Supplementary Information to Chapter 27

KIDNEY FUNCTION TESTS

MARKERS FOR RENAL ASSESSMENT (OLD AND NEW)

Chronic kidney disease (CKD) is common, harmful, and treatable.

Outcomes of CKD
¾¾ ­progression of renal disease to end­stage renal disease (ESDR)
¾¾ ­complications of CKD: Hypertension, anemia, bone and mineral disease
¾¾ ­increased risk of cardiovascular disease (CVD).
CKD Definition - ­KDIGO Guideline
KDIGO = Kidney Disease: Improving Global Outcome
Definition of CKD: GFR < 60 mL/min/1.73 m2 for ≥ 3 months, and/or presence of kidney damage, e.g. albuminuria
> 30 mg/g Creatinine
 88 Textbook of Biochemistry— Case Studies

Five Stages of Kidney Diseases

Stage Description GFR (mL/min/1.73 m2)
1 Kidney Damage with Normal or ↑ GFR ≥ 90
2 Kidney Damage with Mild ↓ GFR 60-89
3 Moderate ↓ GFR 35-59
4 Severe ↓ GFR 15-29
5 Renal Failure < 15(or Dialysis)

Kidney (Renal) Function Tests

1. Maintenance of homeostasis
2. Excretion of metabolic waste products
3. Retention of substances vital to the body
4. Hormonal function:
A. Erythropoietin stimulate Hb synthesis and formation of erythrocytes.
B. Calcitriol (1,25­DHCC) is finally produced in the kidney. It regulates calcium absorption and metabolisum.
C. Renin, proteolytic enzyme liberated by the kidney stimulates the formation of angiotensin II, which leads to
aldosterone production both of which are responsible for electrolyte balance.

Kidney (Renal) Function Tests

Formation of Urine
¾¾ Functional unit of Kidney—­Nephron
¾¾ Approximately one million nephrons in each kidney
¾¾ 1200 mL Blood passes through the kidney in one minute
¾¾ 120 – 125 mL is filtered/minute (GFR)
¾¾ Glomerular filtrate formed in adult 175­180 liters/day
¾¾ About 99% of Glomerular filtrate is reabsorbed by the kidney.
Two steps involved in urine formation
Glomerular filtration and Tubular reabsorption.

Kidney (Renal) Function Tests

Tests to Assess Renal Function
1. Glomerular Function Tests: All clearance tests.
2. Tubular Function Tests: Urine concentration or dilution, acidification tests.
3. Analysis of blood/serum: Estimation of urea, creatinine, protein and electrolytes.
4. Urine examination: Urine pH, volume, specific gravity, osmolality and abnormal constituents.

Clearance Tests
The clearance tests measuring the glomerular filtration rate (GFR) are the most useful in assessing the renal function
1. Urea clearance (≈ 75 mL/min) urea partially reabsorbed and hence less than GFR.
2. Creatinine clearance (≈ 145 mL/min) slightly higher than GFR as it is secreted by the tubules.
 Supplementary Information to Chapter 27 89

3. Inulin clearance (125 mL/min= GFR ) neither secreated nor reabsorbed.

4. Diodrast (diiodopyridone acetic acid)/PAH (para amino-hippuric acid) clearance: It is filtered by the glomerulus
and mostly excreated by the tubules. 700 mL/min represent renal plasma flow.

Global Scenario
↓ INFECTIOUS DISEASES
↑ CHRONIC DISEASES
CHRONIC KIDNEY DISEASE AFFECTS AROUND 10% WORLD POPULATION. HIGH PREVALENCE OVER
THE AGE OF 70 YEARS—16% COMPARED TO 11% IN GENERAL POPULATION CKD IS HIGHER IN PEOPLE
OF LOW SOCIOECONOMIC STATUS AND WITH FAMILY HISTORY OF CKD.

