Proteinuria Plasma proteins - essential components of any living being The kidneys play a major role...

Proteinuria

Plasma proteins - essential components of any living being

The kidneys play a major role in the retention of plasma proteins

glomerular filtration barrier

renal tubules reabsorption of the passing through glomerular filtration barrier proteins

Proteinuria

The glomerular capillary wall - charge and size-selective properties

high-molecular-weight (HMW) plasma proteins can not enter the urinary space

only a tiny fraction of albumin, globulin, and other large plasma proteins cross

LMW proteins (<20,000 Da) cross capillary wall

they are normally reabsorbed by the proximal tubule

Proteinuria

Most healthy individuals

excrete 30 and 130 mg/day of protein the upper limit of normal total urine protein excretion - 150 to 200 mg/day for

adults

the upper limit of normal albumin excretion - 30 mg/day

Proteinuria

Normal tubular protein secretion a very small amount of protein that normally appears in the urine

Tamm-Horsfall protein (uromodulin) HMW glycoprotein (23 × 106 Da) formed on the epithelial surface of the thick ascending limb of the loop of Henle

and early distal convoluted tubule

binds and inactivates the cytokines interleukin-1 and tumor necrosis factor

Immunoglobulin A (IgA)

Urokinase

Proteinuria

Normal urine protein excretion up to 150 mg/d

the detection of abnormal quantities or types of protein in the urine - an early sign of significant renal or systemic disease

Normal urine albumin excretionless than 5 mg/L

low levels of albumin excretion = microalbuminuria

linked to the identification of the early stages of diabetic kidney disease

Microalbuminuria excretion of 30-300 mg/d or 20-200 µg/min

too small to be detected by routine dipstick screening

ProteinuriaAbnormal amounts of protein in the urine – three/four

mechanismsglomerular proteinuria

disruption of the capillary wall barrier a large amount of HMW plasma proteins that overwhelm the limited capacity of tubular reabsorption and cause protein to appear in the urine

tubular proteinuriatubular damage or dysfunction inhibit the normal resorptive capacity of the proximal tubule increased amounts of mostly LMW proteins appear in the urine

increased production of normal or abnormal plasma proteins can be filtered at the glomerulus and overwhelms the resorptive capacity of the proximal tubule (!!)

–rarely - increased urine protein due to increased tubular production of protein

Proteinuria

β2-Microglobulin an LMW (11.8-kDa) protein identified as the light chain of class I major histocompatibility antigens (e.g.,

human leukocyte antigens [HLAs] A, B, and C)

freely filtered at the glomerulus avidly taken up and catabolized by the proximal tubule

detectable urinary levels of β2-microglobulin - associated with many pathologic conditions involving the proximal tubule

•aminoglycoside

•Balkan endemic nephropathy

•heavy metal nephropathies

•radiocontrast nephropathy

•kidney transplant rejection •useful in distinguishing upper from lower urinary tract infection.

Proteinuria

Bence Jones protein

immunoglobulin light chains filtered at the glomerulus plasma cell dyscrasias may produce monoclonal proteins, immunoglobulin, free light chains, and a combination of these

the detection of urine immunoglobulin light chains can be the first clue to a number of important clinical syndromes associated with plasma cell dyscrasias that involve the kidney

plasma cell dyscrasias may also manifest as proteinuria or albuminuria when the glomerular deposition of light chains causes disruption of the normally impermeable capillary wall (!!)

Proteinuria

Selective or nonselective proteinuria

glomerular proteinuria can be further characterized as selective or nonselective

a clearance ratio of immunoglublin G (IgG; an HMW protein)–to-albumin that is less than 0.10 = selective proteinuria

more often seen in patients with minimal change disease predicts a good response to treatment with corticosteroids

IgG-to-albumin clearance ratios greater than 0.50 = nonselective pattern

the cost of the protein separation procedures has limited their widespread clinical use

Proteinuria

Glomerular proteinuria / tubular proteinuria

higher amounts of albumin and HMW proteins suggest glomerular proteinuria nephrotic range >3 g/24 hr certain glomerular source

isolated increases in LMW protein fractions are more suggestive of tubular proteinuriait is unusual for tubular proteinuria to exceed 1 to 2 g/dayonly a small fraction of protein excretion due to tubular damage should be albumin tubular proteins are heterogeneous; α2-microglobulin is often a major constituent

Proteinuria

Techniques to Measure Urine Protein

in random samples

in timed or untimed overnight samples

in 24-hour collections

source of error - inaccurate urine collection particularly 24-hour

collections

ProteinuriaTechniques to Measure Urine Protein

Tests to accurately quantitate total protein concentration in urinerely on precipitation(sulfosalicylic acid is added to a sample of urine, and the

turbidity is measured with a photometer or a nephelometer )

Tests to estimate total protein concentration in urinechemically impregnated plastic strips (dipstick)


Tests to accurately quantitate total protein concentration in urine (precipitation)

proteins detected: γ-globulin light chains, albumin

the method is more sensitive to albumin than to globulins trichloroacetic acid can be used in place of sulfosalicylic acid to increase the

sensitivity to γ-globulin (eg for Bence Jones protein)

