Renal Function Testing Dr. Liu The First Affiliated Hospital, Zhengzhou University
“Urine”: Element of Nature
In ancient times, before the existence of any alphabet, it was common to use symbols to denote important known elements of nature.
Outline
Review physiology Methods to evaluate kidney function
Glomerular filtration rate (GFR)
Inulin Creatinine Cystatin C
Protein Urea and Blood Urea Nitrogen (BUN)
Examples of diseases and their corresponding lab results.
Nephron Physiology I
Glomerulus (5): filters plasma water (~170-200 L/ 24 hrs ultrafiltrate pass)
Size
<50 KD gets filtered
Charge
Basement membrane has negative charge
Electrolytes Small molecules
Negative charged molecules can hardly pass Albumin Most plasma proteins Positive charged molecules can pass easily Light chain Ig
Shape
Linear molecules pass through faster than globular molecules of equal MW
Nephron Physiology II
Proximal Tubule (4)
Reabsorb (isotonic)
Secrete
Water, Cl-, K+, HCO3-, PO4-, Ca2+ proteins, urea, uric acid, amino acids, glucose, and carbohydrates H+, NH4+, uric acid
Loop of Henle (1 & 2)
Descending Limb
Reabsorb water (hypertonic)
Ascending Limb
Reabsorb Na+ and Cl- (hypotonic)
Nephron Physiology III
Distal Tubule (6)
Reabsorb and secrete
Reabsorbs Water - ADH present Reabsorbs Na+ - Aldosterone present Ion exchange - Na+, H+, HCO3-, and NH4+ to maintain acid/ base balance Reabsorb/secrete K+
Collecting Duct (not shown)
Reabsorb and secrete
See Distal Tubule Reabsorb urea
Tests of Renal Function
Practical evaluation of kidney status in renal disease includes examining…
1.) the nephron functions of glomerular filtration. 2.) the secretory capacity for particular endogenous and exogenous compounds. 3.) the kidney’s resorptive capacity for water and electrolytes as manifested by the urine-concentrating ability of the kidneys.
Glomerular Filtration Rate Inulin Creatinine Cystatin C
Glomerular Filtration Rate (GFR)
Rate (mL/min) that substances are filtered through the glomeruli.
Estimate GFR by measuring the clearance of a small molecule that …
http://www.mscd.edu/%7Ebiology/2320course/2320info.htm
Rate (mL/min) = Kf [GHP – (CP + GOP)] Kf = Hydraulic permeability x surface area GHP = Glomerular hydrostatic pressure CP = Capillary pressure GOP = Glomerular Oncotic Pressure
Depends on hydrostatic and oncotic pressures along the afferent arteriole and across the glomerular filter.
Is freely filtered by glomeruli. Has a clearance dependent only on glomerular filtration. Is not secreted or reabsorbed by the tubules. Has a stable production rate.
GFR: Gold Standard
“Gold Standard” – inulin
Exogenous polysaccharide cleared by renal filtration Administered to patient
Bolus injection
Constant infusion (to get steady state concentration)
Measure the plasma disappearance curve
Collect a blood sample and timed urine samples during this period
Disadvantages: time consuming, patient inconvenience, small amount of extrarenal clearance (0.83 mL/min/10 kg) Chourasia MK and Jain SK. Pharm Pharmaceut Sci (www.ualberta.ca/~csps). 6(1):33-66, 2003.
Creatine and Creatinine
Creatine
(creatininase)
Synthesized from glycine, arginine, and S-adenosyl-methionine in the liver. Stored in muscle as part of the energy pool as phosphocreatine, which is used in muscle contraction.
Creatinine
Synthesized from creatine. Release from muscle into the circulation is relatively constant at 1–2% each day of the total creatine pool.
GFR: Creatinine
Is it freely filtered by glomeruli? Yes
Does its clearance depend only on glomerular filtration? Yes Is it reabsorbed or secreted by the tubules? No/Yes
MW: Only 113 Daltons (0.113KD)
Not reabsorbed Small amount (7-10%) secreted; increases with increasing plasma levels
Does it have a stable production rate? Yes
Serum creatinine level is directly proportional to muscle mass. The serum creatinine level should remain constant and normal with normal renal excretory function.
GFR: Renal Clearance “Renal Clearance”: volume of plasma from which a substance is completely removed per unit time. In steady state, rate in = rate out
Rate in = (volume/time)plasma x concentrationplasma
Rate out = (volume/time)urine x concentrationurine
(volume/time)plasma x conc.plasma = (volume/time)urine x conc.urine (volume/time)plasma = ((volume/time)urine x conc.urine )/ conc.plasma (volume/time)plasma = renal clearance
GFR: Creatinine Clearance Ccrea =
(Ucrea ) x (V) ______________ x 1.73/BSA (Pcrea ) x (t)
Ccrea = Clearance in mL of plasma cleared of creatinine per minute (mL/min) Ucrea = Urinary concentration of creatinine V = Volume of urine (mL) Pcrea = Plasma concentration of creatinine = Body Surface Area (m2): estimated from nomograms or formulas using height and weight tBSA = time to collect urine (min) 1.73 = defined standard BSA (m2)
Creatinine: Analytical Techniques
Coupled enzymatic assays
Creatininase – series of four enzymatic steps; measure decrease of NADH absorbance at 340 nm
Poor sensitivity, poor precision, high reagent cost
Creatininase and Creatinase – series of three enzymatic steps that form peroxide; the 4th step measures peroxide, which can be done in a variety of ways including forming colored products Creatinine Deaminase – a series of four enzymatic steps that form peroxide; the 5th step measures peroxide.
