Lessons Learned from Endoscopy

Transcription

1 Mayo Clinic O Brien Urology Research Center Lessons Learned from Endoscopy August 22, 2015 Amy Krambeck, MD Mayo Clinic Department of Urology John C. Lieske, MD Fang Zhao, MD Mayo Clinic Division of Nephrology & Hypertension Nicholas Chia, PhD Department of Surgery

2 New Aims Specific 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.

3 New Aims Specific Aim #2: Define dual energy CT algorithms to predict stone fragility Hypothesis: 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

4 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.

5 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

6 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 Oct;84(4):818-25

7 Ductal plugs are common in CaP stone formers Video Mapping Plug % CaOx CaOx -MAL CaPO4 Stone Type Other Struvite UricAcid Linnes et al: Kidney Int Oct;84(4):818-25

8 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 Oct;84(4):818-25

9 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

10 Results 90% of controls and 75% stone formers had plaque coverage less than 5% (p=0.67) Upper 25% Lower 75% Controls ICSF

11 Clinical Characteristics ICSF with high plaque (n=10) ICSF with low plaque (n=32) P value Age ( yr) 61±7 62± Male 6(60%) 8(25%) BMI 27±2.5 31± 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%) History of UTI 0(0%) 11(34%) 0.04 Hypertension 4(40%) 13(42%) 0.92 DM 1(10%) 10(31%) 0.25

12 Urine Chemistry ICSF with high plaque (n=9) ICSF with low plaque (n=27) P value ph 6.3± ± Osmolality 444± ± Urine volume (l/24h) 2.5± ± Uric acid (mg/24h) 555± ± Sodium (mmol/24h) 170±97 134± Potassium (mmol /24h) 65±22 55± Calcium (mg/24h) 291±99 187± Phosphate (mg/24h ) 980± ± Magnesium (mg/24h) 112±32 91± Chloride (mmol/24h) 148±88 126± Creatinine ( mg/24h) 1369± ± Sulfate (mmol /24h) 21±11 15±5 0.04

13 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

14 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 stones None of high plaque ICSF did (p=0.04)

15 Calcium Deposits by Yasue Staining Punctate Punctate Dense Fraction of ICSF, % Low plaque ICSF High plaque ICSF Low plaque ICSF tended to have lesser amounts of punctate and dense crystallization

16 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

17 Gene Expression Studies Hypothesis ICSFs with low amount of plaque have a different gene expression profile than those with high amount of plaque

18 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

19 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

20 Selected genes for validation study Gene aka logfc Up-regulated in High Plaque group CHI3L1 Chitinase 3-Like 1 (Cartilage Glycoprotein-39) ALOX15B Arachidonate 15-Lipoxygenase, Type B ( 15-LOX-2) EPHA3 EPH Receptor A CRTAC1 Cartilage Acidic Protein ALB Albumin SMOC1 Secreted Modular Calcium-Binding Protein Down-regulated in High Plaques group MMP19 matrix metallopeptidase S100A8 S100 calcium-binding protein A SELE selectin E UPK3A Uroplakin-3a

21 Results of validation study: Genes Up-regulated in HP group CHI3L Normal LP HP ALOX15B Normal LP HP EPHA3 Normal LP HP 1.6 CRTAC ALB 16 SMOC Normal LP HP 0 Normal LP HP 0 Normal LP HP

22 Results of validation study: Genes Down-regulated in HP group 7 MMP S100A Normal LP HP 0 Normal LP HP 2.5 SELE 1.4 UPK3A Normal LP HP 0 Normal LP HP

23 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 extracellularmatrix 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

24 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

25 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 SMOC1 Normal LP HP

26 SMOC1 LC0001 LC0002 LC0003 LP: LS0021 LP: LS0072 LP: LS0088 HP: LS0040 HP: LS0010 HP: LS0049

27 UA Stones: often mixed

28 UA SF: Papillae similar to CaOx Non Stone Forming Control N = 19 CaOx N = 95 Uric Acid N = 23 p-value % Plaque SA 0.9 ( ) 2.0 ( ) 2.0 ( ) (median; IQR) % Plug SA 0.0 ( ) 0.0 ( ) 0.1 ( ) (median; IQR) # with plaque > 0 (%) 17 (90) 95 (100) 23 (100) # with plug > 0 (%) 2 (11) 43 (45) 13 (57) 0.006

