BIOCHEMICAL MARKERS

IN URINE

Prof dr Andrej Veljković

 KIDNEY ANATOMY
the basic role of the kidneys is to maintain the normal composition of blood
and body fluids- MAIN EXCRETORY ORGAN
1L BLOOD /MINUT

– Hilum
– Medulla
– Pyramids
– Papilla
– Renal Pelvis
ROLE OF THE KIDNEY
 STRUCTURE OF THE NEPHRON

• NePHRON
– Glomerule
– Baumanov capsul
– Proximal tubul
– Henles loop
– Distal tubul
– Collection channel
 STRUCTURE OF GLOMERULA
FILTRATION BARRIER

fenestrated endothelium,
glomerular basement membrane (GBM)
pithelial cells
GBM: supporting role, coarse filter. The structure consists of collagen (type IV and VI)
and proteoglycans (heparan sulfate))

Glomerular epithelial cells

(podocytes) on the out side
cytoplasmic processes (toe-foot
processes) they form "slit" pores.
Slit pores are covered with a
membrane consisting of
transmembrane protein nephrine .

fenestre
MECHANISM OF TUBULAR RESORPTION
PROXIMAL TUBULES
80% reabsorption
Water
Electrolyte
(phosphate bicarbonateCL,
Glucose
Amino acid
Small proteins
Urea
Acidum uricum
HENLE'S LOOP
Water reabsorption
DISTAL TUBULES
Water reabsorption,
Na, CL.
Action of ADH and aldostero
COLLECTION CHANNELS
ADH and aldosterone
Water reabsorption,
Na, Cl, K, H,Ammonia.
Impermeable to urea
Three processes are involved in the formation of
urine:
Glomerular filtration
Reabsorption of substances from tubular fluid into
the blood
Secretion of substances from the blood into the
tubular fluid
 BIOCHEMICAL MARKERS IN URINE

Urine (urine) is an excrement, a product of kidney secretion,

in which excess water is excreted , mineral
salts and a number of end products of metabolism.

In 24 hours, 1500 liters of blood pass through the

kidneys. In the form of primary urine, 130-150
liters are created, and as the final urine, about
1.5 liters are excreted.The process of excretion of
primary urine in the glomeruli (ultrafiltration of
blood plasma). Reabsorption processes in renal
tubules (concentration)
Urin is a clear liquid, yellow in color, with a specific odor,
salty-bitter taste.
The daily amount of urine (diuresis) is 18-20ml / kg body
weight, ie.about 1000-1600 ml.
The ratio of day-night rhythm is 2-4: 1.
Urine contains 95-97% water and 3-5% soluble ingredients.
Of the inorganic ingredients, it contains water, sodium,
potassium, calcium, magnesium, ammonia, trace elements,
chlorides, phosphates, sulfates and bicarbonates.
Physiological organic ingredients are: urea, uric acid, purine
bases, creatinine and creatine, hypuric acid, amino acids,
porphyrins, urobilinogen and urobilin, phenols, enzymes (
amylase), hormones and vitamins (C, B groups).
Pathological components of urine are glucose, acetone
bodies, proteins, bile dyes, hemoglobin and its derivatives,
phenyl-keto acids and homogentisic acid.
 PREPARATION OF PATIENTS AND
SAMPLES
ECLM (European Confederation of Laboratory Medicine)
EUG (European Urinaanalysis Group)
NCCLS (National Committee for Clinical Laboratory- America)

The analysis is done in order to:

Diagnosis
Monitoring the course of the disease
Monitoring efficacy and complications during therapy
Screening for asymptomatic,
congenital and hereditary diseases
SAMPLING TIME
Individual samples (first morning, second morning, middle stream,
Random samples taken at a specific time (24 hours, night)
 Routine urine examination - first
morning sample
• the best results are obtained when examining
the first morning sample, which usually has a
high osmolality and low pH, which prevents rapid
decomposition of the formed elements. Urine
testing should be done within two hours of taking
the sample. Standing urine for a long time leads
to a change in pH, and bacterial contamination
and the conversion of nitrates into nitrites are
possible, so false positive tests for bacteriuria
can be obtained.
 Urine collection

