URINARY PROFILES

Giovanni B Fogazzi, MD, Milano, Italy Giuseppe Garigali, BSc, Milano, Italy Nuša Avguštin, MD, Ljubljiana, Slovenia THE FOUR PILLARS OF WISDOM OF URINARY MICROSCOPY 1. Sound laboratory methodology

2. Knowledge of all particles and their clinical meaning

3. Identification of the main urinary profiles (= combining urinary sediment findings with and S-)

4. Placing of urinary findings into a wider laboratory and clinical context MAIN URINARY PROFILES

• Isolated microscopic * • Glomerular diseases • Acute interstitial • Acute injury • BK virus in kidney transplant recipients * • Urological disorders •

* DEALT WITH IN OTHER PARTS OF THIS COURSE GLOMERULAR DISEASES

A WIDE SPECTRUM OF URINARY PROFILES URINARY PROFILES IN GLOMERULAR DISEASES • Isolated dysmorphic microscopic hematuria • Isolated proteinuria • Proteinuria+microscopic hematuria • Gross hematuria (one shot,recurrent,persistent) • The nephritic sediment • The nephrotic sediment • The nephritic+nephrotic sediment

S-CREATININE NORMAL OR INCREASED EITHER ACUTELY OR CHRONICALLY THE ACUTE NEPHRITIC SEDIMENT ACUTE

DEFINITION: A condition characterised by the rapid increase of creatinine associated with hematuria (either microscopic or gross) and variable proteinuria,with/without high blood pressure CAUSES: Proliferative and/or necrotizing (GN), either primary or secondary PROLIFERATIVE GN

GN characterised by increased numbers of cells (either local or from the circulation) within the glomeruli with/without necrotizing lesions PROLIFERATIVE GN IN OUR UNIT (in decreasing frequency)

• IgA nephropathy • Paucimmune crescentic/necrotizing GN • nephritis (class III and IV) • Mesangiocapillary GN • Acute postinfectious • Schönlein-Henoch purpura GN A NORMAL IgA NEPHROPATHY Increased number of mesangial cells, increase of mesangial matrix and dominant IgA deposits) CRESCENTIC GN Crescents, either focal or diffuse, circumferential (A) or segmental ( B)

A B THE NEPHRITIC SEDIMENT DISTINGUISHING PARTICLES: - RBCs (usually >50/HPF up to too many to count) - ERYTROCYTIC/ CASTS

ASSOCIATED PARTICLES: - WBCs (~1-10/HPF) - R-RTECs (0-≥≥≥1/HPF) - OTHER CASTS: epithelial, leukocytic, granular, waxy

S-creatinine: rapid increase * Proteinuria: mild to ≥ 3.5 g/24 hours) THE NEPHRITIC SEDIMENT Many RBCs, often but not always dysmorphic THE NEPHRITIC SEDIMENT RBC casts ACUTE NEPHRITIC SEDIMENT: ORIGIN OF RBCs

Bonsib SM.

Am J Pathol 1985; 119:357-360

Scanning electron microscopy investigation on acellular glomeruli.

Numerous holes and gaps of different size in the glomerular basement membrane of a patient with necrotizing (non proliferative) GN ACUTE NEPHRITIC SEDIMENT: ORIGIN OF WBCs

Burkholder PM Am J Pathol 1969, 56: 251-265

Transmission electron microscopy investigation of glomerular basement membrane in acute proliferative GN.

PMN passage through holes of GBM (arrows) SEQUENCE OF EVENTS LEADING TO NEPHRITIC SEDIMENT

Glomerular Tubular epithelial proliferation ±±± necrosis damage

RBC & WBC passage through gaps Detachment of tubular of GBM and their extravasation cells from tubular into Bowman’s space basement membrane

RBC & WBC passage Tubular cells within into the tubular the tubular lumen lumen

Tubular cells entrapment RBC & WBC entrapment within the matrix of casts within the matrix of casts forming in the tubules forming in the tubules

Tubular cells and Hematuria RBC & WBC cylindruria epithelial casts Leukocyturia in the THE URINE SEDIMENT IN PROLIFERATIVE GNs

The examination over time of the urine sediment is of particular importance in patients with chronic renal diseases which can recur after a phase of remission, such as and small vessel vasculitis WHY? Together with other laboratory tests, the U-sed allows to evaluate whether the disease is quiescent rather than relapsing THE NEPHROTIC SEDIMENT THE

DEFINITION: A condition characterized by proteinuria >3.5 g/24 hours, , hypercholesterolemia and variable CAUSES: Non proliferative glomerular diseases, either primary or secondary NON PROLIFERATIVE GN

