Renal function and tests of renal function 3.pptx

Renal function and
tests of renal function
Dr Akeem Oyeyemi Lawal
MBBS (Ilorin), MPH (Manchester), MWACP (LM), FMCPath (Chemical Pathology)
Department of Chemical Pathology
National Hospital Abuja
Lecturer I, Department of Chemical Pathology
College of Health Sciences
University of Abuja
Visiting Consultant Chemical Pathologist
Federal Medical Centre, Jabi

2
Pre-test 1…
Which of the following is NOT regarded as a function of the kidney?
A. Fluid balance
B. Synthesis of thrombopoitein
C. Excretion of acid
D. Urine formation
E. Clearance

3
Pre-test 2…
Which of the following is most accurate in assessing glomerular
function?
A. Creatinine
B. Cystatin C
C. Para-amino hippuric acid
D. Inulin
E. Iohexol

4
Pre-test 3…
Which of the following kidney functions can be inferred from plasma
Calcium assay?
A. Storage function
B. Endocrine function
C. Synthetic function
D. Excretory function
E. Regulatory function

5
Pre-test 4…
‘Electrolyte/urea/creatinine’ is one of the commonly requested
biochemical investigations. What category of kidney function tests can
it be classified?
A. Homeostatic function
B. Synthetic function
C. Excretory function
D. Homeostatic and synthetic function
E. Homeostatic and excretory function

6
Learning objectives
• To appreciate the functional anatomy of the kidneys
• To understand the physiological roles of the kidneys
• To understand the categories of tests of renal function including
tests of glomerular and tubular function
• To appreciate the application of renal function tests in clinical
practice

7
Outline
• Introduction
• Functional anatomy
• Physiology
• Tests of kidney function
• Clinical applications of renal function tests
• Conclusion
2012©
Encyclopaedia Britannica Inc.

8
Introduction…
OUTLINE
• Introduction
• Physiology
• Clinical applications of renal
function tests
• Conclusion

9
Introduction
• Renal system: kidneys, ureters, urinary bladder and urethra; accompanying
neurovasculature
• Latter play essentially storage or conduit roles hence renal function is largely
synonymous with kidney function
• Kidneys play a wide array of critical functions including excretory, homeostatic,
synthetic and endocrine functions
• Defects in renal function therefore life-threatening
• Kidney function tests rank most requested biochemical investigations in clinical practice

10
Functional anatomy…
OUTLINE
• Introduction
• Physiology
function tests
• Conclusion

11
Functional anatomy
Gross:
• Paired bean-shaped organs
located in the retroperitoneal
space
• Approx. 12cm long, extending
from lower border of T11 to
upper border of L3
• Weight: 150g each
• Superior relations: adrenal
glands
Adapted from https://courses.lumenlearning.com/suny-ap2/chapter/gross-anatomy-of-the-kidney/

12
The Anatomy of the Kidney | Interactive Biology, with Leslie Sa
muel (interactive-biology.com)

13
Functional anatomy:
Gross cont’d
• Blood supply: renal
arteries; branches of abd
aorta.
• Venous drainage - renal vv.
tributaries of IVC
• Nerve supply: Sympathetic
and parasympathetic
ganglia

14
Histology:
• 2 basic parts: cortex and medulla
• Others: renal pyramids, renal
papillae, minor calyxes, major
calyxes, renal pelvis
• Basic structural and functional
unit – nephron
• Nephron dose – no. of nephrons
an individual is born with; each
kidney approx. 600,000 – 1.2
million nephrons
• 5 basic parts: Glomerulus, PCT,
LOH, DCT, CD

16
NEPHRON:
Functional
unit of the
kidney

17
Glomerulus
• Composed of tuft of capillaries
(approx. 40 – 60 loops) enclosed in a
capsule (‘pouch’) known as
Bowman’s capsule lined by epithelial
cells (visceral and parietal)
• Capillary tufts + Bowman’s capsule =
Renal corpuscle
• Functions in filtration of plasma
• The glomerular filtration barrier is
made up of 3 parts:
• Capillary endothelium
• Glomerular basement membrane
• Visceral epithelium (podocytes)
Glomerulus (kidney) - Wikipedia