It is important to avoid contamination of

the sample with bacteria, leukocytes and
desquamated epithelial cells. For a
general examination, a medium stream is
taken in a chemically clean container,
after the morning toilet of the urogenital
tract.
 24-hour urine

Accurately measured
amount and with an
accurately defined
method of collection.
-the first one in the
morning is missed.
-it is taken for the next
24 hours
-the next day's morning
urine is included
 PHYSICAL AND CHEMICAL PROPERTIES
OF URINE
QUANTITY:

COLOR:

URINE pH:

URINARY COMPOSITION:

URINARY SEDIMENT
 QUANTITY OF URINE
• Diuresis is the volume of the urine that is excreted within 24 hours. It depends on fluid
intake and fluid elimination by perspiration, respiration or gastrointestinal tract.
Average daily urine production in men is 1500-2000 ml, and 1200-1600 ml in women.
Normally, daily production of the urine is 2 times greater than production during the
night.
Polyuria is a condition of excessive production of urine, more than 2000 ml per day. It
is present in diabetes mellitus, diabetes insipidus etc.
Oliguria is a condition when urine production is less than 500 ml per day. It can be
caused by low fluid intake, high body temperature, diarrhea, some kidney and heart
diseases.
Anuria is a condition when urine production is less than 100 ml per day. It is present
in uremia and acute kidney inflammation.
Pollakiuria refers to a frequent urination (more than 5 times during the day and more
than 1 time during the night). It is observed with anxiety, excessive consumption of
fluids and also with pregnancy. It may be a symptom of diseases of the bladder,

uterus, or prostate gland .

• Dysuria refers to a burning or stinging
sensation during urination. It is most
often a result of a urinary tract
infection.
Eneuresis refers to a repeated inability
to control urination. It is a result of
inflammation or CNS disorders.
Nocturia or nycturia is defined as the
complaint that the individual has to
wake at night one or more times for
urination.
 APPEARANCE (TRANSPARENCY) OF URINE

Physiological code of healthy TRANSPARENT URIN

Presence of insoluble salts, blood cells, bacteria,
epithelial cells or fats can cause the urine turbidity.
 Urine color
• Urine color is usually a pale
yellow. It is primarily result of
the urobilin presence. The
color principally depends
upon a body's level of
hydration, as well as other
factors.
Bilirubin- dark colored urine,
as bier melanin- black color
hemoglobin- red color
alkapton medicines, bacteria
etc
 pH of urine
• The pH of urine is usually between 5 and 7. Consumption of animal food is
related to more acidic urine reaction, while vegetarian diet is related to more
alkaline urine reaction.
• The pH of urine can be determined using urine test strips or lakmus paper.
Urine pH varies during the day.
• Urine has more alkaline reaction during the stomach phase of food
digestion because (post-prandial alkalosis) because bicarbonate ions is
extensively secreted during the HCl synthesis. During the intestinal
phase of food digestion urine has more acidic reaction because of
phosphate excretion.
• Urine tract infections are associated with alkaline urine pH because
ammonia is produced due to protein degradation by bacteria.
 Specific gravity of urine
• Normal urine density or specific gravity values vary between
1.010 and 1.030.
Hypersthenuria is related to an excretion of urine of unusually
high specific gravity and concentration of solutes, resulting
usually from loss or deprivation of water. The exception is
diabetes mellitus where hypersthenuria and polyuria are
present at the same time.
Hyposthenuria is related to an excretion of urine of low
specific gravity due to an inability of the tubules of the kidneys
to produce concentrated urine.
Isosthenuria is related to an excretion of urine that has not
been concentrated by the kidneys and has the same
osmolality as that of plasma.
Urine density is determined using urinometer.
 URINARY COMPOSITION
It contains 95-97% water and 3-5% dissolved
ingredients
INORGANIC INGREDIENTS
Water, sodium (NaCl), potassium, calcium, magnesium, ammonia,
trace elements, chlorides, phosphates (inorganic and organic), sulpha
(inorganic-free, organic-bound and neutral sulfuramino acid), bicarbo