GNs characterised by a normal number of cells within the glomerulus without necrotizing lesions NON PROLIFERATIVE GN IN OUR UNIT (in decreasing order)

• Membranous nephropathy • Focal segmental • • Renal • Lupus nephritis (class V ) • A NORMAL GLOMERULUS MEMBRANOUS NEPHROPATHY Diffuse thickening of the glomerular basement mebranes associated with subepithelial/intramembranous deposits, mostly of IgG FOCAL SEGMENTAL GLOMERULOSCLEROSIS Areas of glomerular sclerosis with at times increased mesangial cellularity

Sclerotic area DIABETIC NEPHROPATHY Diffuse mesangial expansion + sclerotic acellular nodules in the advanced phase

Sclerotic acellular nodule THE NEPHROTIC SEDIMENT

DISTINGUISHING PARTICLES: - No or few RBCs (usually not exceeding 20 to 30/HPF) - Fatty particles

ASSOCIATED PARTICLES: - RTECs - CASTS: granular, fatty, epithelial (waxy in amyloidosis?) S-creatinine: normal to increased Proteinuria: >3.5 g/24 hours) URINARY LIPIDS IN NS

• Fatty droplets (isolated or in clumps) • Oval fat bodies • Fatty casts • Cholesterol crystals LIPID DROPLETS OVAL FAT BODIES CHOLESTEROL FATTY CAST CRYSTAL MASSIVE LIPIDURIA MECHANISMS OF LIPIDURIA

• Lipid ultrafiltration due to altered GBM permeability

• Lipid reabsorption by proximal tubular cells

• Intracellular transport into lysosomes

• Active expulsion of lysosomes from the cells and tubular cellular detachment from tubular basement membrane LIPIDURIA IN PATIENTS WITH NEPHROTIC SYNDROME Ravignaux M-H et al Significance of cytolipiduria during a nephrotic syndrome Néphrologie 1991;12:12-16

NUMBER % WITH SEVERE LIPIDURIA (%) Membranoproliferative GN 12/15 80 5/12 (42)

Amyloidosis 10/13 77 5/10 (50)

Membranous nephropathy 18/25 72 10/18 (56)

Focal segmental glomeruloscclerosis 14/22 64 14/22 (40)

IgA nephropathy 4/7 57 1/4 (25)

Intra-extracapillary GN 6/11 55 2/6 (33)

Minimal change nephropathy 10/24 42 2/10 (20) FATTY PARTICLES IN GNs

Fairley KF. Urinalysis. In: Diseases of the Kidney 5th Ed. Schrier RW and Gottschalk CW Eds. 1993, Boston, Little, Brown & Company, p 354 THE URINE SEDIMENT IN ACUTE INTERSTITIAL NEPHRITIS WHAT IS AIN?

• It is a condition characterized by AKI associated with acute cellular infiltrate of the renal interstitium (mainly with lymphocytes, monocytes and plasma cells; less frequently also PMNs and/or eosinophils) • With possible extrarenal symptoms such as fever, skin rash, arthralgia, lumbar pain, etc • With different causes CAUSES OF AIN (Praga M & Gonzáles E. Kidney Int 2010; 77: 956-61) URINARY FINDINGS IN AIN COMMON VIEW

”A few and unspecific changes” Namely: • Low-grade (NOT detected by dipstick!!) * • Leukocyturia with/without • Hematuria (either microscopic or macroscopic)

* NSAIDs: glomerular proteinuria in nephrotic range EXPERTS VIEW (1) (Praga M & Gonzáles E. Kidney Int 2010; 77: 956-61)

“Microscopic hematuria…is found in almost two-thirds of the patients, although the presence of casts is rare . A very common finding (>80% of cases) is leukocyturia, frequently accompanied by leukocyte casts.” EXPERTS VIEW (2) (Perazella MA & Markowitz GS. Nat Rev Nephrol 2010;6:461) URINE SEDIMENT FINDINGS IN THE LITERATURE (BIOPSY-PROVEN CASES) *

• Patient number 318 • RBCs 171 (53.7%) • WBCs 211 (66.3%) • RTECs --- • Casts 27 (8.5%) -granular 13 (4.0%) -undefined 10 (3.1%) -”cellular” 2 (0.6%) -leukocytic 1 (0.3%) -erythrocytic 1 (0.3%)

*23 papers, from 1975 to 2010, selected because of adequate information on U-sed (drug-induced 17; general 6, including 2 pediatric series ) RBC CASTS IN THE URINE-1

Nine patients with biopsy-proven drug associated AIN. RBC casts found in the urine sediment of a 19-year-old man (11.1%) with AIN due to penicillin RBC CASTS IN THE URINE-2