18
Glomerulus cont’d
• Capillary endothelium, unlike
others possesses pores (fenestrae)
abt 60nm diameter (size barrier)
• Covered by a ‘gel-like’ substance
(glycocalyx) which possesses
negatively charged
glycosaminoglycans providing
(charge barrier)
• GBM is made up of dense network
of collagen fibres
• Visceral epithelial cells are
referred to as ‘podocytes’ which
have primary and secondary foot
processes that form filtration slits
• Entire structure also covered by
gel matrix and provides a barrier
that is impermeable to substances
≥ 60kDa (Albumin) approx.
diameter 3.5nm

19
PCT
• Most metabolically active
part of the nephron
• Composed of channels and
transporters (active and
passive) which function
largely in reabsorption of
filtered substances
• Some of which are
symporters, others
anteporters
https://www.researchgate.net/figure

20
PCT cont’d
• Responsible for
reabsorption of:
• 60 - 70% of Na and
water
• 97 - 100% glucose,
99% amino acid
• others – HCO3-
, Ca,
Phos, Uric acid
reabsorption etc
Adapted from Comprehensive Nephrology 6th
edition

21
Loop of Henle…
• ‘Hair-pin’ structure
• 2 limbs: Thin descending and thick ascending
• Thin descending limb is permeable to water, possessing Aquaporin 1
channels
• Thick ascending limb is impermeable to both water and solutes but
possesses transport channels for active efflux of electrolytes (Na, K, Cl)
from the tubular lumen into the peritubular space and ultimately into
peritubular capillaries
• Functions in urine formation by Countercurrent multiplier and
exchanger mechanisms

22
LOH
• Countercurrent multiplier
system refers to the opposite
flow of tubular fluid in the
descending and ascending limbs
of the LOH
• It describes the process by which
the urinary filtrate from PCT
initially isotonic to plasma
subsequently becomes more
hypertonic as the urinary filtrate
progresses down the thin
descending limb of the LOH due
to increased permeability to
water but impermeability to
solutes
Adapted from https://courses.lumenlearning.com/suny-ap2/chapter/gross-anatomy-of-the-kidney/

23
LOH cont’d
• As the fluid moves up the thick
ascending limb, it becomes
progressively hypotonic because
the ascending limb is impermeable
to water; in addition, solutes (Na,
K and Cl-
) are actively pumped out
via the NKCC2, ROMK and CLC-Kb
channels inhibited by loop diuretics
• The active efflux of solutes into the
medulla creates an osmotic
gradient that facilitates continuous
movement of water from the thin
descending limb
• Medullary urea concentration is
also contributes in maintaining this
gradient
• Net effect – tubular fluid leaving
the LOH is hypotonic Adapted from Comprehensive Clinical Nephrology 6th
ed.

24
LOH
• Active transporters -
NKCC2, ROMK1, CLC-Kb for
Na, K, Cl
• Paracellular transport – Ca,
Mg, some Na→Claudin (aka
Paracellin) channels
Adapted from https://www.researchgate.net

25
LOH: Vasa recta
• Countercurrent exchanger
• Maintains solute and water
gradient between the two
limbs of the LOH and the
vasa recta
Adapted from Comprehensive Clinical Nephrology 6th
ed.

26
DCT
• Responsible for fine
adjustments in regulation
of fluid and electrolytes
• 5-10% of Na reabsorption
under the influence of the
Thiazide-sensitive NCCT
channel
• Reabsorption of Ca2+,
Phosphate and Magnesium
also occurs here
• Distal portion has
specialized macula densa
cells which are integral
components of the JGA Adapted from Comprehensive Nephrology 6th
edition

27
Collecting duct…
• Composed of 2 types of
cells: principal and
intercalated cells
• Principal cells: Site of
action of Anti-diuretic
hormone (ADH) and
Aldosterone – Na
reabsorption via
mineralocorticoid receptor
• Intercalated cells: H+
exchange and HCO3-
generation
Adapted from https://doctorlib.info/pharmacology/basic-clinical-pharmacology-13/15.html

28
Collecting duct cont’d:
Intercalcated cells
• Intercalated cells: alpha
and beta
• H+ secretion and
Bicarbonate generation