ORGANIC INGREDIENTS
Urea, uric acid, purine bases, creatinine and creatine,hippuric acid,
amino acids (0.4-1 g / 24 hours), porphyrins,urobilinogen and
urobilin, phenols, enzymes (a amylase), hormones and vitamins (C, B
groups).
PATHOLOGICAL INGREDIENTS OF
URINARY TRACT
glucose, acetone bodies, proteins, bile dyes, hemoglobin and
its derivatives, phenyl keto acids, homogentisic acid
URINARY PARAMETER MONITORING
TEST TAPE
 Glucose in the urine
• .Glucose is not usually found in urine. It is reabsorbed by active
transport. The presence of glucose in the urine is called glycosuria
or glucosuria (Glykemia above 8,5 mol/l). Glycosuria leads to
excessive water loss into the urine with resultant dehydration, a
process called osmotic diuresis. Glucose may occur in urine with:
• diabetes- Small increases in urine glucose levels after a large meal
are not always a cause for concern.
• a rare condition in which glucose is released from the kidneys into
the urine, even when blood glucose levels are normal (renal
glycosuria)
• pregnancy - Up to half of women have glucose in their urine at some
time during pregnancy. Glucose in the urine may mean that a
woman has gestational diabetes.
 Bilirubin in urine
• The normal concentration of total bilirubin
(indirect + direct) in plasma is 0-21 µmol / L.
Values for unconjugated bilirubin vary from 9-16
µmol / L, while levels conjugated bilirubin ranges
from 0-5 µmol / L. An increase in the
concentration of bilirubin in the blood is called
hyperbilirubinemia. There is no bilirubin in the
urine of healthy people. When its concentration
in the blood reaches a value of 85 µmol / L, it is
found in urine.
• Only conjugated bilirubin can be found in urine
(hepatic and posthepatic hiperbilirubinemia)
 Acetone bodies

Acetone
Acetoacetic acid
β-hydroxybutyric acid
 Acetone bodies
Acetyl-CoA formed in the process of b-oxidation of fatty acids
in mitochondria is further oxidized in the tricarbon cycle
acid to water and carbon dioxide.
A certain amount of this acetyl-CoA is produced in the
mitochondria of the liver (and to a certain extent kidney) is
involved in the process of ketogenesis, i.e. in the synthesis
of acetone bodies .An important source of energy for many
peripheral tissues Heart, skeletal muscle, brain
acetonemia or ketonemia
acetonuria or ketonuria
The synthesis and breakdown of acetone bodies
represent a way of redistributing fat energy between the
liver and extrahepatic tissues, because excess
acetyl-CoA in the liver is incorporated into acetone
bodies, and they are used as an energy source in other
tissues.Many tissues, except the CNS, use fatty acids for
energy needs. Acetone bodies are the water-soluble
equivalent of fatty acids, and are easily transported
through blood plasma to extrahepatic tissues, where
they are an extremely useful source of energy when
there is no glucose.
All conditions favoring the accumulation of acetyl-CoA
lead to increased synthesis of acetone bodies. An
increase in their concentration in the blood and ketonuria
occur in patients with
unregulated diabetes,
during starvation
and in cases of inadequate nutrition (predominant diet
with fats).
The basis of increased ketogenesis is a disturbance in the
balance between lipolysis and lipogenesis, that is,
simultaneous disorder of fat and carbohydrate
metabolism.
Acetone bodies in Diabetes Mellitus
In the presence of insulin:
Increased entry of glucose into tissues
Decreased lipid mobilization from adipocyte

