

CRTAC1 Cartilage Acidic Protein 1 2.444

ALB Albumin 2.187

SMOC1 Secreted Modular Calcium-Binding Protein 1 2.052

Down-regulated in High Plaques groupMMP19 matrix metallopeptidase 19 -2.033

S100A8 S100 calcium-binding protein A8 -2.629

SELE selectin E -3.492

UPK3A Uroplakin-3a -3.853

Results of validation study:Genes Up-regulated in HP group

00.20.40.60.8

11.21.41.61.8

2

Normal LP HP

CHI3L

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0.4

0.6

0.8

1

1.2

1.4

1.6

Normal LP HP

CRTAC1

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0.5

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1.5

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2.5

Normal LP HP

EPHA3

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Normal LP HP

SMOC1

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ALB

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ALOX15B

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0.6

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1.4

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UPK3A

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1.5

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S100A8

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SELE

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Normal LP HP

MMP19

Results of validation study:Genes Down-regulated in HP group

Summary of biological function of confirmed DE genes

Genes Up-regulated in High Plaques group

CHI3L1 • CHI3L1 is able to bind both proteins and carbohydrates that allows potential interactions with a variety of cell-surface and extracellular-matrix proteins

EPHA3 • Plays a role in cardiac cells migration and differentiation and regulates the formation of the atrioventricular canal and septum during development.

CRTAC1 • May be involved in cell-cell or cell-matrix interactions.

ALB • Serum albumin, the main protein of plasma, has a good binding capacity for water, Ca(2+), Na(+), K(+), fatty acids, hormones, bilirubin and drugs.

• Major zinc transporter in plasma, typically binds about 80% of all plasma zinc

SMOC1 • This gene encodes a multi-domain secreted protein that plays essential roles in both eye and limb development.

• Probable regulator of osteoblast differentiation

Summary of biological function of confirmed DE genes

Genes Down-regulated in High Plaques group

MMP19 • Endopeptidase that degrades various components of the extracellular matrix

• May also play a role in neovascularization or angiogenesis.

S100A8 • Promote vascular calcification by providing necrotic bodies as calcification nidus

UPK3A • Encodes a member of the uroplakin family, a group of transmembrane proteins that form complexes on the apical surface of the bladder epithelium

• Contributes to the formation of urothelial glycocalyx which may play an important role in preventing bacterial adherence

Immunohistochemistry validation study

• SMOC1• NGS result: Up-regulated in high

plaque group, logFC 2.05

• RT-PCR validation result:• Compared to controls, SMOC1

mRNA expression level was 8.6-fold change in LP groups, and 10.9-fold change in HP groups

• Function: • Encodes a multi-domain secreted

protein that plays essential roles in both eye and limb development

• Probable regulator of osteoblast differentiation

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Normal LP HP

SMOC1

LP: LS0021 LP: LS0088LP: LS0072

HP: LS0049

HP: LS0040 HP: LS0010

LC0001 LC0002 LC0003

SMOC1

UA Stones: often mixed

UA SF: Papillae similar to CaOx

Non Stone Forming ControlN = 19

CaOx

N = 95

Uric Acid

N = 23

p-value

% Plaque SA (median; IQR)

0.9 (0.6-1.5) 2.0 (1.0-4.3) 2.0 (1.0-3.1) 0.009

% Plug SA (median; IQR)

0.0 (0.0-0.0) 0.0 (0.0-0.7) 0.1 (0.0-1.0) 0.006

# with plaque > 0 (%) 17 (90) 95 (100) 23 (100) 0.002

# with plug > 0 (%) 2 (11) 43 (45) 13 (57) 0.006

UA vs CaOx Histology

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Punctate BMCalcifications

Dense BM Calcifications Interstial Inflammation Intraluminal Crystals Polarizing Crystals

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UA

CaOx

*

UA Histology by %CaOx

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InterstitialInflammation

Intraluminal Crystals Polarizing Crystals

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Minority UA

Mixed

Majority UA

Struvite SF: less plaque and similar plugStruvite CaOx P Control P

Endoscopic map, N

Plaque, N (%)

Mean SA, % (SD)

Max SA, % (SD)

29

28 (97)

1.5 (±1.4)

3.6 (±4.4)

90

90 (100)

3.7 (±4.3)

8.4 (±11.2)

0.0018

0.0028

17

15 (88)

1.7 (±2.7)

3.1 (±4.1)

0.76

0.61

Plug, N (%)

Mean SA, % (SD)

Max SA, % (SD)

9 (31)

0.3 (±0.8)

0.8 (±1.8)

40 (44)

0.8 (±2.8)

2.1 (±5.5)

0.14

0.16

2 (18)

0 (±0.1)

0.1 (±0.4)