29
Collecting duct
• Action of Vasopressin
• Water reabsorption via
translocation and insertion
of Aquaporin 2 channels

30
Physiology…
OUTLINE
• Introduction
• Physiology
function tests
• Conclusion

31
Functions of the kidney…
1. Homeostatic function: Fluid and electrolyte
balance; Acid-base balance
2. Urine formation
3. Excretory function
4. Endocrine function
5. Synthetic function

32
Homeostatic function…
A. Fluid and electrolyte balance:
• Accomplished via the renin-angiotensin-aldosterone system
• Involves the JGA composed of:
• Juxtaglomerular (granular) cells
• Macula densa
• Extra-glomerular mesangial cells of Lacis
• Baroreceptors in the JGA, carotid sinus and aortic arch,
chemoreceptors in the macula densa and osmoreceptors in the
hypothalamus

33
Fluid and electrolyte balance: Role of JGA
Glomerulus (kidney) - Wikipedia

34
Fluid and electrolyte balance cont’d:
Renin – zinc metalloprotease produced by granular cells of JGA
Stimuli for release: decreased renal perfusion pressure, hyponatraemia, hyperkalaemia,
↓ABP, hypovolaemia
synthesized from its precursor
Pre-prorenin →Prorenin (stored in secretory granules) → Renin
Angiotensinogen → Angiotensin I → Angiotensin II
Renin ACE

35
Fluid and electrolyte balance cont’d:
• Angiotensin II:
• Potent vasoconstrictor
• Stimulates thirst centre
• Stimulates ADH release
• Activates enzyme Aldosterone synthase in the adrenal cortex to synthesize
aldosterone
• ADH – produced in the Supra-optic and paraventricular nuclei (more
in the supraoptic nucleus); stimulus for release in ↑plasma
osmolality >290mOsm/kg
• Aldosterone – produced in the zona glomerulosa of the adrenal
cortex; stimulates Na reabsorption by activating ENaC channel at CD

36
Homeostatic function…
B. Acid-base balance:
• The kidneys play vital roles in acid-base balance via the ability to
secrete H+
into the urinary filtrate and reclaim, as well as generate
bicarbonate (HCO3
-
) in the PCT and DCT respectively
• It’s large concentration of carbonic anhydrase facilitates these
processes making it the major organ in long-term acid-base control
• The presence of important urinary buffers i.e. ammonium/ammonia
buffer system (NH4
+
/NH3) and phosphate buffer system
(H2PO4
-
/HPO4
2-
) also contribute to its role in acid-base balance

37
Bicarbonate reclamation in the PCT

38
Bicarbonate generation in the DCT

39
Role of
Ammonium/Ammonia
buffer system
• In pathological states, the liver
diverts ammonia from Urea
synthesis into generation of
glutamine which is utilized by the
kidneys to generate ammonium
ion
• The Glutamine is hydrolysed in
the kidneys by glutaminase to
form glutamate and subsequently
2-oxoglutarate and ammonium
ion which then dissociates into
NH3 and H+
acting as a urinary
buffer
Adapted from Clinical Chemistry and Metabolic Medicine by M.
Crook

40
Urine formation
• The kidneys are involved in urine formation via the Countercurrent
multiplier and exchanger systems
• Also requires the influence of ADH and Aldosterone

41
Excretory function
• Excretion of metabolic wastes and nitrogenous compounds e.g. Urea,
Creatinine, uric acid, water soluble drugs etc

42
Endocrine function
• Renin
• 1,25-(OH)2vitamin D - 1α hydroxylase enzyme
• Erythropoietin
• Thrombopoietin
• Prostaglandins

43
Synthetic function
• Gluconeogenesis
• Bicarbonate generation

44
Tests of kidney function…
OUTLINE
• Introduction
• Physiology
function tests
• Conclusion

45
Assessment of glomerular function
• Glomerular filtration rate (GFR)
• Concept of clearance
• Markers for clearance studies
• Estimated GFR (eGFR)

46
Glomerular filtration rate
• The kidneys receive 25% of cardiac output
• About 125mL of blood is filtered across the glomerulus per minute
(125mL/min ≡ 180L/day)
• Filtration is dependent on a balance of forces
• Also affected by:
• Renal blood flow
• Renal perfusion pressure