Chronic Kidney Disease (CKD)

CKD is a silent killer, incidence ↑ world wide.
CKD—progressive loss of renal function

Major Risk Factors

Diabetes and hypertension.
Glomerular nephritis, polycystic kidney disease, urinary tract infection, autoimmune diseases
kidney stone and toxic effects of some drugs

Markers for CKD

¾¾ SERUM CREATININE
¾¾ GFR
¾¾ MICROALBUMIN
¾¾ CYSTATIN C

Renal Function Tests

¾¾ Blood urea: 20 – 40 mg/dL
¾¾ Serum creatinine: M: 0.7 – 1.4 mg/dL
F: 0.6 – 1.3 mg/dL
¾¾ Serum uric acid: M: 3.5 – 7 .0 mg/dL
F: 3.0 – 6.0 mg/dL
¾¾ Serum potassium: 3.5 – 5.mEq/L
¾¾ Urine R/E, microalbuminuria
Clearance tests
¾¾ Urea clearance: Maximum: 75mL/min
¾¾ Standard: 54 mL/min
¾¾ Creatinine clearance: M: 85 – 125mL/min
F: 80 – 118 mL/min
¾¾ Inulin clearance: 125ml/min (GFR)
 90 Textbook of Biochemistry— Case Studies

Creatinine is the most widely used biomarker of kidney function. Generally, considered as reflecting GFR correctly.
Color (Jaffe’s) reaction is not very specific.
It is inaccurate due to various reasons.
Creatinine production is dependent on the muscle mass. It is not only filtered by the glomeruli, but also secreted by the
renal tubules. This tubular secretion contributes approximately 20% of the total creatinine excretion by the kidney, and it
can increase as GFR decreases.

Enzymatic Method for Creatinine

Creatininase
Creatinine   Creatine
Creatinase
Creatine Sarcosine
Sarcosine oxidase
Sarcosine Glycine + H2O2
Peroxidase
H2O2 Nascent oxygen

Chromogen

Quinone pigment

¾¾ Normal serum creatinine may be observed in individuals with significantly impaired GFR.
¾¾ Creatinine levels increase until puberty and differ according to gender, making their interpretation difficult in pediatric
patients.
¾¾ Serum creatinine does not increase until the GFR has moderately decreased (about 40 mL/min/1.73 m2).
¾¾ This insensitivity to small to moderate decreases in GFR in the so called creatinine blind GFR area (40 – 70
mL/min/1.73 m2).

Procedure for Creatinine Clearance Test

¾¾ Give 500 mL of water to the patient, to promote good urine flow.
¾¾ 30 minutes, empty the bladder and discard the urine.
¾¾ Exactly after 60 minutes, again void the bladder and collect the urine, and note the volume.
¾¾ Take one blood sample.
¾¾ Creatinine level in blood and urine are tested and calculated.
¾¾ Uncorrected clearance = ( U/P ) × V
¾¾ Creatinine clearance corrected for surface area could be calculated as
U × V × 1.73
P×A

Normal Reference Values

Serum creatinine GFR
Adult M 0.7-1.4 mg/dL 95-115 mL/min
Adult F 0.6-1.3 mg/dL 85-110 mL/min
Children 0.5-1.2 mg/dL
 Supplementary Information to Chapter 27 91

Factors reducing serum creatinine Factors increasing serum creatinine

Low muscle mass Old age
Malnutrition Renal diseases
Medicines  Glomerulonephritis
 Thiazide  Pyelonephritis
 Vancomycin   Renal failure
  Urinary obstruction
Congestive cardiac failure
Medicines
  Amphoteracin B
 Captopril
 Cephalosporins
 Kanamycin

Estimated GFR (eGFR)

A simpler technique of estimating creatinine clearance and thereby GFR is by using serum creatinine level. This would
eliminate the need for timed urine collections.

Cockcroft-Gault equation
Ccr = (140 – age in years) × weight in Kg (0.85 in females)/72 × Pcr in mg/dL
The factor 0.85 is used in females assuming that they have 15% less muscle mass.

