Kidney Function Test (1).pdf

By: Aklilu G.(M.Sc.)
June , 2020
Jimma University
Institute of Health ,Faculty of
Health Sciences ,School of Medical
Laboratory Science
Analytical clinical chemistry, systemic
approach II

Kidney function
Organ function

Objective of the lecture
 At the end of this lecture the students will be able to :-
 Explain about the urinary system and its components.
 Elucidate about anatomy of kidney and some other organs of the urinary
system.
 Discuss the physiology of the renal system.
 Describe non protein nitrogenous (NPN) compounds and how and why are
used as indicators of pathogenesis process in the renal system .
 Discuss about, NPN compounds mainly of Creatinine, urea, and uric acid:
source, metabolism, clinical significance(pathophysiology )
 Explain about different routine and advanced lab technics to diagnose kidney
impairment .
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Lecture outline
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 Introduction to urinary system
 Kidney function
 Overview of kidney anatomy
 Major components of the kidney
 Glomerular Filtration and its impact on the chemical profile
of vascular system
 Renal disease
 Renal function tests
 Routine tests
 Advanced tests

urinary system
The urinary system, also known as the renal system,
The Urinary System is constituted from group of organs in
the body concerned with filtering out excess fluid and other
substances from the bloodstream.
The urinary system consists of the two kidneys, two
ureters, a bladder, and urethra.
The two human kidneys are the main structural part of
urinary system, responsible for the formation of urine.
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urinary system
Kidney:Is the basic structural and functional unit of the
urinary system.
Each kidney consists of millions of functional units called
nephrons. ... Urine is formed in the kidneys through a
filtration of blood.
 Structure
 Bean shaped paired organs.
 Outer layer = cortex; composed primarily of glomeruli, PCT,
DCT
 Inner layer = medulla; composed primarily of the loop of
Henle and collecting ducts.
 Renal pelvis: collects urine into the ureters.
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urinary system
Ureters:
 The ureter is a tube that carries urine from the kidney to the urinary
bladder. There are two ureters, one attached to each kidney. The upper half
of the ureter is located in the abdomen and the lower half is located in the
pelvic area.
 The ureter is about 10 to 12 inches long in the average adult. The tube has
thick walls composed of a fibrous, a muscular, and a mucus coat, which are
able to contract.
Bladder:
 The bladder is a round, bag-like organ that stores urine. It is located in
the pelvic area, just below the kidneys and right behind the pelvic bone.
 While it is basically a fleshy storage tank, it is very complex in its design.
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urinary system
The bladder is connected to the kidneys by two long tubes
called ureters.When urine is produced by the kidneys, it travels
down the ureters to the bladder, where it is stored.The bladder
has four layers.
The other parts of the bladder are located at the bottom of the
sack.An opening at the bottom of the bladder is connected to
the urethra.A circular, muscular sphincter, pinches tight to keep
the opening and the urethra from leaking urine.This is the
stricture which makes urination temporary .
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urinary system
Urethra:
The urethra is the tube that carries urine from the bladder to
outside of the body.
In males, it has the additional function of ejaculating semen
when the man reaches orgasm.
When the penis is erect during sex, the flow of urine is blocked
from the urethra, allowing only semen to be ejaculated at
orgasm.
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Urinary system
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Kidney functions
 The kidney are the two structural units in the urinary system which have the following
function:-
 It filter the blood(passage of small molecules, water, ions through glomerulus).
 It reabsorbs essential substances from the filtrate to the blood (from tubules back into
bloodstream (capillaries)).
 It regulate the concentration of substances in the body(Hormonal regulation:
erythropoietin,ADH, aldosterone).
 It excrete the end product of metabolism and inorganic substances(removal from
body).
 It secretes different substances - in the process regulating the body state (from
bloodstream into tubules (urine)).
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Kidney function
 Every day the kidneys filter nearly 200 liters of fluid from the
bloodstream, allowing toxins, metabolic wastes, and excess ions to
leave the body in urine while returning needed substances to the
blood.
 Much like a water purification plant that keeps a city’s water
drinkable and disposes of its wastes.
 The kidneys are usually unappreciated until they malfunction and
body fluids become contaminated.
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Kidney function
Apart from our kidney there are also organs in our body
which participate in excretion process this include :-
Our lung (respiration)
Our skin (perspiration)
But our kidney plays the most important part in excretion
metabolic waste products from our body.
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Kidney function
 Excretion, regulation and secretion are not the only physiologic
function of our kidney it also act as essential:-
 Regulators of the volume and chemical makeup of the blood, maintaining the
proper balance between water and salts and between acid and base.
 It involves in gluconeogenesis process during prolonged fasting.
 It produces the hormone renin and erythropoietin.
 Metabolizing of vitamin D to its active form.
 But this is really tricky work for chemical engineer, but the kidneys do it
efficiently most of the time.
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Overview of kidney anatomy
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 A kidney is a structural unit in the urinary system which is
designed so that the following important parts of body structure
passes through it.
 Blood vessel
 Nerves
 Ureters
 Lymphatic drainage
About 25% of cardiac out put supplied to kidney through
renal arteries (branches of descending aorta)
Returns back by renal veins (branches of inferior vena cava).
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A large renal artery takes blood to each kidney.The artery
divides into many tiny blood vessels (capillaries) throughout
the kidney.
Tiny blood vessels (capillaries) in the kidneys, will divide
and form gromeruli which , filter the blood contained in the
capillaries.
Water and waste materials will be filtered through the walls
of the capillaries(glomerulus) into the nephrons to form
urine.
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Refreshment questions :
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What are the major components of the renal system ?
Explain their function.