Red Blood Cell Casts In Acute Interstitial Nephritis

Jerald F. Sigala, MD; Claude G.Biava, MD; Henry N. Hulter, MD

Arch Intern Med 138: 1419-1421, 1978

“A 45-year-old woman receiving hydralazine and hydrochlorothiazide therapy was found to have a reduced glomerular filtration rate, a positive antinuclear antibody reaction, and RBC casts in the urinary sediment . Glomeruli with normal morphology were found on ; however a mild interstitial nephritis was observed that predominantly involved the distal tubules…..” “Although RBC casts have been thought to be diagnostic of glomerular diseases, the present case demonstrates that tubulointerstitial disease can be responsible for RBC cast formation.” RBC CASTS IN THE URINE-3

RBC casts in the urine sediment of 4 out of 12 patients with unspecified AIN (33.3%). Neither details nor comments found in the paper AIN: OUR RETROSPECTIVE EXPERIENCE (1997-2010) Number 21 (biopsy-proven) M:F 12:9 Age 55.4 ±±± 19.6 (17-80)

S-creatinine at RB 4.5 ±±± 3.2 (1.4-14.6) Proteinuria 19/21 (90.4%) (g/24hrs) 1.3 ±±± 2.7 (0.2-12.1) 7 NSAID Cause 5 4 Unidentified 3 Salazopyrin 2 Other (PPI: 1; Doxazosin 1) URINE SEDIMENT FINDINGS (1)

U-sed PARTICLE OUR COHORT (21) LITERATURE (318) WBCs 12 (57.1%) 211 (66.3%) RBCs 10 (47.6%) 171 (53.7%) RTECs 3 (14.2%) --- CASTS 20 (95.2%) 27 (8.5%) Hyaline 19 (90.4%) --- Hyaline-granular 17 (80.9%) --- Granular 13 (61.9%) 13 (4.0%) Waxy 0 --- Erythrocytic 6 (28.5%) 1 (0.3%) Epithelial (RTECs) 3 (14.2%) --- Leukocytic 3 (14.2%) 1 (0.3%) URINE SEDIMENT FINDINGS (2)

RBCs + WBCs + casts 8 Only casts 6 WBCs + casts 2 Only WBCs 1 RBCs + casts 1 RTECs + casts 1 RBCs + RTECs + casts 1 WBCs + RTECs + casts 1 TOTAL 21 COMMENT

In our retrospective study, compared to the 23 papers we have reviewed, we found: 1. A much higher frequency of all casts (with the exception of the waxy ones, which we did not apparently find in any sample) 2. An unexpectd high frequency of RBC casts (6/21: 28.5%), which is close to that reported by Köhler et al, but is much higher than that found in the literature POSSIBLE EXPLANATIONS

1. The care with which in our laboratory we examine the urinary sediments

2. For RBC casts, our finding in the urine is supported by their presence in the renal tubules (3/6 pts). Quite surprisingly, RBC casts are usually not reported among the pathological features of AIN, the only paper reporting on them being that by Geevasinga et al (Clin Gastroenterol Hepatol, 2006; 4: 597-604) on AIN due to proton pump inhibitors Interstitial HOW DO inflammation URINARY RBC

Disruption of interstitial CASTS FORM vessel wall IN AIN?

RBC extravasation within the interstitium

Passage of RBC into tubular lumen through Packing of RBC within TBM gaps the tubular lumen

RBC cast cylindruria CONCLUSION

1. In our experience, the urinary sediment findings in AIN are somewhat different from those reported in the literature

2. The finding of RBC casts in the urine sediment DOESDOES NOTNOT rule out the diagnosis of acute interstitial nephritis 2012; 60: 330-332

EOSINOPHILURIA STILL A SENSITIVE AND SPECIFIC MARKER OF AIN?

EOSINOPHILS (BY MGG) EOSINOPHILURIA IN AIN AUTHOR STAIN URINARY EOSINOPHILS Galpin 1975 Wright’s AIN (>33%): 9/9 (100%) Other conditions: 0/43 Linton 1980 Wright’s AIN (>33%): 6/9 (67%)

Corwin 1985 Wright’s AIN (>1%): 8/9 (88%) Other conditions: 27/56 (48%) Nolan1986 Hansel’s AIN ( ≥≥≥1%): 10/11 (91%) vs Wright’s* Other conditions: 11/66 (17%) Corwin 1989 Hansel’s AIN: 5/8 (63%) vs Wright’s* Other conditions: 13/144 (9%) Ruffing 1994 Hansel’s vs AIN (>1%): 6/15 (40%) Wrigh’ts Other conditions: 10/36 (28%)

