Acute kidney injury-pathogenesis and lab monitoring .

2. Ekbal Mohamed Abohashem-MD
Prof. of Clinical Pathology, Mansoura University, Egypt

3. AGENDA :
 Definition and magnitude of the problem
 Characters of an ideal biomarker
 Pathophysiology and mechanisms
 Biomarkers of AKI
 Methods to quantitate biomarkers
 Therapeutic agents
 Literature sources

4. ACUTE KIDNEY INJURY
Definition and Prevalence
Acute kidney injury (AKI) is currently recognized as the preferred
nomenclature for the clinical disorder formally called acute renal failure
(ARF).. The Acute Kidney Injury Network (AKIN), which was formed recently
in an effort to facilitate improved care of patients who are at risk for AKI,
described AKI as “functional or structural abnormalities or markers
of kidney damage, including abnormalities in blood, urine, or
tissue tests or imaging studies present for less than three
months.”

5. AKI has been reported to complicate 1% to 7% of all hospital admissions and
1% to 25% of intensive care unit (ICU) admissions.
Over the past 50 years, mortality rates of patients with AKI in the ICU have
remained high, at approximately 50% to 70%.. A recent large international
study of the epidemiology and outcome of AKI in critically ill adult patients
reported an overall in-hospital mortality rate of 60%.
Of those who survived to hospital discharge, 13% remained dialysis-
dependent., renal insufficiency persisted in 41% and overall 5- year survival
post -discharge was 50%.

6. Apart from prophylactic measures, no effective
treatment for this syndrome is known.
Therefore, early recognition of AKI not only can
provide better opportunities for preventive
interventions, but also opens many gates for
research and development of effective
therapeutic options.

7. Stage GFRa Criteria UOb Criteria
Risk
SCrc increased 1.5-2 times
baseline
or
GFR decreased >25%
UO < 0.5 mL/kg/h < 6 h
Injury
SCr increased 2-3 times
baseline
or
GFR decreased >50%
UO < 0.5 mL/kg/h >12 h
Failure
SCr increased >3 times
baseline
or
GFR decreased 75%
or
SCr ≥4 mg/dL; acute rise
≥0.5 mg/dL
UO < 0.3 mL/kg/h 24 h
(oliguria)
or
anuria 12 h
Loss of function
Persistent acute renal failure: complete loss of kidney
function >4 wk (requiring dialysis)
d
Complete loss of kidney function >3 mo (requiring
RIFLE criteria for AKI :(2002)
DIAGNOSIS OF AKI

8. Abrupt (within 48 h) reduction in kidney function
currently defined as an absolute increase in
serum creatinine of 0.3 mg/dL or more (≥26.4
μmol/L) or
A percentage increase in serum creatinine of 50%
or more (1.5-fold from baseline) or
A reduction in urine output (documented oliguria
The AKIN (Acute Kidney Injury Network) classification
system of acute kidney injury (2004)

9. The AKIN criteria differ from the RIFLE criteria in several
ways. The RIFLE criteria are defined as changes within 7
days, while the AKIN criteria suggest using 48 hours. The
AKIN classification includes less severe injury in the
criteria and AKIN also avoids using the glomerular
filtration rate as a marker in AKI, as there is no
dependable way to measure glomerular filtration rate and
estimated glomerular filtration rate are unreliable in AKI.

10. Stage SCr UO
1
↑ SCr ≥26.5 μmol/L
(≥0.3 mg/dL) or ↑SCr
≥150 a 200% (1.5 a
2×)
<0.5 mL/kg/h (>6
h)
2
↑ SCr >200 a 300%
(>2 a 3×)
<0.5 mL/kg/h
(>12 h)
3b
↑ SCr >300% (>3×)
or if baseline SCr
≥353.6 μmol/L (≥4
mg/dL) ↑SCr ≥44.2
μmol/L (≥0.5 mg/dL)
<0.3 mL/kg/h (24
h) oranuria (12 h)
The AKIN staging for AKI
:aSCr, serum creatinine; UO, urine output.
bStage 3 also includes patients requiring RRT independent of the stage (defined by SCr and/or UO) they are in at the moment they initiate RRT.