In the absence of insulin:

Decreased entry of glucose into tissues (no
oxaloacetate to react with acetyl CoA)
Increased lipid mobilization from adipocytes
(increased production of acetone bodies in the liver)
Acetyl-CoA cannot be included in the cycle of tricarboxylic
acids, due to the lack of oxaloacetic acid, which is obtained
by carboxylation of pyruvic acid produced in the glycolysis
process. Oxalacetic acid deficiency also occurs in conditions
of increased gluconeogenesis when it is used for glucose
synthesis.Acetoacetic and b-hydroxybutyric acid are
medium-strength organic acids that require the consumption
of significant amounts of buffer substances - alkaline
cations, so when these acids are eliminated via the kidneys,
sodium and potassium are eliminated, which significantly
reduces the body's alkaline reserves.
As a result, metabolic acidosis occurs
 PROTEINURIA
• Proteinuria is defined as a presence of the proteins in
urine in a concentration higher than 150 mg/1,73m2/24h.
• Normally, under physiological conditions only the
proteins of molecular weight less than 40 kD (β2
microglubulin (12 kDa), cystatine C (13 kDa), α1
microglobulin (27 kDa)) are filtered. The small amounts
of albumin (68 kDa) and transferrin (80 kDa) are filtered
as well, while high molecular weight proteins (IgG (125
kDa), α2 macroglobulin (250 kDa), IgM (900 kDa)) are
completely retained. More than 99% of filtered proteins
are reabsorbed in proximal tubules by the process of
energy-dependent endocytosis.
• Proteinurias are classified as:
• 1. Functional
• 2. Prerenal
• 3. Renal
• a. glomerular (selective and
non-selective)
• b. tubular
• c. mixed
 Functional proteinuria
• represents a transient increase of the proteins excreted in
urine without the presence of kidney disease. It appears in
conditions such as fever, emotional stress, after hard
physical work, and it is a result of hemodynamic changes
without the damage of glomeruli. Orthostatic proteinuria is
a benign condition which appears in some young men
(3-5% of adolescents). The total protein level in urine is
less than 2 g/day. A change in renal hemodynamics
causes protein (mostly albumin) to appear in urine when
they are in the standing position. There is normal urinary
protein excretion during the night but increased excretion
during the day, associated with activity and upright posture.
The exact cause of orthostatic proteinuria is not known
 Prerenal proteinuria
• It appears when the concentration of the low
molecular weight proteins in the plasma is
increased. These proteins are filtered in the
glomerulus and their concentration in the primary
urine is increased, but the reabsorption by the
tubular cells is not complete. Glomeruli are
functionally and structurally intact. The most
common proteins in prerenal proteinuria are:
light chains of immunoglobulins (Bence Jones
protein in multiple myeloma), myoglobin (Crush
syndrome), hemoglobin and enzymes (amylase
in acute pancreatitis)
 Renal proteinuria
• It is caused by functional or organic damage of
the kidney. Glomerular proteinuria is a result of
capillary damage of glomerular membrane. It can
be selective (increased permeability for
medium-sized negatively charged proteins such
as albumin) or non-selective (increased
permeability for all proteins, including high
molecular weight proteins). Tubular proteinuria is
rare and it appears when the ability for
reabsorption of low molecular weight proteins by
the tubules is decreased (alpha 2-microglobulin).
 Postrenal proteinuria
• Postrenal proteinuria
is caused by the
disorders of the lower
urinary tract (ureter,
urine bladder or
urethra) such as
inflammation,
infections, tumors or
calculosis.
 URINARY SEDIMENT

Urine sediment is formed during the prolonged

standing or urine centrifugation. It is composed of
organic and inorganic substances that are
present in urine.