Major component of the kidney
 Nephron is filtering unit of the kidney.
 The Nephrons is the Fundamental Urine-Producing Unit of the Kidney.
 We have a total of 2 million nephrons in the 2 kidneys.
 Components of the nephron:
1. Glomerulus - tuft of capillaries where filtration occurs .
2. Bowman's capsule- surrounds glomerulus, collects filtrate.
3. Proximal convoluted tubule.
4. Loop of Henle.
5. Distal convoluted tubule.
6. Collecting duct- adjusts volume & concentration of urine.
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 A.Arterioles
 The kidney is supplied with blood via the renal arteries that branch directly from the
aorta, and enter the kidney at the hilus.
 The interlobar artery is the first branch of the renal artery which gives rise to the
arcute arteries .
 The arcuate arteries (which arise from the interlobar arteries) run along the cortico-
medullary junction and can be seen on cross section in histologic renal sections.
 The arcuate arteries give rise to the interlobular arteries which then supply the
glomeruli via afferent arterioles.
 The small branches leaving the interlobular artery will form the afferent
arterioles.
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The efferent arterioles either
1) Carry blood to capillaries in the medulla (Vasa recta) or
2) Form an astomotic capillaries in the cortex (peritubular plexus).
Efferent arterioles of glomeruli in the outer cortex form the
peritubular plexus which surrounds proximal and distal tubules.
Efferent arterioles of glomeruli in the deeper cortex contribute to
the adjacent peritubular plexus and also form the vasa recta which
accompany the loop of Henle into the medulla.
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 Arteriolar sphincters
 There are "feedback" mechanisms which regulate glomerular filtration and
blood flow in each nephron in the kidney .
 Efferent and afferent arterioles constrict and dilate to control blood flow to
the glomerulus.
 The filtration process at the glomerular wall is regulated by auto regulation
of the GFR.
 Auto regulation processes at the glomerulus is a process in which
hydrostatic pressure of glomerulus has to be kept fairly constant.
 Auto regulation of GFR is achieved by auto regulation of renal blood flow
and a feedback mechanism known as "glomerular tubular balance".
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GlomerularTubular balance:
When there is a decrease in GFR, there is a resulting decrease in the fluid flow
rate within the tubule
At the loop of Henle, there is greater time for reabsorption of sodium and
chloride ions.
Therefore there is a decrease in the number of sodium and chloride ions reaching
the distal tubule which is detected by the macula densa.
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 B. Glomerulus
 Each glomerulus consists of a tuft of capillaries that protrudes into
the dilated, blind end of the nephron (the Bowman capsule) .
 The nephron is composed of epithelial cells, and these endothelial
cells of the glomerular capillaries are separated by a basement
membrane.
 visceral basement membrane is continuous with a parietal
basement membrane associated with the epithelial cells forming
the outer part of the Bowman capsule, which are continuous with
the epithelium of the proximal convoluted tubule.
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 C. Bowman’s Capsule
 The parietal layer of Bowman's capsule is usually lined by a layer of simple
squamous epithelium but high cuboidal epithelium similar to that found in
the proximal tubule may be seen in humans, monkeys, mice and rats.
 In the mature male mouse this cuboidal epithelium is commonly seen and its
prevalence is influenced by age and circulating levels of testosterone. Rats
also seem to show this type of Bowman's capsule, males generally with a
higher incidence or greater prominence than females.
 It is also more prevalent in spontaneously hypertensive strains than their
normotensive counterparts and although its prevalence increases with
advancing age it is not clear whether this predisposition is linked to
hypertension.
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The parietal epithelium may show hypertrophy and
hyperplasia in pathological conditions of the glomerulus,
notably in advanced glomerulosclerosis of rats.
In humans, tubular type epithelium has been associated with
malignant disease but it can also be found in normal subjects.
It has been postulated that parietal epithelial cells have an
important role in the development of focal segmental
glomerulosclerosis and matrix production in humans.
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D. Nephron
A nephron is the basic unit of structure in the kidney.A nephron
is used separate water, ions and small molecules from the blood,
filter out wastes and toxins, and return needed molecules to the
blood.
The nephron functions through ultrafiltration. Ultrafiltration
occurs when blood pressure forces water and other small
molecules through tiny gaps in capillary walls.
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 As the heart pumps the blood, the pressure created pushes small
molecules through the capillaries and into the glomerular capsule.
This is the, more physical function of the nephron.
 Next, the ultrafiltrate must travel through a winding series of
tubules.The cells in each part of the tube have different molecules
that they like to absorb.
 Molecules to be excreted remain in the tubule, while water,
glucose and other beneficial molecules work their way back into
the bloodstream.
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As the ultrafiltrate travels down the tubules, the cells become
more and more hypertonic compared to the ultrafiltrate.
This causes a maximum amount of water to be extracted
from the ultrafiltrate before it exits the nephron.
The blood surrounding the nephron returns to the body via
the interlobular vein, free of toxins and excess substances.
The ultrafiltrate is now urine, and moves via the collecting
duct to the bladder, where it will be stored.
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 E.Tubules
 1.Proximal ConvolutedTubule (PCT):
 The proximal convoluted tubule reabsorbs 65% of the filtered water, Na+,
Cl−, and K+.
 The epithelia of the proximal tubule have “leaky” tight junctions and can
maintain only a small trans-epithelial membrane potential.
 Most of the energy consumed by the proximal tubule is tied to Na+
reabsorption. On the apical surface, Na+ enters the cell by facilitated
diffusion and can be inhibited .
 The Na+/K+-ATPase on the basolateral surface prevents intracellular Na+
accumulation.
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 Glucose and amino acids are reabsorbed by Na+-coupled transport in
the proximal tubule . A family of transport proteins on the apical surface
of the epithelial cell uses the diffusion of Na+ down its electrochemical
gradient as the energy source.
 Transport of glucose across the basolateral surface also occurs by
facilitated diffusion
 HCO3
− is reabsorbed as major anion early in the proximal tubule
through a variety of mechanisms. The apical Na+/H+ antiport secretes
H+ into the lumen, where it combines with filtered HCO3
− to form
CO2.
 The CO2 can freely diffuse from the lumen into the cell, where it
dissociates back to H+ and HCO3
−.
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 The H+ is recycled and again secreted into the lumen. The HCO3
− is
transported out of the cell across the basolateral surface by an HCO3
−/Cl−
exchange.
 The H+ secretion causes the luminal pH to drop to 7.2 in the proximal tubule.
 The reabsorption of Na+ and HCO3
− causes a slight drop in the filtrate
osmolarity.
 The osmotic gradient between the filtrate and the renal interstitial fluid,
combined with the “leaky” tight junctions, allow water to be reabsorbed.
 This water reabsorption then causes an increase in the concentration of all the
other filtrate components. This concentration gradient provides a driving force
to allow reabsorption by diffusion.
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K+ reabsorption in the proximal tubule is primarily
paracellular, driven by a concentration gradient caused by
water reabsorption.
A small amount of K+ is actually secreted in late proximal
tubule, but a net 70% of the filtered K+ load is reabsorbed in
the proximal tubule.
Cl− is absorbed passively in later proximal tubule by both a
chemical gradient and a transluminal electrical gradient.
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 The proximal tubule normally reabsorbs 100% of filtered glucose,
amino acids, and small peptides. On the apical surface, this
movement is due to Na+-coupled co-transport.
 Consequently, amino acid and glucose reabsorption show
saturation kinetics .
 The transport maximum for glucose is only about three times
higher than the normal filtered load. If plasma glucose increases
enough to increase the filtered load above this level, some of the
filtered glucose will not be reabsorbed and will be excreted in the
urine.
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Diabetes mellitus results from either a deficiency in insulin
production (type I) or an impaired tissue response to insulin
(type II).
Both forms of the disease are characterized by persistently
high blood glucose levels.
When the glomerular filtered load of glucose exceeds the
reabsorptive capacity of the renal tubules, glucose remains in
the filtrate, where it acts as an osmotic particle causing
diuresis
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Refreshment questions:
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Which one of the above analyte from a figure on slide 44
can be used as kidney biomarker ?
Which one do you think the best ?Why ?