11. KDIGO defines AKI as any of the following:
 Increase in serum creatinine by 0.3mg/dL or
more within 48 hours or
 Increase in serum creatinine to 1.5 times
baseline or more within the last 7 days or
 Urine output less than 0.5 mL/kg/h for 6
hours
The KDIGO has also recommended a staging
KDIGO Clinical Practice Guidelines
In 2012 the Kidney Disease Improving Global Outcomes (KDIGO)
released their clinical practice guidelines for acute kidney injury (AKI),
which build off of the RIFLE criteria and the AKIN criteria.

12. Stage Serum creatinine
1 Increase in serum creatinine by ≥0.3 mg/dl (≥26.5 μmol/l) within 48 h from the onset of the
disease or after cardiac surgery, or increase in serum creatinine of 1.5–1.9-fold of the
baseline level
2 Increase in serum creatinine of 2.0–2.9-fold of the baseline level
3 Increase in serum creatinine of 3.0-fold of the baseline level or increase in serum
creatinine to ≥4.0 mg/dl (≥353.6 μmol/l) or initiation of renal replacement therapy
Staging of AKIKDIGO

13. The excretion of creatinine does not depend on the load
filtered solely by the glomeruli, but also on that secreted by
the kidney tubules, which normally varies from 5 to 20%of
total excretion, and may increase to 50% as a compensatory
mechanism when the glomerular filtration rate (GFR)
decreases.
The other parameter of AKI is oliguria, which is neither
sensitive nor specific, since it could occur as a result of a
kidney injury, but may also reflect an adaptive physiological
response to either intracellular dehydration or hypovolemia.
HOWEVER:
Therefore, the need for the development of new biomarkers for
prediction and diagnosis of AKI among patients at high risk has been
very palpable.

14. Biomarkers of acute kidney injury:
the pathway from discovery to clinical
adoption

15.  allow the early detection of kidney injury before an increase in
serum creatinine and/or BUN;
 differentiate acute tubular necrosis (ATN) from acute
glomerulonephritis or acute interstitial nephritis;
 be able to monitor the effects of an intervention or treatment;
 and would predict the need for dialysis, mortality, and long-
term kidney outcome.
The objectives are to :
Accomplish more timely interventions to achieve the most favourable
outcomes in patients with AKI.
An ideal biomarker of AKI should :

16. Pathophysiology and Mechanisms
 Acute kidney injury can result from
 Decreased renal or intrarenal perfusion,
 A toxic or obstructive insult to the renal tubule,
 Tubulointerstitial inflammation and edema,or
 Primary reduction in the filtering capacity of the glomerulus

17. A Number of pathophysiological mechanisms can contribute
to AKI following an ischemic or toxic insult :
a) Alterations in renal perfusion resulting from loss of autoregulation and
increased renal vasoconstriction,
b) Tubular dysfunction and cell death by apoptosis and necrosis,
c) Desquamation of viable and dead cells contributing to intratubular
obstruction,
d) Metabolic alterations resulting in transport abnormalities that can lead to
abnormalities of tubuloglomerular balance, and
e) Local production of inflammatory mediators resulting in interstitial
inflammation and vascular congestion

18. 18

19. 19

20. 20

22. 1. Functional markers : SCr and plasma/serum CyC
2. Up-regulated proteins : NGAL, KIM-1, L-FABP and
IL-18
3. Enzymes : NAG, a-GST, p-GST, GGT and AP
4. Low-molecular weight proteins : Urine CyC,alfha-1
macroglobulin,beta-2 macroglobulin, RBP, etc….
Biomarkers for AKI :

24. Functional markers
Serum creatinine (SCr). Serum creatinine (SCr) is a degradation product of
muscle cells and represents a surrogate for the efficiency of glomerular
filtration. It has poor predictive accuracy for renal injury, particularly, in the
early stages of AKI. In the case of critical illness, SCr concentrations are
subject to large fluctuations due to a patient’s induced dilutional volume
status, the catabolic effects of critical illness, the likelihood of concentration
decreases in septic conditions and the increased tubular excretion with
diminishing renal function. Furthermore, after an injurious event, the rise in
SCr is slow. Therefore, detection of the earliest evidence of AKI necessitates
the use of other plasma or urinary biomarkers.

