Course of Advanced Diagnostics Laboratory evaluation of kidney function Prof. Giuseppe Castaldo (a.y )

Kidney structure

Structure of the nephron

Glomerular filtration + Low mw Hydrophilic structure High blood concentration Hydrostatic pressure

Renal reabsorption and secretion

Biochemical markers of kidney function Glomerular filtrationGlomerular filtration –Capacity of filter “small” hydrophilic molecules –Holding capacity for “big” molecules Tubular functionTubular function –Capacity to reabsorb essential nutrients

Kidney function Markers of glomerular function Markers of tubular function a) Capacity of filtration Serum creatinine Creatinine clearance b) Selectivity of filtration Proteinuria (high mw) Aminoaciduria Glycosuria Proteinuria (low mw) ….

Capacity of filtration S Urea S Creatinine Creatinine clearance Cystatin C

Urea metabolism

Urea Urea is produced by the urea cycle (liver, kidney, brain) Urea is filtered by glomeruli but about 50% is reabsorbed Thus, Urea serum levels depend on: -The rate of production (protein intake, catabolism, liver function) -The rate of glomerular filtration -The rate of reabsorption

1-2% of muscle creatine is converted in creatinine daily1-2% of muscle creatine is converted in creatinine daily The amount of creatinine produced depends on the muscle massThe amount of creatinine produced depends on the muscle mass Creatinine is filtered by glomeruliCreatinine is filtered by glomeruli There is a minimal tubular secretionThere is a minimal tubular secretion Creatinine

OutputKidney PlasmaPoolContent CreatinineInput NormalMuscleMass NormalKidneysDiseasedKidneys NormalMuscleMass DiseasedKidneysNormalKidneysIncreasedMuscleMassReducedMuscleMass Effect of Muscle Mass on Serum Creatinine =

clearance is the volume of plasma from which a substance is completely removed by the kidney in a given amount of time (usually a minute). C = Clearance P = Plasmatic concentration of the substance U = Urinary concentration of the substance V = Volume/minute ratio of urine Thus, the clearance is: C = (U x V )/P

The most used clearance in clinical biochemistry is creatinine clearance V: (volume/min) is obtained dividing the volume of urine excreted in 24h by 1440 (60 min x 24 h) P: plasma creatinine U: urine creatinine Creatinine clearance = (U x V) / P To better assess the GFR of the subject, the body surface area is kept into account referring to an ideal surface of 1.75 m 2. Thus, finally, the clearance value is: C = ( U x V / P) x (Sc/1.75)

The main limitation of creatinine clearance

An example of miscalculation C = ( U x V / P) x (Sc/1.75) C: 90 – 130 mL/min9769 U: 0.8 – 1.3 mg/dL V: 1400 mL1400/ /1440 P: 0.8 – 1.2 mg/dL1.01.0

Cystatin C Cationic polypeptide of about 13 kDa  Family of cysteine proteases involved in the protein catabolism  Constant synthesis in all cells (independent by inflammation)  Cystatin C is filtered by the kidney, fully reabsorbed and immediately degraded in aminoacids  Thus, serum levels of cystatin C are directly related to glomerular filtration rate (GFR) First evidences suggest that serum cystatin C has a higher diagnostic sensitivity than serum creatinine

Proteinuria Glomerular damage Tubular damage

The diagnostic significance of urine protein depends on the mw of the protein In physiological conditions: -Proteins smaller than albumin are filtered and fully reabsorbed by proximal tubule -A few amount of albumin is filtered and eliminated -Proteins greater than albumin are not filtered

The diagnostic significance of urine protein depends on the mw of the protein In patients with impaired tubular reabsorption: -Proteins smaller than albumin are filtered and not reabsorbed -A few amount of albumin is filtered and eliminated -Proteins greater than albumin are not filtered

The diagnostic significance of urine protein depends on the mw of the protein In patients with impaired glomerular filtration: -Proteins smaller than albumin are filtered and reabsorbed -A higher amount of albumin is filtered and eliminated -Proteins greater than albumin are filtered and eliminated

