2. Why Nephrotoxicity ?
• Kidneys are very sensitive to toxicants since receive large
quantity of blood relative to body weight.
• Presence of drug metabolising enzymes. Extensive
oxidation, reduction, hydrolysis, and conjugation reactions
of xenobiotics can occur in the kidney.
(Lohr et al., 1998)
• Accumulation and concentration of chemicals because of
presence of plasma membrane transport proteins.
3. Some common Nephrotoxic drugs
• Aminoglycosides
• Amphoterecin B
• Isofosfamide
• Foscarnet
Source- Concise review: Current and emerging biomarkers of nephrotoxicity, Current Opinion in Toxicology, June
7. Isolated Perfused Kidney…
Advantages
• Well suited for studies requiring intact renal
morphology.
• Control over the perfusate composition is possible.
Limitations
• Studies are restricted for acute effects only.
• IPK model exhibits several functional abnormalities
which must be considered while evaluating results.
8. Micropuncture and microperfusion
techniques
• In-vivo technique.
• To study the function of individual nephron
segments in intact kidney.
• Drug of interest is directly applied to the renal
tubule in the renal perfusate and effect on
tubular function is determined.
9. Renal slices
• Slices are incubated in Dubnoff shaking bath with
various media/Buffers.
• Krebs-Ringer, Dulbecco’s Modified Eagle’s
Medium(DMEM), Waymouth’s culture medium,
Krebs-HEPES buffer etc., are used.
• Intracellular enzyme leakage, losses in tissue slice
K+ content are indicator of toxicity.
10. Isolated perfused tubules
• Used in transport physiology studies.
• To determine transepithelial transport of nephrotoxicants.
• Rabbit kidney is the choice since tubules are easier to i/d
and isolate without use of digestive enzymes.
• Perfusing and bathing solutions may be simple electrolyte
solutions supplemented with D-glucose,glutamine or
glutamate with pH 7.4.Ultrafilterate of rabbit plasma used
for good results.
11. Primary culture and Renal cell culture lines
• Isolation of tubular segments by perfusion of renal cortical tissues
with collagenase.
• Density gradient centrifugation in percoll or ficoll.
• Incubation
• Evaluation of cell viability by trypan blue exclusion, LDH leakage
assay.
• Addition of test drug to the medium.
• Identification of marker enzymes.
13. Drug induced Nephrotoxicity and its biomarkers
Source-Drug-Induced Nephrotoxicity and Its Biomarkers ;Journal of Biomolecules and Therapeutics
14. Serum Creatinine
• By-product of muscle metabolism that is excreted
unchanged by the kidneys primarily by
glomerular filtration.
• Elevated serum creatinine is due to reduction in
GFR.
• Late marker since observed only with marked
damage to functioning nephrons.
• Not a specific marker for nephrotoxicity.
15. Urinary proteins with enzymatic activity
• The enzymes present in tubular epithelial cells
are spilled into the urine and can be detected as
nephrotoxic biomarkers.
• Alanine aminopeptidase.
• Alkaline phosphatase.
• α-glutathione-S-transferase.
• γ-glutamyl transpeptidase.
• π-glutathione-S-transferase.
• N-acetyl-D-glucosaminidase.
16. Proteinuria
• In normal conditions, the glomerulus restricts the
migration of high molecular weight proteins from
blood to nephron lumen by filtration.
• High molecular weight proteins- albumin,
transferrin, immunoglobulin G.
• Low molecular weight proteins- β2 microglobuluin,
α1-microglobulin,retinol-binding protein,cystatinC.
17. Kidney injury molecule-1 (KIM-1)
• KIM-1 is a type I transmembrane glycoprotein expressed
on de-differentiated renal proximal tubule epithelial cells.
• Urinary KIM-1 is significantly upregulated in both rats and
human after therapy with known nephrotoxic drugs such
as aminoglycoside antibiotics or cisplatin.
• A comparison time course study showed that urinary
KIM-1 was elevated within 24 h after the administration
of nephrotoxicants in rats, whereas there were no
significant changes in SCr and BUN levels (Zhou et al.,
2010).
18. Neutrophil gelatinase-associated lipocalin (NGAL)
• one of the earliest and most robustly induced proteins
in the kidney after ischaemic or nephrotoxic AKI in
animal models.
• Sensitive biomarker for the early diagnosis of acute
kidney injury since NGAL expression is increased in
proximal tubule cells by drug-induced nephrotoxicity.
• Can be detected in both urine and blood samples
19. Cellular Necrosis
• LDH release Assay.
• Trypan blue uptake assay.
Apoptosis
• TUNEL (terminal deoxy nucleotidyl transferase
mediated dUTP-biotin nick end labelling) method.
• Flow cytometry methods.
20. References
• Drug-Induced Nephrotoxicity and Its Biomarkers, Sun Young
Kim and Aree Moon, 2012; Biomolecules and Therapeutics,.
• Neutrophil gelatinase-associated lipocalin (NGAL) A new marker
of kidney disease, Prasad Devarajan ; PMCID: PMC2528839.
• Principles and Methods for Renal Toxicology, Lawrence H. Lash;
Principles and Methods of Toxicology by a.Wallace Hayes.
• Cell- and biomarker-based assays for predicting nephrotoxicity,
Huang et al.,2014 ; Expert Opin. Drug Metab. Toxicol.