25. Cystatin C (CyC) is a 13-kDa non-glycosylated cysteine protease inhibitor produced by all
nucleated cells at a constant rate. In healthy subjects, plasma CyC (pCyC) is excreted
through glomerular filtration and metabolized completely by the proximal tubules.
Furthermore,there is no evident tubular secretion. Several studies claim the superiority of
pCyC against SCr to detect minor reductions in glomerular filtration rate (GFR) .
However, the interpretation of pCyC levels is biased by older age, gender, weight, height,
cigarette smoking and high levels of C-reactive protein (CRP) . In addition, CyC levels are
supposedly influenced by abnormal thyroid function the use of immunosuppressive therapy
and malignancies .
Plasma/serum cystatin C (CyC).

26. Neutrophil gelatinase-associated lipocalin ( NGAL)
NGAL, also known as siderocalin, lipocalin 2, or oncogene 24p, is a
25-kDa protein of the family of lipocalins. Human NGAL exists in
three distinct forms: 25-kDa monomer, 45-kDa homodimer, and 135-
kDa heterodimer. Heterodimeric NGAL is conjugated to gelatinase
and is specific to neutrophils . NGAL is also expressed at steady low
levels in various cell types, such as the uterus, prostate, salivary
gland, lung, trachea, stomach, colon, and kidney . Its production
increases with age and is higher in women than men.
UP-REGULATED PROTEINS

27. NGAL is a small secreted polypeptide that is protease resistant
and thus may be easily detected in the urine.
The appearance of NGAL in the urine preceded the appearance of
other urinary markers such as N-acetyl-beta-D-glucosaminidase
and beta2- microglobulin.
Although NGAL is represented in some human tissues, it is one of
the most upregulated transcripts in the kidney early after
ischemic, septic, or toxic AKI in animal models and human
neonates, children, and adults, implying its role as an early marker
of structural renal tubular damage .

28. Physiologically, NGAL binds to iron-siderophore complexes. Then
it exerts a bacteriostatic role of the innate immune system by
sequestering iron-siderophore complexes and thereby limiting iron
uptake by bacteri. In addition to bacteriostasis, NGAL also
provides antiapoptotic effects and enhances proliferation of renal
tubular cells, which establishes its potential pathways in kidney
protection during AKI
The thick ascending limb of the loop of Henle and the intercalated
cells of the collecting duct are the primary sites of NGAL
production in the kidney.

29. There are commercial kits to measure uNGAL (urinary NGAL)
or pNGAL (plasmatic NGAL).Under stable conditions, plasma
and urine concentrations are around 20 ng/mL. The marker
increases 2–4 h after the kidney damage occurs. The NGAL has
been associated with a probable kidney protective effect, since
it is released by nephron segments where they can form a
complex with siderophores that binds to iron; thus, the iron
released by the damaged tubular cells is chelated which
prevents the formation of hydroxyl radicals and superoxide
anions .