* = Hansel’s more sensitive than Wright’s MURIITHI AK et al (Clin J Am Soc Nephrol 2013; 8: 1857-62)

• Retrospective study on 566 pts, all with a RB and the search for Ueos by Hansel’s stain Ueos >1% = 179 pts

AIN: 28/91 Other conditions: 151 (31%) Ueos IN BIOPSY-PROVEN KIDNEY DISEASES <1%

>1%

>5% Ueos IN AIN Ueos >1%: 28/91 (1-5%: 10; >5%: 18)

>1% Cutoff >5% Cutoff Sens 30.8 19.8 Spec 68.2 91.2 PPV 15.6 30.0 NPV 83.7 85.6 Pos LR 0.97 2.3 Neg LR 1.01 0.9

Positive LR >10 = good evidence to rule in a disease Negative LR <0.1 = good capacity to rule out a disease CONCLUSIONS

Clin J Am Soc Nephrol 2013; 8: 1841-3 ACUTE KIDNEY INJURY (AKI) DEFINITION

Decline of renal function occurring over hours or days with/without reduced urine output in a variable clinical context CAUSES

• PRE-RENAL Intravascular volume depletion of whatever cause • RENAL Glomerulonephritis Acute interstitial nephritis Large or small vessel disease Intratubular obstruction Acute tubular necrosis (ATN) POST-RENAL Acute urinary tract obstruction

In clinical practice it is very important to distinguish prerenal AKI from AKI associated with ATN URINARY MARKERS IN AKI: PRE-RENAL vs ATN PRE-RENAL ACUTE TUBULAR NECROSIS Osmolality >500 <500

Specific >1.020 <1.020 gravity Sodium <20 >40

FE Na <1 >2

NEW BIOMARKERS: KIM-1; NGAL; IL-18,etc The two conditions can also be distinguished on the basis of the urinary sediment findings as suggested by a number of studies THE URINE SEDIMENT IN AKI: RECENT STUDIES Clin Pract 2008; 110: c145-c150

The study, performed on a small number of patients (No = 18), shows that a cast score index based on the number of granular and epithelial casts , is useful in identifying patients with more severe AKI associated with ATN Clin JASN 2008; 3: 1615-9

231 hospitalized patients with AKI investigated by the means of a urine sediment score system based on the number of: GRANULAR CASTS & RTECs CONCLUSION S The study, based on a large cohort of patients, shows that a urine sediment score based on granular casts and RTE cells is useful in differentiating AKI patients with ATN from those without (prerenal AKI)

However, it must be noted that some overlaps are found between the two conditions especially when the number of granular casts and RTECs are 1-5/HPF Clin J Am Soc Nephrol 2010; 5: 402-408 Prospective study on 197 hospitalized patients with AKI (63 pre-renal, 134 ATN)

AIMS: 1. To investigate the correlation between U-sed score and AKI stage (based on S-creat level only) 2. To investigate a U-sediment score (based on the No of granular casts and RTECs) as predictor of AKI worsening (= S-creat increase from the time of consultation, need for dialysis, or in-hospital death) CONCLUSIONS

U-score based on the number of RETCs and of GCs is associated with AKI stages and with the risk of worsening of AKI U-score is a better predictor of worsening of AKI than changes of S-creati from baseline U-score reflects cell death and apoptosis in the renal tubules Urine microscopy should remain as a standard practice and a valuable tool in the diagnosis and assessment of AKI Clin JASN 2011; 6: 2740-9

• Prospective study on 249 patients with AKI • Aims: to test the utility of new and traditional urine biomarkers of AKI in predicting primary and secondary outcomes • New biomarkers: N-GAL, KIM-1, IL 18 • Traditional biomarkers: FeNa, FeUrea, urine microscopy (No of granular casts and RTECs) CONCLUSIONS

1. A single marker measurement or microscopy score at the first rise in Screat is able to risk stratify patiens for progression to higher AKI stage or in-hospital death irrespective of AKI aetiology

2. FeNa and FeUrea are not useful for AKI prognosis in this cohort of patients IN CONCLUSION

All these studies demonstrate that

• Granular casts • Renal tubular epithelial cell casts • Renal tubular epithelial cells Are good markers of AKI associated with ATN THE ATN MARKERS (1)

RTECs and renal tubular Renal tubular epithelial fragments cell casts THE ATN MARKERS (2)

Granular casts granular casts granular ± ± ±± ATN overlaps! Pre-renal AKI RETC casts RETC ± ± ±±