Other Proposed Formulas for eGFR

Hoek formula (Hoek FJ, Nephrol Dial Transplant 2003)
eGFR = -4.32 + 80.35 × 1/CysC
body-surface adjusted
Larsson formula (Larsson A, Scand J Clin Lab Invest 2004)
eGFR = 77.24 × CysC-1.2623
body-­surface not adjusted
Arnal formula (Beauvieux MC, Diabetes Care 2007)
eGFR = 74.83 × CysC-1.333
body­-surface adjusted
Stevens formula (Stevens LA, Am J Kidney Dis 2008)
eGFR = 76.7 × CysC-1.19
= 127.7 × CysC­1.17 × age-0.13 × (0.91 if female)
body-­surface adjusted

Disadvantages of creatinine as a marker for GFR

1. About 10% is tubular component.
2. When the GFR is reduced, the secretion component is increased, and will vitiate the results. The creatinine clearance
will overestimate GFR by about 10-20 mL/min.
3. Early kidney failure is not detected:
creatinine blind GFR area
(40–70 mL/min/1.73 m2)
 92 Textbook of Biochemistry— Case Studies

So, serum creatinine is not be a good parameter for assessing of GFR, especially at lower rates.
In 80% cases, a delay exists between the decline in GFR and that in serum creatinine.
Creatinine is generally assessed by non-specific color reaction.
Moreover, creatinine is not a good marker especially in the creatinine blind GFR area.
Hence, we have to search for better and more sensitive markers.

Cystatin C
First described as ‘Gamma-trace’ by Butler and Flynn in 1961.
Grubb and Löfberg first reported its amino acid sequence in 1982.
First proposed as a measure of glomerular filtration rate by Grubb et al in 1985.

¾¾ High concentrations in biological fluids: Breast milk, tears, saliva.

¾¾ Expressed in virtually all organs of the body.
¾¾ The most abundant extracellular cysteine protease inhibitors.
¾¾ Inhibits lysosomal cathepsines, metalloproteinases, proteases of parasites and microbes.
¾¾ Produced at a constant rate by all nucleated cells.
¾¾ No diurnal variation.
¾¾ Levels are not altered by inflammatory conditions.
¾¾ Not influenced by diet and muscle mass.
¾¾ Free renal filtration.
¾¾ No tubular secretion.
¾¾ No re­entry into circulation.
¾¾ Tubular re­absorption follows degradation.
Serum levels of cystatin C are a more precise test of kidney function (GFR) than serum creatinine levels.
Multiple studies have found that Cystatin C to be more sensitive to actual changes in GFR in the early stages of CKD
than creatinine based GFR estimates.

Advantages of Cystatin C (over Creatinine)

Unaffected by non­renal factors:
• Muscle mass
• Weight/height
• Gender
• Less inter­individual variation
Sensitive to changes in the so­called creatinine blind GFR (40–70 mL/min/1.73m2).
So, Cystatin C enables early detection and treatment of CKD.
 Supplementary Information to Chapter 27 93

Cystatin C does not have a blind area and will therefore show a true positive reduction of GFR.

Cystatin C – sensitive in creatinine blind range

General Population—Mortality risk

PREVEND study:
¾¾ About 8592 individuals from the PREVEND study, aged 28–75
years (community-based longitudinal study).
¾¾ Three years median follow-up for death and cardiovascular
events.
¾¾ Cystatin C predicts mortality while creatinine does not.
¾¾ Adjustment of CysC for age, sex, weight, smoking, CRP and
creatinine did not change the relationship.
 94 Textbook of Biochemistry— Case Studies

Cystatin C – Prognostic Marker

What is Microalbuminuria?
Repeated appearance of low but above normal levels of albumin in urine. It indicates progressive Diabetic nephropathy.
¾¾ If diabetic Nephropathy is diagnosed at the microalbuminuria stage, the further progress of nephropathy can be
arrested with
• Tight glycemic control
• Well controlled blood pressure

When to screen for microalbuminuria?

Should be screened immediately after diabetes is diagnosed.
Many patients might have diabetes for several years before it is diagnosed and might have a well-progressed diabetic
nephropathy.
The patients found to be negative for microalbuminuria at the time of diagnosis of diabetes, should be screened at
least once every year.
Patients found to be positive for microalbuminuria should be tested for microalbumin, 2 to 3 times every year.

Methods of Screening
There are 3 methods for screening of microalbuminuria.
1. Microalbumin in 24 hour urine specimen.
2. Microalbumin in the 1st morning urine specimen.
3. Microalbumin to creatinine ratio in random or 1st morning urine specimen.

“Microalbumin in 24 hour urine specimen” is the gold standard.