ORGANIZED- (morphologic part) which

consists of different cells such as
epithelial cells, leukocytes,erythrocytes,
bacteria etc
UNORGANIZED- (chemical part) which
consists of crystals and other compounds.
• Normally, organized urine sediment consists of epithelial cells, few
erythrocytes (up to 3 in the microscopic field) and leukocytes (up to
5 in the microscopic field). Increased number of these cells or
presence of bacteria are pathologic and symptoms of different
diseases of urogenital tract.
• Urinary casts are cylindrical structures produced by the kidney and
present in the urine in certain disease states.
• Depending on their composition urine casts are classified as:
hyaline, granular, fatty, bacterial, epithelial, red blood cell casts,
white blood cell casts etc.
• Unorganized sediment is classified as sediment that appears in
acidic urine and sediment that appears in alkaline urine.
 leukocytes Erythrocy
Erythrocytes and leukocytes tes

Epithelial cells
Bacteria
Ammonium biurate Uric acid
The most common crystals that
Ammonium biurate are present in acidic urine are
crystals are uric acid crystals. They look
yellowish-brown and like barrels, rosettes, rhomboids,
can be seen in a needles or hexagonal plates.
“thorn apple” shape They are yellow because of
(round with thorny urochrome which is present on
projections) or in their surface. Uric acid crystals
spherical form are usually present in urine of
people who eat more meat, in
concentrated urine and in urine of
patients with gout
 Calcium phosphates Calcium sulfates

Dicalcium phosphate looks

Calcium sulfate crystals are
like long, wedge-shaped,
present in very acidic urine.
whitish-gray needles that
They look like long white
can be seen as single
needles.
crystals or grouped in a
Rarely calcium sulfate crystals
form of star-piles where the
can have a prismatic shape.
spike is oriented to the
center of the pile.
Calcium oxalate

Calcium oxalate crystals can be found in acidic,

neutral or alkaline urine. They are colorless
and have a form of letter envelope, sand watch or
biscuit.
 ENZIMURIA

Try to define a specific marker of the progressive course of

the disease as early as possible

Sensitive
Non-invasive indicator
test renal impairment

Proteomic technology identifies more than 1000 proteins and

peptide in urine and allows simultaneous examination of
samples
and correlation with individual diagnosis, response to therapy
and prognosis.
 ENZIMURIA: A TUBUL DAMAGE
MARKER

N-acetyl-b-D-glucosaminidase(NAGA)

Leucine aminopeptidase(LAP)

Gamma-glutamyl transferase(g-GT)

Retinol binding protein(RBP)

Dipeptidyl aminopeptidase IV (DPA IV)

Alanine aminopeptidase(AAP)

Alpha 1 microglobulin(α1M)

transferrinuria
 AMINOACIDURIES
RENAL AMINOACIDURIES
•CYSTINE LYSINURIA (cysteine, lyzin, arginine and ornithine)
•HOMOCYSTINURIA- (disorder in activity of cistation b sintetase)
•HARTNUP SYNDROME (disturbance in transport, ie resorption
• amino acids)
•HEREDITARY GLYCINURIA
•FANCONI SYNDROM (renal aminoaciduries, mental retardation,
eye anomalies)
•GALACTOSEMIA followed by amino aciduria

PRERENAL METABOLIC DISORDERS

•ALKAPTONURIA- homogentisic acid
•TYROSINOSIS-tirozine
•PHENYLKETONURIA- fhenil alanine and acyds
 STEROID HORMONES

17 KETOSTEROIDS (important for monitoring male sex hormon

17 HYDROXYSTEROIDS (products of the metabolism of

glyco and mineral corticosteroids)

TESTOSTERONE (activity Leydig's glands)

ESTROGENS (important for pregnancy)

PROGESTERONE (endometrium disorders and pregnancy)

CHORION GONADOTROPIN (functional state of the placenta)