47
Glomerular filtration rate cont’d
• GFR is considered the most reliable measure of functional capacity of
the kidneys
• Reflection of number of functioning nephrons and most sensitive and
specific marker of overall changes in renal function
• Implicated in major classification systems of Acute kidney injury (AKI)
and Chronic kidney disease (CKD) ie. RIFLE/AKIN criteria and CKD-
EPI/KDIGO classification respectively

48
Concept of clearance
• Definition: volume of plasma from which a substance is completely
cleared (removed) per unit time
• Is an index for measurement of GFR
• Requirements for an ideal marker for clearance:
• Stable concentration in plasma – no extra-renal elimination
• Physiologically inert – negligible plasma protein binding
• Freely filtered
• Not reabsorbed
• Not secreted
• Not synthesized
• Not metabolized

49
Formula for clearance
• If criteria fulfilled, for clearance of a substance ‘S’
• GFR x Ps = Us x V (i.e. amount of substance ‘S’ in plasma = amount in
urine)
• Therefore, GFR =
• Where:
• Us – Urine concentration of substance “S”
• Ps – Plasma concentration of substance “S”
• Vt – timed urine volume

50
Classification of markers…
A. Based on source:
• Exogenous
• Non-radioisotopic e.g. Inulin, Iohexol
• Radioisotopic e.g. I-iothalamate, Cr-EDTA, Tc-DTPA
• Endogenous e.g. Creatinine, cystatin C, Urea
B. Based on accuracy of GFR measurement:
• Gold standard – continuous Inulin infusion
• Silver standard – bolus inulin infusion
• Bronze standard – Plasma Creatinine, cystatin C, etc

51
Markers for clearance studies
Marker Chemistry Advantages Disadvantages
1. Exogenous
Continuous inulin infusion
(aka sinistrin)
Polymer of fructose Gold std; closest known ideal
marker
Exogenous, prone to
anaphylactic reactions;
time-consuming; laborious;
requires urethral
catheterization; prone to
errors due to incomplete
bladder emptying; complex
laboratory analysis
Inulin (bolus) Polymer of fructose Silver std; acceptable
agreement with gold std
As above; also some
extrarenal clearance
I-Iothalamate continuous
infusion
Labeled with Iodine
radioisotope
Silver std; simple
measurement with
radioactive detector;
acceptable agreement with
gold std
Exogenous; risk of ionizing
radiation; time consuming,
laborious procedure;
anaphylactic reactions;
positive bias with gold std
probably due to secretion
I-Iothalamate (bolus) Labeled with Iodine
radioisotope
As above As above

52
Markers for clearance studies cont’d
51
Cr-EDTA
continuous infusion
Labeled with
Chromium, 51
radioisotope
Simple measurement;
acceptable agreement with
gold std
Ionizing radiation; time
consuming complex procedure;
probable tubular reabsorption;
less readily available than 99m
Tc
51
Cr-EDTA (bolus) As above As above As above; extra-renal clearance
Iohexol continuous
infusion
Non-isotopic contrast
medium
Widely available;
inexpensive; acceptable
Laborious procedure; complex
measurement requiring ID-MS
or HPLC; anaphylaxis
Iohexol (bolus) As above As above As above; extrarenal clearance
99m
Tc-DTPA
continuous infusion
Labeled with
Technitium radioisotope
Widely available; acceptable
Protein-binding leading to
underestimation of GFR;
complex procedure; variable
dissociation of 99mTc from
DTPA resulting in imprecision
and bias
99m
Tc-DTPA (bolus) As above Acceptable agreement with
silver stds
Poor agreement with gold std

53
Markers for clearance studies cont’d
Cystatin C Bronze std; endogenous
proteinase inhibitor
Not secreted or reabsorbed; less
influenced by physiological
variation than creatinine;
unaffected by recent meat intake;
internationally standardized assays;
applied in GFR estimating
equations
More expensive than creatinine;
influenced by obesity and thyroid
function; possible influences by
genetic factors
Creatinine
(serum/plasma)
Bronze std; endogenous
nitrogenous substance
synthesize from muscle
protein
Inexpensive; internationally
standardized assays; applied in GFR
estimating equations
Physiological/racial/pathological
variations in GFR-Creatinine
relationship; affected by diet and
meat intake; variable tubular
secretion; variable intestinal losses;
analytical interferences
Creatinine
clearance
As above Inexpensive As above; also requires a timed
urine collection; challenges with
adequacy of urine volume;
cumbersome
Urea Nitrogenous waste product
from ammonia synthesized
in the liver
Endogenous; inexpensive Affected by hydration status, diet;
tubular reabsorption; poor
sensitivity and specificity
Others of uncertain clinical use: β-trace protein, retinol-binding protein, α1-macroglobulin, β2-macroglobulin, Symmetric
dimethyl arginine (SDMA)