 2. The loop of Henle carries filtrate from the proximal tubule to
the renal medulla and back to the renal cortex.
 There are three functional divisions: the thin descending limb, thin
ascending limb, and thick ascending limb.
 The thin descending limb of the loop of Henle has leaky “tight”
junctions.
 This allows water to leave by passive diffusion as the tubule
segment enters the hypertonic renal medulla. In addition, urea
and Na+ diffuse from medullary interstitial fluid into the lumen of
the tubule.
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The thin ascending limb of the loop of Henle is distinguished
from the descending limb in that the tight junctions are now
“tight” and water impermeable.
As the name suggests, at the transition from descending to
ascending, the tubule segment makes a 180-degree turn and
the filtrate is now being carried back toward the cortex
The epithelia of the thick ascending limb of loop of Henle
also contain “tight” junctions.
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The solute transport without water movement results in a
drop in the filtrate osmolarity to 100 mOsm, so the
ascending limb of the loop of Henle is sometimes called the
diluting segment.
The solute transport into the interstitial space without water
movement also is one of two mechanisms causing
hypertonicity of renal medullary interstitial fluid.
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3.Distal and convoluted tubules
Potassium-Sparing DiureticsThe entry of Na+ through the
apical channel on the distal tubule principal cell creates the
electrical gradient that causes K+ secretion.
Any diuretic that increases the amount of Na+ delivered to the
distal tubule will cause K+ loss in the urine.
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The tight junctions of the cells lining the distal tubule are
“tight,” so water and electrolytes cannot diffuse across the
tubule and the filtrate remains hypotonic.
In the early portion of the distal tubule, an apical Na+/Cl−
transporter causes further reabsorption of ions.
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The principal (most common) cells of later distal tubule and
cortical collecting duct have a complex mechanism mediating
the aldosterone-sensitive secretion of K+.
The apical surface has an Na+ channel, allowing the
absorption of Na+.
The apical and basolateral cell membranes have identical K+
channels.
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As Na+ enters across the apical membrane, the transepithelial
potential becomes negative (up to —50 mV). This
transepithelial potential is the driving force for K+ secretion.
The magnitude of the transepithelial potential determines
whether potassium is secreted back into the lumen across the
apical surface or K+ moves across the basolateral surface.
The net effect of these transport processes is that as Na+ is
reabsorbed, K+ is secreted.
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 Distal tubule K+ delivery is low because of the K+ reabsorption in the
thick ascending limb of Henle, so active K+ secretion in the distal tubule
determines urinary K+ loss.
 Blockade of electrogenic Na+ reabsorption decreases transluminal
potential, so K+ secretion is impaired. This is the mechanism of action of
K+-sparing diuretics such as amiloride.
 Another type of cell found in the cortical collecting duct is the
intercalated cell. These carbonic anhydrase—rich cells secrete H+ and
decrease transluminal potential.
 The loss of the negative transluminal potential caused by H+ secretion
accounts for the decreased K+ secretion in acidosis .
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What are the important things for the kidney for the
transport process to happen ?
Do you think those things are important for kidney function?
Can they be used as kidney biomarker ?
Can they be used as kidney function test ?