29 UA vs CaOx Histology 100 * 90 % Patients with Histologic Feature Present UA CaOx 10 0 Punctate BM Calcifications Dense BM Calcifications Interstial Inflammation Intraluminal Crystals Polarizing Crystals

30 UA Histology by %CaOx % Patients with Histologic Feature Present Minority UA Mixed Majority UA 10 0 Punctate BM Calcifications Dense BM Calcifications Interstitial Inflammation Intraluminal Crystals Polarizing Crystals

31 Struvite SF: less plaque and similar plug Struvite CaOx P Control P Endoscopic map, N Plaque, N (%) 28 (97) 90 (100) 15 (88) Mean SA, % (SD) 1.5 (±1.4) 3.7 (±4.3) (±2.7) 0.76 Max SA, % (SD) 3.6 (±4.4) 8.4 (±11.2) (±4.1) 0.61 Plug, N (%) 9 (31) 40 (44) 2 (18) Mean SA, % (SD) 0.3 (±0.8) 0.8 (±2.8) (±0.1) 0.12 Max SA, % (SD) 0.8 (±1.8) 2.1 (±5.5) (±0.4) 0.14

32 Struvite histology: Prominent Inflammation

33 Current Enrollment Modifying factor Confirmed Stone category Number Mapping data Malabsorption Renal Tubular acidosis Hyperpara thyroidism Primary Hyperoxaluria Calcium oxalate Apatite Brushite Uric Acid Struvite/infection Other Control Total

34 N Age %F Weight Stone %DM CaOx (12) (21) 1 (0; 4) 23 CaOx MAL (9) (31) 1 (0, 4) 33 CaOx HPT (0, 40) 0 CaOx PH 3 40 (24) (13) 5 (5, 15) 0 HA (19) (13) 2 (0; 20) 14 HA MAL HA HPT BR (13) (25) 4.5 (2.5; 27) 0 BR MAL BR RTA 3 50 (16) (45) 30 (13; 60) 0 UA (12) (21) 1 (0, 3) 48 UA MAL ST (16) (27) 1 (0; 3) 26 OTHER* 6 49 (24) (28) 10 (3; 14) 0 CONTROL (19) (24) 0 6 *Cystine (3); DHA; Matrix; HA? Median (25:75)

35 Future Direction Differences between stone formers, high and low plaque Urine biomarkers Urine exosomes Micro RNA Characterization of icaox SF with plug

36 icaox with Plug Median Sodium, (U mmol/24hr) (IQR) Median Calcium, (U mg/24hr) (IQR) Median Urine ph (IQR) OH Apatite DG (IQR) plug <1% N= (100.5, 214.0) (137.0, 292.0) 6.0 (5.5, 6.4) 3.7 (1.6,4.9) plug >1% N= (98.5, 237.0) (142.5, 327.5) 6.5 (6.2, 6.7) 5.4 (3.9,5.7) p value icaox with plug 55 icaox without plug

37 Recent Project 2 publications 1: 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 Apr;85(4): doi: /j.urology Epub 2015 Feb 11. PubMed PMID: ; PubMed Central PMCID: PMC : de Cógáin MR, Lieske JC, Vrtiska TJ, Tosh PK, Krambeck AE. Secondarily infected nonstruvite urolithiasis: a prospective evaluation. Urology Dec;84(6): doi: /j.urology Epub 2014 Oct 11. PubMed PMID: ; PubMed Central PMCID: PMC : 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 Oct 7;9(10): doi: /CJN Epub 2014 Aug 4. PubMed PMID: ; PubMed Central PMCID: PMC : 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 Dec;20(12): doi: /j.acra PubMed PMID: ; PubMed Central PMCID: PMC : 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 Aug;82(2): doi: /j.urology Epub 2013 Jun 20. PubMed PMID: ; PubMed Central PMCID: PMC

38 Mayo Clinic O Brien Urology Research Center