54
Creatinine
• Anhydride product of muscle protein creatine
• MW 113Da
• Endogenously produced at a fairly constant rate (1 – 2%
per day)
• Freely filtered at the glomerulus, not reabsorbed
• Cheap, convenient, easy to measure
• Incorporated into many eGFR formulae

55
Creatinine
• However affected by age, gender, race, muscle mass, nutritional
status, other preanalytical and analytical factors
• Secreted by renal tubules
• Some extrarenal clearance in GIT
• Wide intraindividual variation

56
Cystatin C
• Low MW (12.8kDa) protein synthesized by all nucleated cells
• Functions as a cysteine protease inhibitor
• Endogenously produced at a fairly constant rate
• Freely filtered at the glomerulus, completely reabsorbed and
catabolized by renal tubules hence can also be used as a marker of
tubular function
• Not affected by muscle mass
• However affected by age, gender, weight, height, obesity, thyroid
hormone concentration

57
Urea
• Nitrogenous waste product of protein catabolism
• Synthesized exclusively in the liver
• CO(NH2)2
• Freely filtered at the glomerulus
• Significant tubular “back-diffusion” (passive reabsorption) about 40 –
70% from the tubules into renal interstitium and ultimately into
plasma
• Back-diffusion is reduced in high-flow states e.g. pregnancy due to
increased GFR; reduced in low flow states e.g pre-renal AKI

58
Urea
• Back-diffusion limits its use as a marker of GFR→ underestimation
• Also significantly affected by protein diet, hydration status, increased
protein catabolism, GI haemorrhage which leads to reabsorption of
blood proteins and treatment with cortisol (or its synthetic
analogues)
• Overall has poor sensitivity and specificity as a marker for GFR and
thus no longer used

59
Urea
• May however find utility in the urea-creatinine ratio to crudely
differentiate pre-renal from intrinsic AKI
• Normal urea-creatinine ratio in a normal individual with normal
dietary intake = 49 – 81 mmol Urea/mmol Creatinine (12 – 20
mg/mg)
• Ratio >81 mmol/mmol said to be suggestive of pre-renal AKI
• Also useful in assessing adequacy of dialysis using the urea reduction
rate ((pre dialysis urea – post dialysis urea)/pre dialysis urea x 100%)

60
Tests of tubular function
• Involves evaluation of proximal tubular function (i.e. tubular handling
of sodium, glucose, phosphate, calcium, bicarbonate and amino acids)
and distal tubular function (urinary acidification and concentration)
• Increased levels in urine indicate tubular dysfunction
• Examples:
• Urinary β2-macroglobulin
• Urinary N-acetyl-β-D-glucosaminidase
• Urinary Cystatin C

61
Estimated glomerular filtration rate (eGFR)
• Developed due to limitations of collection of timed urine specimen for
clearance studies
• Estimate of GFR using mathematical formulae derived from
comparison with clearance studies

62
Estimated glomerular filtration rate (eGFR)
• Cockcroft-Gault equation
• Modification of diet in renal disease (MDRD)
• CKD-EPI Creatinine (2009)
• CKD-EPI Cystatin C (2012)
• CKD-EPI Creatinine-Cystatin C (2012)
• CKD-EPI Creatinine (2021)
• Schwartz formula (children)

63
Cockcroft-Gault equation
• Oldest equation
• Estimates GFR using weight, age, serum creatinine
• Cohort used did not include women
• Requirement of weight made utility cumbersome
• Was derived using creatinine clearance rather than a reference
method for creatinine
• Did not account for body surface area (BSA)