Glomerular Filtration and its impact on
the chemical profile of vascular system
 Glomerular filtration is a physiological function of kidney
nephrons. The ultrafiltrate, which appears in the lumen of the
proximal convoluted tubule, is composed of water and solutes that
can pass through the filtering membrane of the capillaries.
 Under physiological conditions, the large molecular weight
proteins and blood cells do not pass through the capillary wall and
hence do not appear in the luminal fluid.
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 Therefore, glomerular filtration is relatively nonselective. The
benefits of the filtration process are that it disposes of excess fluid,
solutes, and metabolism byproducts and that it serves to detoxify
the system by disposing of chemicals identified as foreign.
 The balance of pressures in the glomerular capillaries causes
filtration of 20% of the plasma entering the kidney (filtration
fraction 20%).
 Normal renal plasma flow is 625 ml/minute, 20% of which yields
a normal GFR of 125 ml/min.
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 About 99% of the glomerular filtrate is reabsorbed in the tubules,
so normal urine production is 1 ml/minute.
 A high GFR makes it possible for the kidney to process (filter and
reabsorb) body fluids many times a day.
 In effect, the total body plasma can be processed about 60 times a
day, which permits tight control of the volume and composition of
body fluids.
 This estimate is based on a total plasma volume of about 3 l and a
GFR of about 180 l/day.
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Factors that influence(positive or negative ) the
filtration of solutes and large molecules:
(I) Size: Small electrolytes (e.g., sodium) and small organic
molecules (e.g., glucose) are freely filtered by the glomerular
capillary membrane, and they are assigned a filterability factor
of 1.0.
The filterability factor significantly decreases as the molecular
weight increases to a level comparable to, or exceeding, the
molecular weight of albumin.
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(II) Electric charge: Although the size of the molecule
determines, to a large extent, the filterability of the
molecule, electric charge makes a difference as well.
For example, the diameter of the plasma protein albumin is
somewhat smaller than the pores of the glomerular
membrane (6 vs. 8 nanometer).
Nonetheless, albumin is not filtered into the lumen mainly
because of the negative charge shared by albumin and the
basement membrane, resulting in electrostatic repulsion.
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This is clearly demonstrated by the significantly greater
relative filterability of positively charges , compared with
negatively, charged dextran for any given molecular radius.
 The relative filterability of neutral dextran falls between the
two values but is closer to the positively charged dextran.
In general, for a given molecular weight, negatively charged
molecules are less readily filtered than are positively charged
or even neutral molecules.
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 (III) Glomerular capillary hydrostatic pressure: Under
normal conditions, this pressure amounts to approximately 60
mm Hg. There is a direct relationship between this pressure and
GFR, with an increase in the glomerular capillary hydrostatic
pressure increasing GFR and vice versa.
 (IV) Physiologic Control of GFR:
1- Sympathetic neural activity: Under normal conditions, in a
resting healthy individual, the sympathetic nervous system exerts
little effect on the renal vascular beds. Renal sympathetic nerves
innervate mostly the preglomerular vessels and the afferent arteriole.
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An intense sympathetic discharge causes a reduction in renal
blood flow and fall in GFR. Nonetheless, if such a change is
short-lived, e.g., following moderate unloading of
baroreceptors, the effect of the increased sympathetic tone on
GFR is negligible.
2- Circulating hormones and autacoids: Vasoconstricting
hormones, e.g., adrenal medullary norepinephrine and
epinephrine, increase the vascular resistance of afferent and
efferent arterioles, resulting in a reduced GFR. Similar to the
sympathetic nervous system, these hormones have little effect
on GFR under normal physiological conditions
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3- The renin-angiotensin system: Angiotensin II plays a
significant role in the autoregulation of GF R.
The overall effect of angiotensin II, acting through the
activation of its AT1 receptor, depends on the predominance
of its modulation of vascular responses at the efferent and, to
a smaller degree, afferent arteriolar resistance vessels.
In physiological condition most of the systems above will be
kept constant and so the person condition
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 But if there is any pathological process in the renal system or
gromerulus will cause significant change in the chemical
content of the blood and so to the urine .
Most of the causative agents that result in pre – renal or renal
cases are associated with gromerular dysfunction.
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Tubular Reabsorption and its impact on the
chemical profile of the vascular system
 Reabsorption or tubular reabsorption is the process
by which the nephron removes water and solutes from the
tubular fluid (pre-urine) and returns them to the circulating
blood.
 It is called reabsorption both because these substances have
already been absorbed once (particularly in the intestines)
and because the body is reclaiming them from a
postglomerular fluid stream that is well on its way to
becoming urine (that is, they will soon be lost to the urine
unless they are reclaimed).
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Renal Threshold
 Defined as the plasma concentration of a substance that when
exceeded, the kidney tubules will not reabsorb any more into the
bloodstream, resulting in the substance being excreted into the
urine.
 Example: glucose ~160-180 mg
 Substances are reabsorbed into the bloodstream dependent upon
their blood concentration and the body’s needs.
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Renal Threshold
 When the plasma concentration of a substance is higher than
a certain ‘threshold value’, reabsorption of the substance is
no longer possible
The substance is then spilled into the urine
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Tubular Secretion and its impact on the
chemical profile of the vascular system
 Substances in the peritubular capillary blood are secreted
into the filtrate for excretion through urine.
 Elimination of waste products not filtered by the glomerulus
a. Medications bound to proteins (proteins remain in blood
stream)
b. Organic waste: urea, uric acid, creatinine
 Uromolulin (Tamm-Horsfall) protein is secreted (PCT)
 Regulation of acid-base balance: H+ , HCO3- secretion
dependent upon acid-base status of body
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Refreshment questions
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 why is secretion?
What are the physiologic functions of the kidney ?
Is there circumstances for this to be halted ?What are those
circumstances ?
As a laboratoriest how that can diagnosed ?