30. KIM-1 is a 38.7-kDa type l transmembrane glycoprotein with an extracellular
immunoglobulin-like domain topping a long mucin-like domain . It has a
transmembrane domain and a short intracellular domain that contains a signaling
protein for tyrosine phosphorylation (KIM-1b) .The extracellular domain of KIM-1 is
shed from the cell surface by a metalloproteinase-dependent Process .
KIM-1 expression is mainly upregulated in the proximal tubule cells, especially the
S3 segment .Urinary KIM-1 levels linearly increase with age in healthy individuals,
and higher KIM-1 values are noted in males than in females . KIM-1 is thought to
participate in both kidney injury and healing processes. In situ hybridization
indicated KIM-1 as a marker of proliferation and regeneration in proximal tubules .
Kidney injury molecule 1 (KIM-1)

31. KIM-1 is also a sensitive biomarker of tubular injury in other renal diseases
besides AKI, for example focal glomerulosclerosis, immunoglobulin A
nephropathy, membranoproliferative glomerulonephritis, membranous
glomerulonephritis, acute rejection, chronic allograft nephropathy, systemic
lupus erythematosus, diabetic nephropathy, hypertension and Wegener's
granulomatosis comared with normal kidney tissue.. Urinary KIM-1 was
increased in the same group of patients and correlated positively with tissue
KIM-1 and macrophages and negatively with kidney function but not with
proteinuria.

32. KIM-1 might be useful for the detection of nephrotoxicity in
preclinical and early phase 1 and 2 clinical studies , therefore,
KIM-1 has been approved by the US Food and Drug
Administration (FDA) as an AKI biomarker for preclinical drug
development . Also, a lateral flow dipstick for KIM-1 has been
developed, providing a simplified way of assessing KIM-1 levels
that yields semi quantitative results in 15 min .

33. L-FABP, also known as fatty acid-binding protein 1 (FABP1), is a 14-
kDa protein from the large superfamily of lipid-binding proteins .
It pertains to a family of carrier proteins for fatty acids and aids in
regulation of fatty acid uptake and intracellular transport . L-FABP is
expressed not only in the liver but also in the stomach, intestine,
lung, and kidney . L-FABP binds and transports fatty acids to the
mitochondria and peroxisomes to generate energy via β-oxidation .
L-FABP also plays a cell-protective role by mitigating H2O2-induced
oxidative stress . In the kidney, L-FABP is located in the proximal
tubule and is excreted into the tubular lumen along with bound toxic
peroxisomal products .
Liver-type fatty acid-binding protein

34. IL-18, also known as interferon-gamma inducing factor, is a
24-kDa cytokine from the IL-1 family of cytokines, which
regulates innate and adaptive immunity. IL-18 is
synthesized by multiple tissues, including monocytes,
macrophages, proximal tubular epithelial cells, and the
intercalated cells of the collecting ducts, as an inactive
precursor. It resides intracellularly until it is processed into
an active form by caspase 1.
Its level rises approximately 6 h after the ischemic injury,
24 to 48 h before the AKI diagnosis, and peaks about 12 h
later at values up to 25 times normal levels .
Interleukin 18

35. Cell cycle arrest in G1 phase may be a cellular mechanism to emerge
from circumstances when dormant DNA breakage can happen . Renal
epithelial cells have been shown to undergo G1 cell cycle arrest in
the setting of ischemic or septic AKI . The cyclin-dependent kinase
inhibitor p21 halts cell cycle succession from G1 to S phase. cell
cycle arrest is critical in restricting the consequences of AKI. Both
IGFBP7 and TIMP-2 are involved in G1 cell cycle arrest during the
very early phases of cellular injury .
TIMP-2, a 21-kDa protein, is a member of the TIMP family. TIMP-2 is an
endogenous inhibitor of metalloproteinase activities. IGFBP7, a 29-
kDa secreted protein, is known to bind and inhibit signaling via
insulin-like growth factor 1 receptors.
Insulin-like growth factor-binding protein 7
and tissue inhibitor of metalloproteinase 2

37. Urine [TIMP-2] × [IGFBP7] was superior to other biomarkers (plasma NGAL
and cystatin C and also urinary NGAL, KIM-1, IL-18, pi-glutathione S-
transferase, and L-FABP) and to TIMP-2 and IGFBP7 alone in forecasting AKI
stage 2 or 3, with an AUC of 0.8. Also, follow-up studies ascertained an AUC
of 0.82 and 0.79 for the prediction of AKI stages 2 and 3, respectively.
These findings indicated that, in the ICU setting, [TIMP- 2] × [IGFBP7] is a fair
to excellent biomarker in predicting moderate to severe AKI within 12 h.