“Microalbumin to creatinine ratio in random or 1st morning urine specimen” is the most practical method due to its
convenience to the patients and labs.
 Supplementary Information to Chapter 27 95

Methods of Screening
Micro­albumin in a 24 hr
urine specimen
Normal range <30 mg/24 hours
Micro­albuminuria 30­-300 mg/24 hours
Macro­albuminuria >300 mg/24 hours
Micro­albumin in the Micro­albumin to
1st morning urine Creatinine ratio
<20 mg/L <30mg/g for women
<20mg/g for men
20-­200 mg/L 30-­300mg/g for women
20-­200mg/g for men
>200 mg/L >300mg/g for women
>200mg/g for men

Acute Kidney Injury (AKI)

Abrupt kidney dysfunction.
Fluid and electrolyte imbalance.
Renal tubular cell injury and death.
Increased serum creatinine and low urine output.

Acute Renal Failure

Prevalence.
¾¾ ­5–10% in hospital patients.
¾¾ ­30–50% in intensive care patients.
¾¾ ­Mortality > 40% unchanged in last 30 years.
¾¾ Poor outcome, most cost intensive renal disease in the hospital.
¾¾ ­Chronic kidney disease in 40% of survivors.
¾¾ Chronic dialysis in 10% of survivors.
¾¾ ­Accurate detection of small decreases/changes of GFR.

Markers for AKI

¾¾ Neutrophil Gelatinase-Associated Lipocalin (NGAL)
¾¾ Interleukin 18 (IL-18)
¾¾ Human Kidney Injury Molecule-1 (KIM-1)
¾¾ Liver Fatty Acid Binding Protein (L-FABP)

NGAL
Neutrophil Gelatinase – Associated Lipocalin (NGAL)
¾¾ Protein belonging to the lipocalin superfamily.
¾¾ Initially found in activated neutrophils.
¾¾ Small molecule of molecular weight 25 kDa.
¾¾ Also found in certain epithelia, such as renal tubules where its expression is dramatically increased in ischemic or
nephrotoxic injury.
 96 Textbook of Biochemistry— Case Studies

¾¾ Increase in the production of NGAL have self defensive properties.

¾¾ Suggested to act as growth and differential factor in multiple cell types including developing and major renal epithelia.
¾¾ Suitable as a marker for the early identification of acute kidney injury (AKI).
NGAL can be used to detect AKI in the following cases:
¾¾ Pediatric and adult cardiopulmonary bypass operations.
¾¾ Percutaneous coronary interventions (PCI).
¾¾ Critically ill patients presenting at the emergency department or in the intensive care unit (heart failure, sepsis, multi-
organ failure).
¾¾ Renal transplantation.
¾¾ Patients with chronic kidney disease.
¾¾ Novel predictive marker for AKI and its outcome.
¾¾ Plasma NGAL is influenced by coexisting variables such as chronic kidney diseases (CKD), chronic hypertension,
inflammatory conditions and malignancies.
¾¾ Urine NGAL is elevated in subjects with CKD, lupus nephritis and urinary tract infections.
¾¾ But the elevation in plasma and urine NGAL are significantly lower in the above conditions than AKI.
Reference values:
  Serum: 20 – 150 ng/mL
  Urine: 3 – 850 ng/mL

Interleukin 18 (IL – 18)

¾¾ A proinflammatory cytokine induced and cleaved in the proximal tubules and hence easily detected in urine following
ischemic AKI.
¾¾ The levels are largely unaffected by nephrotoxins, CKD and urinary tract infections.
¾¾ Plasma levels are influenced by endotoxemia, immunological injury and cisplatin toxicity.

Kidney Injury Molecule–1(KIM-1)

¾¾ KIM-1 is one of the most highly induced protein in kidney after AKI and can be easily detected in urine soon after
AKI.
¾¾ The levels in urine is influenced by a number of nephrotoxins as well as in a variety of chronic proteinuric, inflammatory
and fibrotic disease in human.

Liver Fatty Acid Binding Protein (L-FABP)

¾¾ It is 14 kDa protein normally expressed in the proximal convoluted and straight tubules of the kidney.
¾¾ Urinary levels elevated prior to increase in serum creatinine in patients who developed AKI.
¾¾ Urinary levels influenced in CKD, diabetic nephropathy, glomerulosclerosis and polycystic kidney disease.