Why to test the renal function
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Many disease affects the renal function
In some, several functions are affected.
In others, there is selective impairment of glomerular function
or one or more of tubular functions.
Most types of renal diseases cause destruction of complete
nephron.

Renal disease
 Renal disease (kidney failure): also known as end-stage kidney
disease, is a medical condition in which the kidneys no longer
function. It is divided into acute kidney failure (cases that develop
rapidly) and chronic kidney failure(those that are long term).
 Signs and symptoms of renal failure Nausea, vomiting, edema,
pain, shock, Micturia, Urine volume change, urine composition
change and mainly change in the concentration of NPN substances
in the blood.
 Types of renal failure
 Acute renal failure
 Chronic renal failure
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Renal disease
 Acute renal failure: most commonly occurs in hospital setting
as a result of ischemic or nephrotoxic insults.
 Rapid loss of renal functions which is reversible. (CRF is irreversible
with slow loss of renal functions)
 It develops rapidly, and laboratory results show electrolyte, acid-
base, and fluid imbalances.
 Depending on where the damage has occurred, classified as pre
renal, renal, or post renal.
 When causes removed, recovery may occur with days and weeks.
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Renal disease
Chronic renal failure: progressive loss of functioning
nephrons.
The rate that CRF progresses depends on the number of
episods orARF.
Min causes include Diabettis, renal vascular disease,
glomerulrnephrites.
Currently diagnositic tools include in situ hybridization, PCR
techniques.
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What are the basic kidney function
tests
I, Routine kidney function test
 Evaluation of NPNS
 Evaluation of NPNS concentration
 Evaluation of NPNS clearance
 These are compounds that contain nitrogen, but are not proteins
 End products of metabolism
 The kidneys play an essential role in the excretion of these metabolic waste
products.
 Thus, the measurement of these compounds can be used to assess kidney
function
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 Most clinically significant NPN compounds are:
Amino acids: from protein catabolism (breakdown)
Ammonia: from amino acid catabolism
Urea: from ammonia catabolism
Creatinine: from creatine breakdown in the muscle
Uric acid: from nucleic acid catabolism
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 why we Evaluate of NPNS for measuring kidney ability ?
Freely filtered at glomerular barrier
Not reabsorbed by the tubules
Not secreted by tubules
present at stable plasma concentration
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Factors affecting the level of NPNS:
Size of the nephrone:
Pregnancy:
D.M.:
Kidney disease:
Diet
Vomiting ,
Dehydration and fever
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Evaluation of NPNS concentration
Types of NPNS concentration evaluation tests
1. Serum creatinine level
2. Serum urea level
3. Serum uric acid
4. Urinary urea and cratinine concentration
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1- Creatinine is the end product of creatine catabolism
98% of the body creatine is present in the muscles where it
functions as store of high energy in the form of creatine
phosphate.
About 1-2 % of total muscle creatine or creatine phosphate
pool is converted daily to creatinine through the
spontaneous, non enzymatic loss of water or phosphate.