Examination of urine in nephrology

Urine examination plays an important role in the diagnosis

and monitoring of kidney disease therapy.
The earliest biochemical markers are detected in the urine
even when the patient has no clinical or subjective
symptoms, which enables doctors to make an early
diagnosis and provide adequate treatment and follow-up.
 Microalbuminuria
• An early marker of essential hypertension,
positive family history of kidney diseases
and diabetic nephropathy is
microalbuminuria. Adequate therapy of the
underlying disease will lead to the control
of micro-albuminuria, which slows down
the progression of kidney damage.
Pyelonephritis, glomerulonephritis
• In pyelonephritis, in addition to this finding
in the urine, it is possible to find granulated
cylinders, but it is necessary to perform a
blood test. With glomerulonephritis and
nephrotic syndrome, it is most important to
monitor the amount of protein in the urine
(24-hour proteinuria or albumin/creatinine
ratio) in order to monitor the effectiveness
of therapy.
 New markers of kidney diseases
• With the methods of two-dimensional and capillary electrophoresis,
mass spectrometry, HPLC, it is possible to determine in urine even
very small polypeptides and protein fragments that arise after the
process of exocytosis in the tubules or as a result of cell damage.
The identification and quantification of very small proteins that have
been known for a long time, but whose presence could not be simply
proven until now, offers the possibility of early diagnosis of kidney
damage. Nephrin, as a protein component of the glomerular
membrane, appears in urine as an early marker of diabetic
nephropathy. New technologies will enable faster, simpler and more
accurate diagnosis of kidney damage, which with early detection,
good therapy and adequate monitoring will extend the time until the
end stage of the disease appears .
 URINARY TRACT INFECTIONS
Characteristic clinical picture with dysuria, polakizuria and
suprapubic pain in cystitis. Significant bacteriuria (more than
100,000 / ml urine), an increase in Le number and the presence
of nitrite were observed in the urine. Due to the presence of a
mass of bacteria and leukocytes, the protein finding in the urine
may be positive, but does not exceed 1 g / 24h. A normal
laboratory finding is present in the blood, while in
Pyelonephritis there is an increase in the number of
leukocytes, accelerated sedimentation, an increase in CRP and a
possible slight increase in nitrogen products. At the same time,
the patient's clinical condition is much more severe, with fever,
groin pain, fever, nausea and vomiting, with or without dysuric
disorders. In acute pyelonephritis, in the sediment, in addition
to the mass of Le and bacteria, there are also leukocyte
cylinders, which are a common finding in chronic pyelonephritis.
For safe diagnosis and adequate treatment in both cases, it is
best to do a urine culture in order to identify the cause.
 New markers of UI
• In order to find newer markers, today procalcitonin
(PCT), interleukin 6 (IL-6) and interleukin 8 (IL-8) are
determined as early markers of acute pyelonephritis. The
TNFalpha/creatinine ratio is determined in the urine,
which significantly decreases after therapy. C-reactive
protein (CRP) as a marker of inflammation is not specific
and has no prognostic significance, except in conditions
of hospital recurrent infections. Macrophage migration
inhibitory factor (MIF) is a pro-inflammatory cytokine that
is determined in the urine and expressed as
MIF/creatinine ratio serves to differentiate acute
pyelonephritis from cystitis; determines persons with a
high risk of permanent renal damage in the future.
Calculosis of the kidneys and urinary tract can be diagnosed
by native graphy, ultrasound, intravenous pyelography and
examination of urine and blood. They are most often present
calcium, oxalate, urate and rarely cystine stones.

Determine the concentration of calcium, phosphorus and uric acid

in the serum,

Determine the total amount of calcium, uric acid and oxalate in

24-hour urine,

Determine urine pH and test for acidification of urine.

 Renal colic
• In the event of acute pain (renal colic) due to the
movement of stones, it is possible to identify:
• erythrocytes in the urine (micro- or sometimes
macro-hematuria),
• leukocytes (if infection is also associated)
• and the appearance individual crystals (which
would indicate the origin of the stone itself).