38. excellent performance, and having been validated, in different
cohorts of critically ill patients with various pathologies (sepsis,
shock, major surgery and trauma) . Also, recent publications
highlight the ability of these biomarkers in the ICU environment to
identify patients with AKI who are at increased risk of mortality or
need for RRT in the next 9 months.
The most notable properties of these urinary biomarkers, and for
which they were selected from more than 300 biomarkers, include:

39. The international multicenter Sapphire study,, showed that the increase in
performance by the combination of these biomarkers was AKI-specific (i.e., it
is not caused by other comorbidities such as sepsis or CKD) and provided a
strong signal as a “kidney alarm”to identify patients who are at imminent risk
of developing AKI . These urinary biomarkers are believed to increase in
response to the kidney tubule cell stress or early injury associated with
factors known to cause AKI.

40. The Food and Drug Administration(FDA) has recently approved the
marketing of the Nephrocheck Test and Astute 140 Meter (Astute Medical
Inc., San Diego, CA, USA), a rapid test that quantitatively measures TIMP-2
and IGFBP-7 cell cycle arrest biomarkers, which block the effect of cyclD-
CDK4 and CyclE-CDK2 in cell cycle promotion, making them ideal G1 cell
cycle arrest markers. The Astute 140 meter automatically multiplies the
concentrations of the 2 biomarkers together and divides this product by
1000, reporting a single test result with units in (ng/ml)2/1000.

41. In clinical practice,after AKI there is activation of cell division and
cell proliferation in order to repopulate the denuded tubular
epithelium.Early cell cycle arrest in G0/G1 could protect the
kidney from further damage, by blocking DNA damage and
caspase 3/7 activation, thereby limiting the extent of damage and
avoiding a potentially dangerous maladaptive process caused by
poor repair. These phenomena occur 24–48 h before the increase
in sCr which occurs after a significant drop in the GFR .

42. Discovery of cell cycle arrest biomarkers of AKI has
led to new investigations and growing knowledge
regarding the role of cell cycle arrest in development
of AKI, impact of cell senescence in AKI on CKD
progression, and advent of new concepts such as
acute kidney stress, subclinical AKI, and acute kidney
disease.

43. The sodium/hydrogen exchanger isoform (NHE3) is the most abundant
apical sodium transporter in the renal tubule, responsible for the proximal
reabsorption of 60%−70% of filtered sodium and bicarbonate . NHE3
localizes to the apical membrane and intracellular vesicular compartment
of renal proximal tubular cells and the apical membrane of the thick and
thin ascending limb cells.
Semiquantitative immunoblotting on urine membrane fractions found
urinary NHE-3 excretion to be a useful marker in discriminating between
control patients, those with prerenal azotemia, those with acute
glomerular disease, and those with ischemic/nephrotoxic ATN.
Sodium/Hydrogen Exchanger Isoform (NHE3) :

44. Calprotectin is a 24-kDa heterodimer composed of the two
monomers S100A8 (10,835 Da) and S100A9 (13,242Da) . It
was initially recognized as an antimicrobial protein in the
cytoplasm of neutrophil granulocytes.
Intracellular calprotectin's principal function is to associate
with the cytoskeleton, whereas when secreted by activated
immune cells, it serves as a danger-associated molecular
pattern protein .
Calprotectin

45. Elevation of calprotectin Levels is reported in a few
medical conditions, including rheumatoid arthritis
inflammatory bowel disease ,myocardial infarction ,
urothelial carcinoma , prostate cancer ,and others . In
addition, since calprotectin is predominantly derived from
neutrophils and monocytes, pyuria can substantially
increase urine calprotectin. Thus, elevated urine
calprotectin levels need to be cautiously interpreted .