Tests to estimate total protein concentration in urine(dipstick)pH-sensitive colorimetric indicator that changes color when negatively charged

proteins bind to itpositively charged proteins are less detected immunoglobulin light chains may

escape urine dipstick detection even when present in large amounts in the urinesensitive to very small urine protein concentrations -the lower limit of detection 10-

20 mg/dL (could be Tamm-Horsfall protein )

Proteinuria

Techniques to Measure Urine Protein

quantitative determinations of albuminas gold standards

Radioimmunoassay

Immunoturbimetric technique

Enzyme-linked immunosorbent assay (ELISA)


Screening methods

dipstick = qualitative or semiquantitative for total protein excretion and microalbuminuria

sensitivity and specificity - markedly influenced by fluid intake, state of diuresis, and the resulting urine concentration

protein-to-creatinine or albumin-to-creatinine ratio in random, or timed urine collections

there is a high degree of correlation between 24-hour urine protein excretion and protein-to-creatinine ratios in random, single-voided urine samples in patients with a variety of kidney diseases


Screening methods - protein-to-creatinine or albumin-to-creatinine ratio

more quantitative than a simple dipstick screening procedure

a protein-to-creatinine ratio of greater than 3.0 or 3.5 mg/mg or less than 0.2 mg/mg indicates protein excretion rates of greater than 3.0 or 3.5 g/24 hr or less than 0.2 g/24 hr

Proteinuria

Analytic tools - to separate and identify individual urinary proteins

agarose gel electrophoresis column gel chromatography polyacrylamide gel electrophoresis immunoelectrophoresisisoelectric focusing proteomic techniques employing mass spectrometry peptide mass fingerprinting

have been used in clinical laboratories to determine the selectivity of urine protein to identify monoclonal immunoglobulin heavy and light chains

Proteinuria

β2-Microglobulin commonly measured in urine using radioimmunoassay or ELISA

Bence Jones proteinimmunoglobulin light chains may not be detected by dipstick a proteinuria or albuminuria may also exists when the glomerular deposition of light chains causes disruption of the normally impermeable capillary wall monoclonal proteins are best detected using serum and urine immunoelectrophoresisthe diagnosis of a plasma cell dyscrasia can be suspected when a tall, narrow band on electrophoresis suggests the presence of a monoclonal γ-globulin or immunoglobulin light chain

Proteinuria

Screening methods established diagnosis of kidney disease

Measuring the amount of urine protein (usefull in IgA nephropathy, membranous nephropathy, type I membranoproliferative glomerulonephritis, nephrotic syndrome, mild renal insufficiency )

additional prognostic information

can be used to monitor the response to immunosuppressive therapy

Proteinuria

There are variations of concentration of albumin in the urinewith: physical activity level, acute illnesses or fevers, menstruation, pregnancy, vaginal discharge, diet, blood pressure, volume status, degree of glycemic control, and urine collection method (eg, 24 h, overnight or timed, short-term)

Albumin excretion - 25% higher during the day than overnight, with a day-to-day variation of 40%

before a patient is classified as having microalbuminuria - at least 3 urine samples over a 6-month period that satisfy the above range criterion are recommended

Proteinuria

The presence of abnormal amounts or types of protein in the urine reflects :

A defective glomerular barrier abnormal amounts of proteins of intermediate molecular weight enter the Bowman space

Tubular diseases inability of the kidneys to normally reabsorb the proteins through the renal tubules

Overproduction of plasma proteins pass through the normal glomerular basement membrane (GBM) enter the tubular fluid in amounts that exceed the capacity of the normal proximal tubule to reabsorb them

Proteinuria

Symptoms

in most patients - asymptomatic proteinuria

detected upon screening in patients with hypertension, diabetes

frequently in the absence of serious underlying renal disease

Proteinuria

The more common and benign causes of proteinuria

transient proteinuria associated with physical exertion and fever

orthostatic proteinuria (albuminuria < 1 g/d, normal renal function)

typically - in tall thin adolescents or adults younger than 30 years, +/- severe lordosis

overnight urine collection shows normal protein excretion (ie, <50 mg during 8-h period)

nonrenal disease (proteinuria < 1 g/d, normal renal function) severe cardiac failure, sleep apnea

Proteinuria

Microalbuminuria (30-300 mg/d or 20-200 µg/min) frequently observed in association with:

Hypertension

Early stages of diabetic nephropathy

Proteinuria

Proteinuria in significant glomerular disease

in excess of 500 mg/dparticularly associated with:

Changes in urine appearance: red/smoky, frothyEdema: ankle, periorbital, labial, scrotalElevated blood pressure Elevated cholesterol, lipiduria, hypoalbuminemiaActive urine sediment (dysmorphic red blood cells,+/-red cell casts)Abnormal renal function

Proteinuria Proteinuria in significant glomerular disease

Joint discomfort, skin rash, eye symptoms, or symptoms of Raynaud syndrome

Multisystem disease : systemic lupus erythematosus, rheumatoid arthritis

History of kidney disease (including pregnancy related)

Medication, including over-the-counter or herbal remedies

Past health problems, such as jaundice, tuberculosis, malaria, syphilis, endocarditis

Systemic symptoms, such as fever, night sweats, weight loss, or bone pain

Risk factors for HIV or hepatitis

Symptoms that suggest complication(s) of nephrotic syndrome

Loin pain, abdominal pain, breathlessness, pleuritic chest pain, rigors

Associated upper respiratory tract infection

How dose proteinuria damage the kidney?