Proteins most frequently analyzed in urine PROTEIN mw, kDa Significance Retinol binding protein 21 Tubular proteinuria  1 -microglobulin 33 Tubular proteinuria Albumin66,5 Glomerular proteinuria Transferrin90 Glomerular proteinuria (severe) IgG160  2 -macroglobulin 750 Glomerular proteinuria (severe)

ProteinNormalGlomerularTubular diseasedisease Total Protein 2.5< 2.5 g/24 h Albumin 500< 500 mg/24 h  2 microglobulin0.10.1> 20 mg/L Glomerular and tubular proteinuria

physiological “ microalbuminuria ” nephropathy 20 mg/L200 mg/L 30 mg/g creat 300 mg/g creat 30 mg/24 h300 mg/24 h Albuminuria

Protein analysis in urine today is performed by nephelometry (quantitative assay) while the electrophoresis of urinary protein is not more used

Urinalysis (Esame delle urine)

Physical examination Volume Specific weight Colour

Physical examination Volume: not relevant on a single sample ! ???

Physical examination Specific weight: strongly depends on diuresis + Diabetes- Hyperhydration + Dehydration- Diabetes insipidus + Enhanced production of ADH ???

Physical examination Colour: strongly dependent on the diet a) Intensity (Vogel score)

Physical examination b) Colour: dark yellow … pink... red... brown Hematuria

Physical examination b) Colour: orange-red Heamoglobin ???

Physical examination b) Colour: “marsala – like” Porphyria

Physical examination b) Colour: orange Bilirubin

Chemical analysis - Glucose - Bilirubin - Urobiline - Ketones - Proteins - pH - Hemoglobin - Nitrites

Urine glucose: normally absent Its presence indicates that the tubular carrier is saturated GLUCOSE -Diabetes -Pregnancy (reduced tubular theshold or gestational diabetes) -Other causes (adrenal hormones, cortisol, steroid therapy, tubular damage) Hyperglicemia (> 170 mg/dL) ???

+ Hepatic jaundice ++ Post hepatic jaundice (cholestasis) BILIRUBIN (only conjugated) Normally absent ???

UROBILINOGEN Normally: < 0.2 mg/24h + Pre hepatic jaundice (hemolysis)

Ketones Normally absent + Ketoacidosis (alcoholic, diabetic, denutrition) ???

PROTEINS (Albumin) Normally “absent”

pH > 7.5 diet rich in fruits or alkalosis pH < 4.5 diet rich in proteins or acidosis pH Normal range 4.5 a 7.5

HEMOGLOBIN (Hb) Normally absent - Hemoglobinuria - Hematuria ???

Nitrite Infection by nitrite-producing bacteria (E. coli, Proteus, Klebsiella, Pseudomonas, Stafilococco, Enterococco, ecc.) More than colonies/mL

Microscopic analysis Urinary sediment

Semiquantitative score Scala numerica >30 Scala nominale RariPochiAlcuniNumerosiA Tappeto Report

Scala numerica >30 Scala nominale RariPochiAlcuniNumerosiA Tappeto Cells Reference intervals + Trauma (i.e., stones, neoplasia, catheterism)

Urinary cells

Scala numerica >30 Scala nominale RariPochiAlcuniNumerosiA Tappeto Leukocytes Reference values ++ Urinary tract Infections

Leukocytes (ureter, bladder, urethra) Leukocyte casts (renal)

Reference values Scala numerica >30 Scala nominale RariPochiAlcuniNumerosiA Tappeto Erythrocytes

Erythrocyte casts (renal) Normal morphology (bladder, ureter)Altered morphology (pre-glomerular)

What does it means? U Leukocytes+++ U Nitrite- Culture-

What does it means? U Leukocyte+++ U Nitrite- U Culture+++

What does it means? U Erythrocytes- U Hemoglobin+

What does it means? U Urobilinogen +++ U Bilirubin+++ S total Bilirubin++ Other tests ?