46. The detection of proteins, especially enzymes, released from
damaged proximal and/or distal tubular cells has also been used as
a biomarker of AKI.
Glutathione S-transferase isomers are cytoplasmic enzymes found
in proximal and distal tubular cells. In urine, these enzymes are
normally not present.
NAG is a lysosomal enzyme found mostly in proximal tubules.
Alkaline phosphatase (AP), γ-glutamyl transpeptidase (γGT), and ala-
(leu-gly)-aminopeptidase are brush border enzymes that increase in
urine in cases of tubular injury with loss of micovillous structure .
Tubular enzymuria may be very sensitive to tubular injury from
multiple causes ,e.g tubulointerstitial nephritis , chronic
glomerulonephritis , and contrast nephropathy. Hemodialysis
exacerbates tubular enzymuria in patients with AKI.
Tubular Enzymes

47. Is a lysosomal enzyme (>130 kDa) that is localized in the proximal renal
tubules. Due to its large molecular weight, it precludes glomerular
filtration, implying that urinary elevations have a tubular origin.
Increased activity suggests injury to its cells but may also reflect
increased lysosomal activity without cell disruption. NAG catalyses the
hydrolysis of terminal glucose residues in glycoproteins . Has proven
to be a sensitive, persistent, and robust indicator of tubular injury.
Increased NAG levels have been reported with nephrotoxicant
exposure , delayed renal allograft function, chronic glomerular disease,
diabetic nephropathy , as well as following cardiopulmonary bypass
(CPB ) procedures
N-acetyl-b-D-glucosaminidase: (NAG)

48. The two advantages of using NAG are:
a) sensitivity, subtle alterations in the epithelial cells in the brush border of
the proximal tubules result in shedding of NAG into the urine and the
amount of shed enzyme can be directly correlated to tubular injury; and
b) quantitation, simple and reproducible enzymatic assays are well
established to measure the analyte colorimetrically using a
spectrophotometer.
Nonspecificity may limit the use of NAG levels as a biomarker of AKI.

49. Butler and Flynn in 1961 studied the urine proteins of 223 individuals by
starch gel electrophoresis and found a new urine protein fraction in the post
γ-globulin fraction. This protein was named cystatin C. Cystatin C is a 13-kd
protein produced by all nucleated cells. It is a polypeptide chain with 120
amino acid residues. It is freely filtered by the glomerulus, completely
reabsorbed by the proximal tubules, and is not secreted by the renal tubules.
The urinary excretion of CyC (uCyC) specifically reflects tubular damage
because systemically produced cystatin C is normally not found in urine
Low molecular weight proteins : Cystatin C

50. The measurement of serum Cys-C before 1994 was performed by
using an enzyme amplified single radial immunodiffusion
technique that required at least 10−20 h and had a relatively high
coefficient of variation (>10%). Subsequently, automated rapid
particle enhanced immunoturbidimetric and immunonephelometric
methods were developed that were more precise and were thus
approved by the FDA . Recently, an automatic quantitative assay to
measure urinary Cys-C has also been developed using an N-Latex
Cystatin-C kit with a nephelometer

51. β2-microglobulin (β2M) is an 11.8-kDa protein that is the light
chain of the major histocompatibility class (MHC) I molecule
expressed on the cell surface of all nucleated cells.
β2M dissociates from the heavy chain in the setting of cellular
turnover and enters the circulation as a monomer. β2M is
typically filtered by the glomerulus and almost entirely
reabsorbed and catabolized by the proximal tubular cells , a
process that may be impeded in AKI.
β2-microglobulin

52. Increased urinary β2M excretion has been observed to
be an early marker of tubular injury in a number of
settings, including nephrotoxicant exposure, cardiac
surgery and renal transplantation preceding rises in
serum creatinine by as many as 4−5 days
Unfortunately, the utility of β2M as a biomarker has been
limited by its instability in urine, with rapid degradation
observed at room temperature and in urine with a pH less
than 6.0