* Enzymatic methods usually used in dry chemistry systems, like Ortho-Clinical Diagnostics’ Vitros® 950 at CHRMC.
GFR: Cockroft and Gault Equation
Data needed: Plasma creatinine, age, weight, height, and sex
Cockroft and Gault Equation: GFR (ml/min/1.73 m2) = 2.12K x [140 – age] x W Pcrea x BSA where K = 0.85 for women and 1.00 for men age = years W = weight (kg) Pcrea = serum or plasma creatinine BSA = Body Surface Area (m 2) based on weight and height
GFR: MDRD Prediction Equation
Recommended by NKDEP over other GFR calculations. Data needed: Plasma creatinine, age, sex, and race (weight and height not needed since normalized to average adult surface area)
MDRD equation for adults:
GFR (ml/min/1.73 m2) = 186 x (Pcre )-1.154 x (Age)-0.203 x (0.742 if female) x (1.210 if African American)
Disadvantage: This is most accurate for GFRs 60 ml/min/1.73 m2 or less. Advantage of these type of formulas: No need for 24 hour urine collection. http://www.nkdep.nih.gov
GFR: Creatinine Clearance
Limitations
Difficult in obtaining an adequate 24hr urine collection. Varying amounts of creatinine are secreted into the urine at the proximal tubule. The elderly and young children normally have LOWER creatinine, which can mask renal disease in these patients. Creatinine is increased with diets high in meat and and exercise. Poor sensitivity and specificity
Cystatin C
Low MW weight protein A potent and abundant inhibitor of cysteine proteases Measured by immunoassay
Latex particle-enhanced turbidimetric or nephelometric immunoassay
Serum concentration is not influenced by muscle mass, diet, or apparently, by sex.
www.man.poznan.pl/CBB/GIF/hcc-dim.gif
GFR: Cystatin C
Is it freely filtered by glomeruli? Yes
MW: Only 13 KD and high pI(9.2)
Does its clearance depend only on glomerular filtration? Yes Is it reabsorbed or secreted by the tubules? No Does it have a stable production rate? Yes
Synthesized by all nucleated cells GFR Marker ? Yes, a very good one!!
GFR: Cystatin C vs. Creatinine
0 1
0.5 1-specificity
Sensitivity = TP/(TP+FN) Specificity = TN/(FP+TN)
Nonparametric ROC plots were constructed to assess the diagnostic accuracy of serum concentrations of cystatin C and creatinine in distinguishing between normal and reduced GFR (<80 mL/min/1.73 m2) in 51 patients with various renal conditions (From Kyhse-Andersen J, et. al. Clin Chem. 40: 1921, 1994).
Protein and Nonprotein Nitrogenous Compounds Proteins Urea Blood Urea Nitrogen
Protein and Proteinuria
In healthy kidneys, the glomerulus retains proteins >50KD, partially retains proteins between 5 and 50 KD, and freely filters proteins <5 KD. Since the plasma albumin concentration is greater than other proteins and its mass is 66KD, a small amount in the urine is normal. In normal urine, total protein is 30% albumin, 30% immunoglobulins, and 40% Tamm-Horsfall glycoprotein. Proteinuria is an increase of protein in the urine.
Increased filtered load Decreased tubular reabsorptive capacity
Proteinuria – Increased Filtered Load
Increased filtered load: saturation of tubular reabsorptive capacity with essentially normal tubular function
Glomerular Proteinuria
Glomerular permeability increased
Inflammation or basement membrane damage High MW Proteins (Albumin or IgG) in urine
Overflow Proteinuria
Selective increase of low MW proteins.
Suggests production rate has increased (plasma levels higher). Example: Bence Jones proteins in Multiple Myeloma
Proteinuria – Decreased Tubular Reabsorptive Capacity
Damaged tubular function with essentially normal glomerular permeability
Proximal tubular damage
Decreased nephron number
Toxic agents (ie., drugs, heavy metals) or anoxia [Low MW proteins] > [Albumin] in urine Conditions like chronic renal failure can cause a decrease in nephron number. Increase in protein load per nephron
Enzymuria
Damage to tubular cells causes increased cell lysis and release of enzymes (ALP, LH, and NAG). [Low MW proteins] > [Albumin] in urine
Proteinuria
Relative ratio of low and high proteins can help determine pathology of disease.