64
Cockcroft-Gault equation
• Formula: males…
[(140 - age) x weight x 1.23]/serum creatinine (in µmol/L)
OR [(140 - age) x weight/72 x serum creatinine (in mg/dL)]
• Multiply by a factor of 0.85 in females

65
MDRD
• Modification of diet in renal disease
• Does not require weight
• Also adjusts eGFR with BSA
• Original formula used age, race, gender, serum creatinine, urea and
albumin values
• Modified formula eliminates urea and albumin
• However underestimated eGFR when ≥ 60mL/min/1.73m2

66
CKD-EPI Creatinine
• Developed in 2009 by the Chronic Kidney Disease Epidemiology
Collaboration (CKD-EPI)
• Does not require weight
• Developed against standardized reference assay for creatinine
• Better estimate of GFR than MDRD
• Requires use of age, race and serum creatinine
• Modified formula in 2021 removed race

67
CKD-EPI cystatin C/CKD-EPI Creatinine-cystatin C
• Developed in 2012 by CKD-EPI
• Incorporates cystatin C to overcome or obviate limitations of
creatinine

68
Modified Schwartz formula
• Utilized in individuals ≤ 18 years
• Requires height and serum creatinine

69
Novel biomarkers of kidney disease
• Newer markers to detect early kidney damage
• Neutrophil gelatinase associated lipocalin (NGAL)
• Kidney injury molecule 1 (KIM-1)
• Interleukin 18 (IL-18)
• Liver fatty acid binding protein (L-FABP)

70
Clinical application of renal
function tests…
OUTLINE
• Introduction
• Physiology
function tests
• Conclusion

71
Interprete this result. What renal function is being evaluated?

72
Calculate the eGFR using Cockcroft-Gault and
CKD-EPI equations. What stage of CKD is this?
• 55 year old Nigerian male diagnosed to have diabetes mellitus 5 years
prior to admission. Weight 72kg; serum urea = 18.1mmol/L (2.5 – 6.4)
creatinine = 502µmol/L (50 - 106).
• For CKD-EPI Creatinine, demonstrate with on-line calculator
• https://www.mdcalc.com/calc/3939/ckd-epi-equations-glomerular-filt
ration-rate-gfr

73
Review of learning objectives…
• To appreciate the functional anatomy of the kidneys
• To understand the physiological roles of the kidneys
• To understand the categories of tests of renal function including tests
of glomerular and tubular function
• To appreciate the application of renal function tests in clinical practice

74
Conclusion…
OUTLINE
• Introduction
• Physiology
function tests
• Conclusion

75
Conclusion…
• The kidneys are vital organs which play a myriad of clinical functions
• Defects in normal function of the kidneys have debilitating effects
• Kidney disease is quite common, being a complication of many prevalent non-communicable and
communicable diseases i.e diabetes mellitus, hypertension, sepsis/infections, drugs and toxins etc
• Laboratory investigation of kidney function requires comprehensive understanding of renal physiology
• Assessment of glomerular function is an invaluable index of overall kidney function through GFR and
clearance studies
• Understanding categories of tests of kidney function will guide clinicians in prompt and appropriate
management of kidney disease
KEY
MESSAGES

76
Pre-test 1…
Which of the following is NOT regarded as a function of the kidney?
A. Fluid balance
B. Synthesis of thrombopoitein
C. Excretion of acid
D. Urine formation
E. Clearance

77
Pre-test 2…
Which of the following is most accurate in assessing glomerular
function?
A. Creatinine
B. Cystatin C
C. Para-amino hippuric acid
D. Inulin
E. Iohexol

78
Pre-test 3…
Which of the following kidney functions can be inferred from plasma
Calcium assay?
A. Storage function
B. Endocrine function
C. Synthetic function
D. Excretory function
E. Regulatory function

79
Pre-test 4…
‘Electrolyte/urea/creatinine’ is one of the commonly requested
biochemical investigations. What category of kidney function tests can
it be classified?
A. Homeostatic function
B. Synthetic function
C. Excretory function
D. Homeostatic and synthetic function
E. Homeostatic and excretory function

80
References
• Tietz textbook of Clinical Chemistry and Molecular diagnostics
• Comprehensive clinical nephrology
• Wikipedia
• Encyclopaedia Britannica
• www.researchgate.net

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