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 Creatinine in plasma is filtered freely at the glomerulus and
secreated by renal tubule (10% of urinary creatinine ) .
 Creatinine is not reabsorbed by the renal tubules.
 Plasma creatinine is an endogenous substance not affected by diet.
 Plasma creatinine remains fairly constant throughout adult life.
Serum creatinine (55-120 mol/L in adult)

Refreshment questions
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What is the chemical nature of creatinine and urea ?
Which one is endogenous and why ?
Is it possible for this substances to be freely reabsorbed?
What type of filtration and re absorpation system it utilizes ?
Plasma creatinine remains fairly constant throughout adult
life ? If true , How?

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 2- Urea is formed in the liver from ammonia released from
deamination of amino acids.
 As a kidney function test, serum urea is inferior to serum
creatinine because:
High protein diet increases urea formation.
Any condition of increment in proteins catabolism (Cushing
syndrome, diabetes mellitus, starvation, thyrotoxicosis) will in turn
increase urea formation.
 50 % or more of urea filtered at the glomerulus is passively
 Reabsorbed by the renal tubules.
Serum urea ( 2.5-6.6 mmol/L) in adult:

3- Uric acid:
Causes of hyperuricemia:
1- increased production:
2-decreased elimination:
Methods of determination:
I-chemical method:
Phosphotungestic acid reduction technique:
PTA + uric acid alkali allantoin + tungistin blue (680 nm)
II-enzymatic methods:
1- one step uricase method (Reference method)
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Uric acid + O2 uricase alantoin + H2O2
2- Coupled enzymatic reaction:
a) Uric acid + O2 uricase alantoin + H2O2
H2O2 + ethanol catalse acetaldehyde
acetaldehyde + NAD aldehyde dehydrogenase NADH +
compound acetate measured at 340 nm
b) Uric acid + O2 uricase alantoin + H2O2
H2O2 + methanol formaldehyde.
formaldehyde + acetyl acetone 3-5 diacetyl 1-4dihydro-
lutidin (yellow color at 410 nm)
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Evaluation of clearance
clearance : volume of blood from which a sub. is
completely cleared by the kidney per unit time.
why we use clearance test for measuring kidney ability ?
Freely filtered at glomerular barrier
Not reabsorbed by the tubules
Not secreted by tubules
present at stable plasma concentration
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Factors affecting clearance:
Size of the nephron:
Pregnancy:
D.M.:
Kidney disease:
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Types of clearance tests
1-Creatinine clearance:
2-Urea clearance:
3-Estimated:
4-Calculated:
5- Inulin clearance:
6- Iohexol clearnace:
7-Radio labeled substance clearnce:
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 Creatinine clearance:
 The glomerular filtration rate (GFR) provides a useful index of the
number of functioning glomeruli.
 It gives an estimation of the degree of renal impairment by
disease.
 Creatinine clearance is usually about 110 ml/min in the 20-40
year old adults.
 It falls slowly but progressively to about 70 ml/min in individuals
over 8o years of age.
 In children, the GFR should be related to surface area, when this is
done, results are similar to those found in young adults.

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Accurate measurement of GRF by clearance tests requires
determination of the concentration in plasma and urine of a
substance that is:
 Freely filtered at glomeruli.
 Neither reabsorbed nor secreted by tubules.
 Its concentration in plasma needs to remains constant
throughout the period of urine collection.
 Better if the substance is present endogenously.
 Easily measured.
Creatinine meets most of these criteria.