53. α1-microglobulin (α1M) is a 27−33-kDa protein synthesized by the
liver with approximately half of the circulating protein complexed to
IgA. The free form is readily filtered by the glomerulus and
reabsorbed by proximal tubule cells. Unlike β2M, urinary α1M is
stable over the range of pH found in routine clinical practice,
making it a preferred marker of tubular proteinuria in human
bioassays . It has been found to be a sensitive biomarker for
proximal tubular dysfunction even in the early phase of injury when
no histologic damage is observable .
α1-microglobulin

54. A new area of study involves evaluation of the value of microRNAs,
endogenous and noncoding RNA molecules containing 18 to 22
nucleotides, in AKI .These short strands of RNA regulate gene
expression by inhibiting protein translation. In cardiac surgery
populations, it has been shown that both urine and plasma miR-21
concentrations, which orchestrated a microRNA-controlled apoptosis of
renal tubular epithelial cells and promoted cellular proliferation in
response to renal ischemia-reperfusion injury ,may be helpful in
detection of AKI .
Urine microRNA

55. Microalbuminuria, defined as the pathologic excretion of urinary
albumin at levels (30 to 300 mg/L) below the threshold of detection by
conventional urinary dipstick, has long been established as a useful
marker of the development and progression of renal disease,
particularly diabetic nephropathy. Historically, microalbuminuria has
been assumed to result from alterations in glomerular filtration
secondary to changes in intraglomerular pressure and/or structural
changes of the podocyte or glomerular basement membrane.
Microalbuminuria, however, may also be caused by vigorous exercise,
hematuria, urinary tract infection, and dehydration.
Microalbumin

56. Retinol binding protein (RBP) is a 21-kDa protein that is hepatically
synthesized and responsible for transporting vitamin A from the
liver to other tissues. It is freely filtered by the glomerulus and
subsequently reabsorbed and catabolized by the proximal tubule.
Increased RBP levels during the first two days of life were
predictive of clinically significant AKI in infants following birth
asphyxia, a setting where interpretation of serum creatinine is
particularly problematic as it reflects maternal serum concentration
to a significant extent.
Retinol Binding Protein

57. The utility of low-molecular-weight filtered
proteins, such as RBP, β2M, α1M, Cystatin C, and
Microalbuminria , as biomarkers in the setting of
AKI is limited by concomitant significant
glomerular proteinuria or hyperfiltration, situations
where the tubular reabsorptive pathways may be
saturated .

58. AGT is a 453-amino-acid-long protein with 10 N-terminal amino acids that
are cleavable by renin, leading to the formation of angiotensin I .
Angiotensin I is further converted to angiotensin II by angiotensin-
converting enzyme and exerts its robust biologic effects .
In healthy kidneys, circulating AGT should not be filtered into the urine in
any significant amounts because its molecular size is 65 kDa .
Furthermore, efficient tubular reabsorption or degradation of any filtered
AGT could preclude it from appearing in the urine. Urine AGT arises
principally from local kidney sources .Urinary AGT outperformed 3 AKI
biomarkers, including NGAL, IL-18, and KIM-1, in the prediction of AKI
progression.
Urine angiotensinogen

61. The classical biomarker paradigm is that one test detects
one disease, eg, troponin for acute myocardial infarction.
However, AKI is a complex disease with multiple causes, and
it is possible that one biomarker will not be sufficient to
make an early diagnosis. Thus, a panel of biomarkers may
be necessary in AKI.
Both urinary NGAL and IL-18 were measured in children who
developed AKI after cardio –pulmonary bypass (CPB).
Panels of AKI Biomarkers

62. NGAL increased 25-fold within 2 hours and declined within 6
hours after surgery. In contrast, urine IL-18 increased at 4 to 6
hours after CPB, peaked at over 25-fold at 12 hours, and
remained markedly elevated up to 48 hours after CPB. Both
IL-18 and NGAL were independently associated with number
of days in AKI among cases . The combination of these 2
biomarkers may allow for the reliable early diagnosis and
prognosis of AKI at all times after CPB, much before the rise
in serum creatinine..