1. glomerulosclerosis is explained by the hyperfiltration theory

2. tubulointerstitial injury is olso induced by proteinuria; this is a final common pathway to eventual kidney failure





Albumin - Albumin-bound fatty acids

albumin = a major components in proteinuric urine

albumin-bound fatty acids = important mediators of tubulointerstitial injury apoptosis in proximal tubular cells



Tubulointerstitial injury induced by proteinuria

molecular and cell biological changes

up-regulation of certain proinflammatory and profibrogenic gene: MCP-1, osteopontin

transdifferentiation of tubular cells into myofibroblasts fibrosis of the kidney

a large number of apoptotic cells in the tubulointerstitial compartment

apoptosis of tubular cells atubular glomeruli and decreases the number of functional nephrons


Tubulointerstitial injury induced by proteinuria

=

atubular glomeruli and interstitial fibrosis


What mediates tubular and interstitial damage

induced by proteinuria?



Effects of filtered macromolecules on tubular cells



Various components in proteinuric urine damages tubular cells directly:

growth factors transferrin albumin albumin-bound fatty acids

complement components in proteinuric urine


Protein overload in tubular cells is also associated with ammonium production

Complement component C3 modified by ammonia = amidated C3 amidated C3 forms the alternative pathway convertase of the complement cascade

Preferential secretion of ammonia into the tubular lumen inappropriate activation of the alternative pathway at the brush border in proteinuric conditions


Complement components in proteinuric urine = crucial mediator of tubulointerstitial damage

complement is activated in urine of patients with various glomerular diseases

the degree of intratubular complement activation correlated with the level of non-selective proteinuria



accumulation of inflammatory cells around tubules of high filtered protein load

C3 staining in the brush border or within the cytoplasm at sites of high protein reabsorption

!! renoprotective effects of ACE inhibitor by limiting tubular activation of complement


proteinuria tubular injury

fibrotic changes duration of proteinuria, quantity of proteinuria, quality of proteinuria, other factors

other factors: hypertension, smoking, aging, chronic hypoxia


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Podocytes

Podocytes

Podocytes

Podocytes

Podocytes

there are quite a number of podocyte diseases = podocytopathies

in the podocytopathies the orderly structure of the podocytes and the foot processes interlinked by the slit diaphragms is lost (effacement of the podocytes

Podocytes

the filtration barrier = an amazing structure filtering something like 200-500 l plasma per day and producing something like 200 l of primary urine

the podocyte which is really standing or sitting on top of the capillaries is one of the key players there in the final regulation of the passage of the proteins or actually preventing the leakage of the proteins

Podocytes

there is a number of molecules located in the slit diaphragm domain

transmembrane

or intracellular

these molecules adjust the shape of the podocyte foot processes

PodocytesNephrin - neph-1, neph-2, filtrin, hemoglobulin group of molecules, an immunoglobulin family of cell adhesion proteins

They are clustering together with podocin for the functionality of the slit diaphragm

The nephrin molecules extend from neighbouring podocytes and foot processes and induce the interaction between the different podocyte foot processes

Nephrin really forms the framework of the slit diaphragm

Podocytes

The nephrin damage determines lack of normal foot processes and the filtration slit organisation

Specific administration or generation of specific nephrin antibodies lead to proteinuria

In diabetes and diabetic nephropathy there are found circulating anti-nephrin antibodies many months before the actual attack to the kidney

Proteinuria

Microalbuminuria an independent predictor of cardiovascular disease an independent predictor all-cause mortality in both diabetic andnondiabetic men and women a stronger indicator for future cardiovascular events than systolic BP or

serum cholesterol

Detecting microalbuminuria is an important screening tool to identify people

who are at high risk for cardiovascular events and progression of kidney disease

who need more intensive therapy compared with subjects with normal albumin excretion rates

Proteinuria According to the American Diabetes Association (ADA), the gold

standard for measuring urine albumin excretion is a 24-h urine collection

A more convenient method to detect microalbuminuriais the albumin (g)/creatinine (mg) ratio (ACR) measured in a random urine specimen

Currently, the National Kidney Foundation recommends the use of spot urine ACR obtained under standardized conditions (first voided, morning, midstream specimen) to detect microalbuminuria

The ACR is a more convenient test for patients and may be less prone to errors due to improper collection methods and variations in

24-h protein excretion compared with a random urine specimen

Proteinuria

The ADA and the National Kidney Foundation define microalbuminuria as an ACR between 30 to 300 g/mg in both men and women

Proteinuria

Proteinuria


Proteinuria

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Proteinuria Plasma proteins - essential components of any living being The kidneys play a major role...

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