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Clearance is the volume of plasma cleared from the substance
excreted in urine per minute.
It could be calculated from the following equation:
Clearance (ml/min) = U * V
P
U = Concentration of creatinine in urine mol/l
V =Volume of urine per min
P = Concentration of creatinine in serum mol/l

GFR estimation (Cockcroft-Gault Formula
for) Estimation of GFR
As indicated above, the creatinine clearance is measured by
using a 24-hour urine collection, but this does introduce the
potential for errors in terms of completion of the collection.
An alternative and convenient method is to employ various
formulae devised to calculate creatinine clearance using
parameters such as serum creatinine level, sex, age, and
weight of the subject.
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GFR estimation (Cockcroft-Gault
Formula for Estimation of GFR
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An example is the Cockcroft-Gault Formula:
K  (140 – age)  Body weigh
GFR= ──────────────────
Serum creatinine (mol/L)
 Where K is a constant that varies with sex:
1.23 for male & 1.04 for females.
 The constant K is used as females have a relatively lower
muscle mass.

Cockcroft-Gault Formula
for Estimation of GFR: Limitations
It should not be used if
Serum creatinine is changing rapidly
The diet is unusual, e.g., strict vegetarian
Low muscle mass, e.g., muscle wasting
Obesity
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 Serum Cr is a better KFT than creatinine clearance
because:
 Serum creatinine is more accurate.
 Serum creatinine level is constant throughout adult life
 Creatinine clearance is only recommended in the following
conditions:
 Patients with early ( minor ) renal disease.
 Assessment of possible kidney donors.
 Detection of renal toxicity of some nephrotoxic drugs.

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Normal adult reference values:
 Urinary excretion of creatinine is 0.5 - 2.0 g per 24 hours in a
normal adult, varying according to muscular weight.
Sérum creatinine : 55 – 120 mol/L
Creatinine clearance: 90 – 140 ml/min (Males)
80 – 125 ml/min (Females)

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A raised serum creatinine is
A good indicator of impaired renal function
But normal serum creatinine
Does not necessarily indicate normal renal function as
Serum creatinine may not be elevated until GFR has fallenn by
as much as 50%

Urea Clearance:
 If urine flow >2 ml/min: Maximum urea clearance:
 UxV/P
 If urine flow <2 ml/min: Standard urea clearance:
 U x √V /P
Result is given in ratio: Clearnce obtained
Max. or Std. clearnce
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From the above information what did you understand about
GFR, eGFR and CrCl
Which one do you think the best in signifying kidney
function

New Markers for Evaluating Glomerular
functions
II,Advanced markers
B2-Microglobulin:
Small non glycosylated peptide (M.W.: 11.800 daltons) found
on the surface of most nucleated cells.
Levels of B2-microglobulin remain stable in normal persons.
Elevated levels in serum indicate increased cellular turnover as
im myeloproliferative and lymphoproliferative disorders,
inflam., and renal failure.
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functions
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As a small, endogenous peptide, B2-M is easily filtered by
the glomerulus and about 99.9% is then reabsorbed by
the proximal tubules.
Measurement of B2-M is used clinically to assess renal
tubular function in renal transplant patients.
elevated levels of B2-M in renal transplant patients
indicate organ rejection.
It is more efficient marker of renal transplant rejection
than serum creatinine as it does not depend on lean
muscle mass.

functions
II- Cystatin C:
It is a low molecular-wt protein produced by
nucleated cells.
It is freely filtered by the glomeruli and catabolized by
the proximal tub.
It is produced at a constant rate and remain stable if
kidney function is normal.
It is useful in indicating early changes in kidney functions
but expensive and time consuming as it is measured by
immunoassay methods.
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functions
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Arrangement of markers of GFR:
1. Gold standard marker: Inulin continuous infusion urinary
clearance.
2. Silver standard marker: Inulin single bolus pl. clearnce –
Radioisotope labeled markers - Iohexol.
3. Bronze standard marker: Cystatin C – Creatinine.
4. Of uncertain clinical use: Cr. Cl., urea, RBPr,A1-MG
Note : you can comment on this and also see articles .

III, Evaluation of Renal Ability to Regulate Acid-base
Balance & Electrolyte Balance:
1- Specific Gravity:
 Def: Ratio of wt of certain volume of urine to the wt equal volume of water at a fixed
temp.
 It measures the ability of kidney to concentrate the glomerular filtrate.
 It varies directly with the grams of solutes excreted / lit.
 The physiological range is 1004-1035 but for 24 hs collected urine =
1015-1025
 Factors affecting: Solutes (pr & glucose) – urine vol.
 In renal tubular diseases, the concentrating ability of the kid. is one of the
first functions lost.
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Evaluation of Renal Ability to Regulate Acid-base
Methods of determination:
1- Hydrometer: urinometer is a hydrometer designed to
fit into and float in a narrow cylinder filled with urine
with a specific gravity scale arround it from 1000 to
1040
It should be calibrated by testing it with a sol. of known specific
gravity (1000 with water and 1030 with 75 ml zylene and 25 ml
bromozene)
How to adjust Sp.G. (temp – Pr – glucose)
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Evaluation of Renal Ability to Regulate Acid-
base Balance & Electrolyte Balance:
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2- Refractometer:
Principle: refraction of light in air differs than in solutes.
3-Dipsticks: the strip consists of a polyelectrolyte & an indicator
(bromothymol blue) that changes color as H+ is displaced by Na+
or K+ in the patient’s urine.
4- Osmolality:
Def.: M.W. of a sub. In grams dissolved in 1 Kg water.