GENERAL RULE GENERAL and severe the tubular the severe and damage RETCs Only hyaline andhyaline-granular casts The higherThe number the likely more the Without forgetting exceptions and/or IN ADDITION

ATN can be present in all diseases which affect the renal parenchyma acutely such as Glomerulonephritis Acute interstitial nephritis Large or small vessel disease Intratubular obstruction In these conditions granular casts, RTEC casts and RTECs can be found in association with other particles PARTICLES ASSOCIATED WITH THE PARTICLES OF ATN IN ACUTE DISEASES OF RENAL PARENCHYMA

Many RBCs & Active proliferative GN RBC casts

WBCs ±±± Acute interstitial nephritis WBC casts

Myoglobin Rhabdomyolysis casts

Crystalline Crystals nephropathy ATN ASSOCIATED WITH INTRATUBULAR PRECIPITATION OF CRYSTALS

H&E stain Polarized light UROLOGICAL DISORDERS UROLOGICAL DISEASES

• Urothelial adenoma/carcinoma • Urolithiasis • Urinary tract obstruction

• Other disorders Share the same U-sed findings (+ specific features in some conditions) SHARED U-sed FINDINGS

• Isomorphic hematuria (mild to gross H) with/without • Leukocyturia with/without • Transitional cells S-creatinine normal or increased, either acutely or chronically Proteinuria usually absent unless there is gross haematuria. In this case proteinuria is due to the loss of whole blood, which also contains (up to ++++ by disptick) SPECIFIC U -sed FINDINGS IN UROLOGICAL DISEASES

• Crystals (urolithiasis)

• Atypical/malignant transitional cells (urothelial neoplasia)

• Eggs of Schistosoma haematobium A UROLOGICAL SEDIMENT (Many WBC, a few isomorphic RBC and some deep transitional cells) NORMAL TRANSITIONAL CELLS

DEEP SUPERFICIAL ATYPICAL/MALIGNANT UROEPITHELIAL CELLS Clin Chim Acta 2015, 439:107-111 URINARY TRACT INFECTION THE URINARY SEDIMENT OF URINARY TRACT INFECTION • WBC + bacteria Without ±±± isomorphic RBCs kidney ±±± superficial transitional cells involvement

• WBC + bacteria ±±± isomorphic RBCs With kidney ±±± leukocytic casts involvement ±±± RTECs

S-Creatine normal or increased Proteinuria absent to mild (tubular) WBC & BACTERIA UTI vs URINE CONTAMINATION FROM GENITAL SECRETIONS

INFECTION CONTAMINATION WBC + Bacteria WBC ±±± Bacteria with/without with/without Isomorphic RBCs Isomorphic RBCs Superficial Massive amounts of transitional cells squamous cells Candida and/or Trichomonas vaginalis LARGE AMOUNTS OF SQUAMOUS EPITHELIAL CELLS DUE TO GENITAL CONTAMINATION TRICHOMONAS CANDIDA VAGINALIS URINARY TRACT INFECTION DUE TO PARASITES: URINARY SCHISTOSOMIASIS (Schistosoma Haematobium )

Endemic in many geographic areas (colored areas), from the Middle- East to Northern, Western and South-Eastern Africa LIFE CYCLE CLINICAL MANIFESTATIONS

• Isolated isomorphic microscopic hematuria • Recurrent bouts of gross hematuria (which in rural Africa are also known as “male menstruation ”) • Obstructive uropathy due to granulomas/fibrosis • Bladder carcinoma • Glomerulonephritis BLADDER SCHISTOSOMIASIS

Eggs of Schistosoma haematobium surrounded by intense infiltrates of eosinophils and other inflammatory cells. From: CDC/ Dr. Edwin P. Ewing, Jr. as reported in: Public Health Image Library (PHIL) URINARY FINDINGS Data collected at Tanguiéta Hospital, Bénin Republic, West Africa DIAGNOSTIC PARTICLE: - Eggs of Schstosoma H

ASSOCIATED PARTICLES: - Isomorphic RBCs (100%) - WBCs (including eosinophils)(92%) - Bacteria ( 34%) - Superficial transitional cells (28%) S-creatinine: normal or increased Proteinuria: absent/present (variable amount) SCHISTOSOMA HEMATOBIUM EGGS

The shell Terminal spike Miracidium

Terminal spike

Size 115-170 x 40-70 µµµm DIAGNOSIS OF URINARY SCHISTOSOMIASIS

To increase the egg yield (hence sensitivity): 1) The patient is asked to perform a physical effort (e.g., a run) 2) The urine is collected between 10 am and 2 pm

The quantitation of the eggs is used to estimate the severity of the infection THANK YOU FOR YOUR KIND ATTENTION