Introduction

The Biotrin SMARTASSAYS Nephrotoxicity biomarkers comprise a series of biomarkers that are localized to specific cell types in the nephron. They are readily release upon injury making it possible to localise injury to distinct parts of the nephron. The Biotrin SMARTASSAYS nephrotoxicity biomarker comprise biomarkers for the proximal tubules, distal tubules, loop of Henle and collecting ducts, enabling a broad picture of renal injury to be obtained from a simple urine sample. The Biotrin SMARTASSAYS Nephrotoxicity provide information, earlier and at lower doses than traditional biochemical tests, e.g., serum creatinine. The Biotrin SMARTASAAYS Nephrotoxicity Biomarkers comprise alpha GST, Pi GST/GSTYb1 and RPA-1 EIAs and RPA-1 and RPA-2 antisera.

Glutathione S-transferases and Nephrotoxicity Testing

Glutathione S-transferases (GSTs) comprise a multigene family of proteins found in high concentrations in the cells of the renal tubules (up to 3% of soluble protein) (1). They are readily released into the urine during injury and are excellent biomarkers for renal injury resulting from, for example, nephrotoxicity (2), ischaemic injury (3), transplant rejection (2), proteinuria (4) and diabetic renal tubular injury (5). Different GST subclasses are localised to specific parts of the renal tubule (alpha GST in the proximal tubule and pi GST in the distal) and their appearance in urine is an early indicator of the site and extent of renal injury. By the simultaneous assay of different GST subclasses, different sections of the nephron can be simultaneously and independently monitored. Biotrin Urinary GST EIA kits are available for in invitro (6), preclinical (7-8) and human studies (9)

The renal tubules are affected by many nephrotoxins and GSTs are sensitive and specific biomarkers for injury to them. By monitoring the release of GSTs following exposure to potential nephrotoxins the site of injury and extent of injury can be identified. Commonly used tests for nephrotoxicity, e.g., serum creatinine and BUN may be inappropriate tests for nephrotoxicity monitoring, as they reflect compromised glomerular function, while most nephrotoxins tend to affect the renal tubules. Similarly, proteinuria may reflect decreased or saturated resorbtive capacity. However, the presence of GSTs in the urine indicates that the renal tubular cells themselves are being injured. Monitoring Proximal Tubular InjuryAlpha GST is a very accurate biomarker for proximal tubular damage.

For example:

• Bass et al. showed that the temporal release of alpha GST was closely related to the time course of necrosis (8).
• Kharasch et al. demonstrated a close relationship between the amount of alpha GST released and the extent of tubular necrosis (7).
• Goldberg et al showed a close correlation between the amount of alpha GST released and the dose of toxin (9).

Monitoring Distal Tubular Injury:

Urinary pi GST and GSTYb1 are specific urinary biomarkers for distal tubular effects in humans and rats respectively.

• Pi GST release has been demonstrated following nephrotoxicity resulting from, for example, Amphotericin B therapy (10)
• Simultaneous monitoring of alpha and pi GST enables renal injury to be localised to distinct parts of the renal tubule.

PI GST YB1 GST

Renal Papillary Antigens 1 and 2 and Nephrotoxicity Testing

Renal Papillary Antigen 1 (RPA-1) is a protein specifically found in high concentrations in the cells of the collecting ducts of the rat kidney (11,12.) It is rapidly released into the urine in the event of injury to them. The collecting ducts and the loops of Henle are found in the renal papilla and they are damaged during injury to it. RPA-1 is a potentially very useful biomarker for the serious condition of Renal Papillary Necrosis (RPN). This leads initially to the loss of the ability to concentrate urine and eventually renal failure. Identifying papillary structures in renal biopsy material can be difficult but the Biotrin RPA-1 & 2 monoclonal antibodies (for the collecting duct and loop of Henle respectively) will enable them to be more easily identified by immunohistological procedures (11).

Monitoring Collecting Duct Injury / Renal Papillary Necrosis

Urinary RPA-1 has been shown to be sensitive biomarker of renal collecting duct injury due to type papillotoxins including bromoethanamine, propyleneimine and iodomethacin (12,13).

RPA -1

Monitoring Proximal Tubular Injury

αGST is a very accurate biomarker for proximal tubular damage. For example,

• Bass et al. showed that the temporal release of αGST was closely related to the time course of necrosis (8)
• Kharasch et al. demonstrated a close relationship between the amount of αGST released and the degree of tubular necrosi(7).
• Goldberg et al showed a close correlation between the amount of αGST released and the dose of toxin (9).

References:

1. Corrigal, A.V. et al. (1988). Glutathione S-transferase distribution and concentration in human organs. Biochem. Int. 16: 443-448.
2. Sundberg A.G.M. et al. (1994). Urinary pi class glutathione S-transferase as an indicator of tubular damage in the human kidney. Nephron 67: 308-316.
3. Daeman, J.W. H.C. et al. (1997). Glutathione S-transferase as predictor of functional outcome in transplantation of machine preserved non-heart-beating donor kidneys. Transplantation 63 (1):89-93.
4. Branten, A.W.J. et al. (2000). Urinary excretion of isoenzymes of glutathione S-transferase alpha and pi in patients with proteinuria. Reflection of the site of tubular injury. Nephron 85 120-126.
5. Maxwell P.R. et al. (2004). Differentiation between renal injury and compensatory responses by the use of specific biomarkers. Poster presented at the 43rd Annual meeting of the American Society of Toxicology, Baltimore March 21-25, 2004.
6. Vickers, A.E.M. (1994). Use of human organ slices to evaluate the biotransformation and drug-induced side-effects of pharmaceuticals. Cell Biology and Toxicology 10: 407-414.
7. Kharasch, E.D. et al. (1997). Role of renal cysteine conjugate ß-lyase pathway in inhaled compound A nephrotoxicity in rats. Anesthesiology 88(6): 1624-1633.
8. Bass N.M. et al. (1979). Radioimmunoassay measurement of urinary ligandin excretion in nephrotoxin treated rats. Clinical Science, 56: 419-426.
9. Goldberg, M.E. et al. (1999). Dose of compound A, not Sevoflurane, determines changes in the biochemical markers of renal injury in healthy volunteers. Anesthesia and Analgesia 88: 437-445. Toxicology and Applied Pharmacology 131:94-107.
10. Pai, M.P. et al (2005). Assessment of effective renal plasma flow, enzymuria, and cytokine release in healthy volunteers receiving a single dose of Amphotericin B desoxycholate. Antimicrob Agents Chemother. 49(9):3784-8.
11. Falkenberg, F. et al. (1996). Papillary antigens as markers of papillary toxicity. I Identification and characterisation of rat papillary antigens with monoclonal antibodies. Arch. Toxicol. 71, 80-92
12. Hildebrand, H. et al. (1999). Urinary antigens as markers of papillary toxicity. II Application of monoclonal antibodies for the determination of papillary antigens in rat urine. Arch. Toxicol. 73, 233-2
13. Kilty, C.G. et al (2006) Identification of renal papillary necrosis using an EIA for urinary Renal Papillary Antigen-1 (RPA-1), A new biomarker of collecting duct pathology. Poster presented at the 45th annual meeting of the American Society of Toxicology, San Diego, California