63. In children undergoing cardiopulmonary bypass (CPB )
surgery ,combind measurement of urinary levels of matrix
metalloproteinase-9, NAG, and KIM-1 achieved a perfect
score for diagnosis of AKI, as determined by the area under
the ROC curve.
AKI was defined as a greater than 50% increase in the serum
creatinine within the first 48 hours after surgery.

64. The traditional method to quantitate urinary enzymes has
been enzyme-substrate-based colorimetric assays followed
by measurement using a spectrophotometer. As urinary
proteins were identified as potential biomarkers of AKI,
however, the assay of choice became ELISA, which is
based on the detection of an antigen using two epitopically
distinct antibodies.
METHODS TO QUANTITATE BIOMARKERS

65. There are, however, multiple disadvantages of the ELISA assay:
(a) Only one antigen can be detected in one plate;
(b) the dynamic range of the assay is usually low, requiring
repeat measurements with dilution or concentration of urine
samples so that the antigen concentration will fit in to the linear
range of the standard curve;
(c) the urine sample volume requirement is at least 200 μl per
assay (100 μl in duplicate); and
(d ) at least 5−7 h are required to get the results.

66. A recent technique that is an adaptation of an ELISA,
using principles similar to a flow cytometer, is a particle-
based flow cytometric assay developed by Luminex®
that uses a microfluidics platform incorporating 5.6-μm
beads coupled with the primary (capture) antibody . Each
microsphere is labeled with a precise ratio of red- and
orange-emitting fluorochromes, giving it a unique
spectral signature.

67. This technique has also been used to measure beta-amyloid,
total tau (T-TAU), and hyperphosphorylated tau (PTAU) in
cerebrospinal fluid as biomarkers for Alzheimer's disease.
Both the ELISA and the microbead-based assay require two
epitopically distinct, high-affinity antibodies
Researchers have thus far successfully used this technology
to quantitate 1 5−18 different analytes in the same biological
sample.

68. A multiplexed electrical detection nanowire sensor array was used to measure some of
the AKI markers ,the same technique was also used to detect prostate-specific antigen
(PSA), PSA-α1-antichymotrypsin, carcinoembryonic antigen, and mucin-1 in serum for
the diagnosis of prostate cancer .
This technology is in its initial stages and requires additional evaluation and
validation, but this general approach offers significant advantages over the existing
technology:
(a) its sensitivity is in the femtomolar range,
(b) it requires only one antibody as opposed to two epitopically distinct antibodies;
(c) it produces a read-out within minutes as opposed to hours; and
(d ) it can be adapted as a bedside technology for patient care in hospitals.

70. Anti-inflammatory agents
b1 Integrin antagonist, adenosine receptor
antagonist, mesenchymal stem cells, C5a
receptor antagonist, IL-10, IL-6 antagonist,
statins, erythropoietin, a melanocyte stimulating
hormone, haeme oxygenase-1 inducers
(rapamycin), activated protein C, toll like receptor
Anti-apoptotic agents
NGAL, adenosine receptor antagonist,
mesenchymal stem cells, erythropoietin,
a-melanocyte stimulating hormone, caspase
inhibitors, minocycline, guanosine, pifithrin-a,
poly ADP ribose polymerase (PARP) inhibitor
Therapeutic agents for the treatment of AKI

71. Iron scavengers
NGAL, apotransferrin, deferoxamine
Reactive oxygen species scavengers
Anti-oxidants Edavarone, stobadine, deferoxamine
Vasodilators Endothelin receptor antagonist, CO-releasing
compounds, fenoldopam, anti natriuretic peptide
Growth factors Erytropoetin, hepatocyte growth factor

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