Albumin has replaced total protein for glomerular permeability changes. The low MW proteins (i.e., retinol-binding protein and β2-microglobulin) can be used as markers of glomerular filtration and tubular damage.
Insert table pg 1218 Tietz
Tietz Textbook of Clinical Chemistry. 3rd ed. Philadelphia: WB Saunders, 1999. pg 1218.
Urea Protein
Urea is formed in the liver as the end product of protein catabolism.
Directly related to the metabolic function of the liver and the excretory function of the kidney.
>90% Urea excreted by the kidneys.
Proteolysis, Principally enzymatic
Amino Acids Transamination and Oxidative deamination
NH3 Enzymatic synthesis in “The Urea Cycle”
Urea Blood Urea Nitrogen
Blood Urea Nitrogen (BUN) is confusing.
1.) Sample is serum or plasma and not whole blood. 2.) BUN is in units of urea nitrogen. Urea MW = 60 2 moles of nitrogen per urea molecule N MW = 14 N:Urea = 0.467 Urea:N = 2.143 0.467
mg Urea dL
mg Urea N dL
X X
1g 1000 mg
1g 1000 mg
X X
mole Urea 60 g Urea
mole N 14 g N
X
X
2 moles N 1 mole Urea
1 mole Urea 2 moles N
X
2.143
X
14 g N mole N
60 g Urea mole Urea
X
X
1000 mg 1g
1000 mg 1g
=
Urea N (mg/dL)
=
Urea (mgl/dL)
Urea
Is it freely filtered by glomeruli? Yes Does its clearance depend only on glomerular filtration? Yes Is it reabsorbed or secreted by the tubules? No/Yes
Neither actively secreted or reabsorbed by tubules (No) 40-70% diffuses passively from the tubule in a healthy kidney to interstitium, ultimately to reenter the plasma (Yes)
Back-diffusion dependent on urine flow rate; diffusion increases as flow rate decreases
Does it have a stable production rate? No
Production is too dependent on several nonrenal variables
High-protein diet, malnutrition, dehydration, hepatic synthesis, etc.
***Poor indicator of GFR – Underestimates GFR***
Clinical Utility of BUN BUN:Plasma Creatinine ratio (normal 1220)
Azotemia: Increased BUN
Ratio discriminates between pre-renal, renal, or postrenal causes.
Prerenal- diet, dehydration, and liver disease high ratio (> 20), normal plasma creatinine renal – disease at any level of the nephron or interstitium normal ratio, elevated BUN and plasma creatinine postrenal – obstruction to urine outflow high ratio, elevated plasma creatinine
Low ratio: ATN, low protein diet, liver disease
Analytical Techniques: Direct
Fearon Reaction: Urea can be measured in both plasma and urine (not used very much or at all anymore).
O O
O
H3C
CH3 NOH
Diacetyl monoxime
O
H+
CH3
H3C
H+, ∆
+ H2N
http://www.hscj.ufl.edu/path/lectures/Review_of_Analytical_Methods_1.pps
N
NH2
O
Diacetyl
N
H3C
Urea
CH3
Diazone λmax= 540 nm
Pathophysiology and Corresponding Lab Results Glomerular Diseases Tubular Diseases
Filtration by The Healthy Glomerulus
Glomerulus is a filter with a 50 KD MW cutoff. Only small MW proteins and molecules are filtered into the tubules.
Glomerular Diseases
Damaged Filter.
Loss of glomeruli Damaged filter = larger molecules like albumin, Ig, and red blood cells can get through. Diseases include:
Acute Glomerulonephritis Chronic Glomerulonephritis Nephrotic Syndrome
Glomerulonephritis Acute: rapid onset
Often caused by infection Large, inflamed glomeruli
Chemistry Tests for Both Elevated
Hematuria Proteinuria (Albumin) BUN Serum creatinine Red blood cell casts (UA)
Decreased
GFR
Chronic: long term
Lengthy inflammation leads to scarring and loss of nephrons
Other signs or symptoms
Sodium and water retention Anemia Hypertension CHF (sometimes)
Healthy Renal Tubules
Function is to reabsorb and secrete molecules. Water, electrolyte, and acid-base homeostasis Damaged tubules can no longer perform these tasks well. http://www.nda.ox.ac.uk/wfsa/html/u09/u09_016.htm
Tubular Disease
Occurs to some extent in all renal diseases. In some cases, it is the predominant problem. Results…
Decreased reabsorption and secretion of certain substances. Reduced urinary concentrating capability Affects acid-base balance
Examples of causes: analgesic drug or radiation toxicity, renal transplant rejection, or infections
Tubular Disease
Lab findings include:
Decreased GFR Decreased urinary concentrating ability Decreased metabolic acid excretion Leukocyte casts in the urine Inappropriate control of Na+ balance Increased low MW proteins in the urine
Summary
Kidney function is evaluated in several ways.
GFR
Estimated by measuring inulin, creatinine, or cystatin C.
Protein in the urine can evaluate both glomerular and tubular function. BUN can estimate the amount of non-protein nitrogenous metabolites in the urine.