Osmolarity: M.W. of a sub. In grams dissolved in 1 lit.
Osmolality = Ǿ nc
Ǿ = osmotic coefficient (% of particles dissolved)
n = number of atoms after dissolving
c = conc. In mol.
Calculated osmolality= 1.86 (Na) + glucose/18 +
BUN/2.8
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Osmolal gap (delta osmolality): difference between
measured & calculated osmolality.
Normally, measured osmolality > calculated osmolality
by up to 30 mosmol/Kg (the difference is due to
accumulation of osmotically active metabolites other
than Na, gluc. & urea).
Causes of increased osmolal gap:
Lactic acidosis – ketoacidosis – ethanol overdose – CRF –
methanol, isopropanol & ethylene glycol poisoning.

Measured osmolality:
Serum: 285-310 mosmol/kg
Urine: 300-900 mosmol/kg
Calculated osmolality: 275-290 mosmol/kg (only
serum)
One osmolal sol.: sol. Containing 1 mol of solute in 1 kg
water
One osmolar sol.: sol. Containing 1 mol of solute in 1
lit. water
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 When substances are dissolved in a solvent, 4 physicochemical
parameters are altered:
1- ↑ Osmotic pressure. 2- ↑ In boiling pont
3- ↓ In vapour pressure 4- ↓ In freezing point
Methods of determination of osmolality:
1- Measurement by Osmometer:
2- Calculation (serum only)
U/S osmol ratio: 1-3

3- Urine concentration test:
 For the kidney to concentrate urine we must have active
secretion ofADH and good response of the kidney toADH.
Causes of abnormal conc.Test:
4- Urine dilution test:
 Represent ability of kidney to dilute urine.
5- Fractional Na excretion:
Urine Na X Plasma creatinine
Plasma Na Urine creatinine
6- Renal failure index:
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Plasma creatinine
Urinary Na X
Urine creatinine
<1 = prerenal impairment
>2 = tubular impairment
7- Ability of kidney to acidify urine:
The kidney acidify urine by:
a- Reabsorbtion of HCO3
-
b- Excretion of H+ produced during tissue metabolism as:
NH4
+ - H+ exchange – with buffer anions as inorganic
phosphate
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T.A.=The amount of base needed to titrate a specimen
of urine to PH 7.4.
So, the net excretion of H+ by kidney=T.A. + NH4
+ - HCO3
-
Ammonium chloride test:
Patient is fasting overnight.
At 8 a.m., empty the bladder.

 At 10 a.m. give ammonim chloride by mouth (0.1
gm/kg) in gelatin capsule.
Urine is collected every hour (8 collections)
PH is determined in each urine collection.
 Blood sample before amm. Chloride and 2 hours after
are taken to determine HCO3
-
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 Normal response: (contraindicated in pt.s with liver
disease)
In response to amm. Chloride load, the PH of urine falls to
<5.3 in at least one sample but be sure that satisfactory
acidosis has been induced by amm.
Chloride (plasma total HCO3
- decrease by about 4
mmol/L) in 2 hours period after amm. Chloride.

SODIUM 135 to 145 mEq/L
POTASSIUM 3.5 to 5.5 mEq/L
CHLORIDES 100 to 110 mEq/L
BICARBONATE 24 to 26 mEq/L
CALCIUM 8.6 to 10 mg/dl
MAGNESIUM 1.6 to 2.4 mg/dl
PHOSPHORUS 3.0 to 5.0 mg/dl
URICACID 2.5 to 6.0 mg/dl
pH 7.4
CREATININE 0.8 to 1.4 mg/dl
Normal values of Internal Chemical Environment controlled by
the Kidneys:
15 to 20 mg/dl
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Refinement questions:
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What does complete metabolic panel mean ?
What does kidney metabolic panel mean?

Reference
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1. Bishop LM: 2010, Clinical ChemistryTECHNIQUES,PRINCIPLES, CORRELATIONS(6th Edition
),wolters kluwer health ,philadelphia , USA. 1-700
2. Carl A. burts, Edward R. Ashwood BGS. Tietz Fundementals of Clinical Chemistry. Six edition. USA: SAUNDERS
ELSEVIER; 2008. 1–970 p
3. Harrsan Internal medicine 16th edition
4. C. Guyton. Textbook of Medical Physiology. Eleventh edition
5. Kenneth s. Saladin. Anatomy & physiology: the unity of form and function. Sixth edition. McGraw-Hill
6. Arneson W,Brickell

Thank You !!
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