0.12

0.14

Struvite histology: Prominent Inflammation

Current EnrollmentModifying factor

Confirmed Stone category

Number Mapping data

Malabsorption Renal Tubular acidosis

Hyperparathyroidism

Primary Hyperoxaluria

Calcium oxalate 124 123 21 0 2 3

Apatite 16 16 1 0 1 0

Brushite 14 14 1 3 0 0

Uric Acid 28 28 1 0 0 0

Struvite/infection 32 32 4 0 0 0

Other 7 7 1 0 0 0

Control 19 17 - - - -

Total 240 237 29 3 3 3

N Age %F Weight Stone events@ %DMCaOx 97 61 (12) 44 87 (21) 1 (0; 4) 23

CaOx MAL 22 61 (9) 52 101 (31) 1 (0, 4) 33CaOx HPT 2 54 100 106 20 (0, 40) 0CaOx PH 3 40 (24) 33 73 (13) 5 (5, 15) 0

HA 13 45 (19) 86 78 (13) 2 (0; 20) 14HA MAL 1 42 100 57 7 0HA HPT 1 39 100 117 8 0

BR 10 49 (13) 50 86 (25) 4.5 (2.5; 27) 0BR MAL 1 60 0 93 1005 0BR RTA 3 50 (16) 33 51 (45) 30 (13; 60) 0

UA 27 62 (12) 48 100 (21) 1 (0, 3) 48UA MAL 1 61 0 88 5 0

ST 31 55 (16) 74 92 (27) 1 (0; 3) 26OTHER* 6 49 (24) 57 80 (28) 10 (3; 14) 0CONTROL 17 64 (19) 28 87 (24) 0 6

*Cystine (3); DHA; Matrix; HA ? Infection @ Median (25:75)

Future Direction

• Differences between stone formers, high and low plaque

• Urine biomarkers• Urine exosomes• Micro RNA

• Characterization of iCaOx SF with plug

iCaOx with Plug

• 41 iCaOx with plug• 55 iCaOx without plug

plug <1%N=80

plug >1%N=16

p value

Median Sodium, (U mmol/24hr)(IQR)

145.5

(100.5, 214.0)

169.0

(98.5, 237.0)

0.5

Median Calcium, (U mg/24hr)(IQR)

217.5

(137.0, 292.0)

162.0

(142.5, 327.5) 0.7Median Urine pH

(IQR)6.0

(5.5, 6.4)6.5

(6.2, 6.7)0.02

OH Apatite DG(IQR)

3.7(1.6,4.9)

5.4(3.9,5.7)

0.04

Recent Project 2 publications1: Viers BR, Lieske JC, Vrtiska TJ, Herrera Hernandez LP, Vaughan LE, Mehta RA, Bergstralh EJ, Rule AD, Holmes DR 3rd, Krambeck AE. Endoscopic and histologic findings in a cohort of uric acid and calcium oxalate stone formers. Urology. 2015 Apr;85(4):771-6. doi: 10.1016/j.urology.2014.12.036. Epub 2015 Feb 11. PubMed PMID: 25681832; PubMed Central PMCID: PMC4380657.

2: de Cógáin MR, Lieske JC, Vrtiska TJ, Tosh PK, Krambeck AE. Secondarily infected nonstruvite urolithiasis: a prospective evaluation. Urology. 2014 Dec;84(6):1295-300. doi: 10.1016/j.urology.2014.08.007. Epub 2014 Oct 11. PubMed PMID: 25306481; PubMed Central PMCID: PMC4257060.

3: Wang X, Krambeck AE, Williams JC Jr, Tang X, Rule AD, Zhao F, Bergstralh E, Haskic Z, Edeh S, Holmes DR 3rd, Herrera Hernandez LP, Lieske JC. Distinguishing characteristics of idiopathic calcium oxalate kidney stone formers with low amounts of Randall's plaque. Clin J Am Soc Nephrol. 2014 Oct 7;9(10):1757-63. doi: 10.2215/CJN.01490214. Epub 2014 Aug 4. PubMed PMID: 25092598; PubMed Central PMCID: PMC4186508.

4: Liu Y, Qu M, Carter RE, Leng S, Ramirez-Giraldo JC, Jaramillo G, Krambeck AE, Lieske JC, Vrtiska TJ, McCollough CH. Differentiating calcium oxalate and hydroxyapatite stones in vivo using dual-energy CT and urine supersaturation and pH values. Acad Radiol. 2013 Dec;20(12):1521-5. doi:10.1016/j.acra.2013.08.018. PubMed PMID: 24200478; PubMed Central PMCID: PMC3963806.

5: Krambeck AE, Lieske JC, Li X, Bergstralh EJ, Rule AD, Holmes D 3rd, McCollough CM, Vrtiska TJ. Current computed tomography techniques can detect duct of Bellini plugging but not Randall's plaques. Urology. 2013 Aug;82(2):301-6. doi: 10.1016/j.urology.2013.04.028. Epub 2013 Jun 20. PubMed PMID: 23791212; PubMed Central PMCID: PMC3744363.

Mayo Clinic O’Brien Urology Research Center