Hsp72 is an early and sensitive biomarker to detect acute kidney injury

Jonatan Barrera-Chimal, Rosalba Pérez-Villalva, Cesar Cortés-González, Marcos Ojeda-Cervantes, Gerardo Gamba, Luis E Morales-Buenrostro, Norma A Bobadilla, Jonatan Barrera-Chimal, Rosalba Pérez-Villalva, Cesar Cortés-González, Marcos Ojeda-Cervantes, Gerardo Gamba, Luis E Morales-Buenrostro, Norma A Bobadilla

Abstract

This study was designed to assess whether heat shock protein Hsp72 is an early and sensitive biomarker of acute kidney injury (AKI) as well as to monitor a renoprotective strategy. Seventy-two Wistar rats were divided into six groups: sham-operated and rats subjected to 10, 20, 30, 45 and 60 min of bilateral ischemia (I) and 24 h of reperfusion (R). Different times of reperfusion (3, 6, 9, 12, 18, 24, 48, 72, 96 and 120 h) were also evaluated in 30 other rats subjected to 30 min of ischemia. Hsp72 messenger RNA (mRNA) and protein levels were determined in both kidney and urine. Hsp72-specificity as a biomarker to assess the success of a renoprotective intervention was evaluated in rats treated with different doses of spironolactone before I/R. Renal Hsp72 mRNA and protein, as well as urinary Hsp72 levels, gradually increased relative to the extent of renal injury induced by different periods of ischemia quantified by histomorphometry as a benchmark of kidney damage. Urinary Hsp72 increased significantly after 3 h and continued rising until 18 h, followed by restoration after 120 h of reperfusion in accord with histopathological findings. Spironolactone renoprotection was associated with normalization of urinary Hsp72 levels. Accordingly, urinary Hsp72 was significantly increased in patients with clinical AKI before serum creatinine elevation. Our results show that urinary Hsp72 is a useful biomarker for early detection and stratification of AKI. In addition, urinary Hsp72 levels are sensitive enough to monitor therapeutic interventions and the degree of tubular recovery following an I/R insult.

Copyright © 2011 EMBO Molecular Medicine.

Figures

Figure 1. Renal functional parameters in rats…
Figure 1. Renal functional parameters in rats underwent which different periods of bilateral renal ischemia (10, 20, 30, 45, and 60 min) and 24 h of reperfusion compared to sham-operated rats (white bars)

  1. Serum creatinine levels.

  2. Creatinine clearance.

  3. Renal blood flow.

  4. Mean arterial pressure.

  5. Urinary NAG excretion.

  6. Protein excretion.

n = 6, *p < 0.05 versus sham-operated rats and ¥p < 0.05 versus 45 min I/R group.
Figure 2. Representative images and morphometry of…
Figure 2. Representative images and morphometry of subcortical histopathological lesions induced by different periods of ischemia and 24 h of reperfusion

  1. Sham-operated rats.

  2. 10 min.

  3. 20 min.

  4. 30 min.

  5. 45 min.

  6. 60 min.

  7. Mean cast number per field.

  8. Percentage of tubular affected area.

*p < 0.05 versus sham-operated rats, ¤p < 0.05 versus 10 min, €p < 0.05 versus 20 min, £p < 0.05 versus 30 min and ¥p < 0.05 versus 45 min of ischemia group.
Figure 3. Renal Hsp72 expression in rats…
Figure 3. Renal Hsp72 expression in rats subjected to different periods of ischemia

  1. Total RNA was individually extracted from the renal cortex of all studied groups (n = 5) and Hsp72 mRNA levels were determined by real time RT-PCR.

  2. Renal cortex proteins were individually extracted from three rats of each group, and Hsp72 protein levels were assessed by western blot. Upper inset shows a representative image of the autoradiography of the membrane and the lower graph depicts densitometric analyses of the ratio of Hsp72 to β-actin. *p < 0.05 versus sham-operated rats, £p < 0.05 versus 30 min and ¥p < 0.05 versus 45 min of ischemia group.

Figure 4. Urinary Hsp72 mRNA and protein…
Figure 4. Urinary Hsp72 mRNA and protein levels from rats subjected to different periods of ischemia

  1. Total RNA was individually extracted from the urine of six rats per group and Hsp72 mRNA levels were determined by real time RT-PCR.

  2. Relationship between mRNA levels and tubular affected area.

  3. Urinary Hsp72 levels assessed by ELISA.

  4. Relationship between urinary Hsp72 levels and the % of tubular affected area.

  5. Urinary Hsp72 levels assessed by WB analysis from four rats of each group.

  6. Relationship between urinary Hsp72 levels detected by WB and tubular injured area. *p < 0.05 versus sham-operated rats, ¤p < 0.05 versus 10 min, €p < 0.05 versus 20 min, £p < 0.05 versus 30 min and ¥p < 0.05 versus 45 min of ischemia group.

Figure 5. Representative images and morphometry of…
Figure 5. Representative images and morphometry of subcortical histopathological lesions induced by 30 min of ischemia and different periods of reperfusion

  1. 3 h

  2. 6 h

  3. 9 h

  4. 12 h

  5. 18 h

  6. 24 h

  7. 48 h

  8. 72 h

  9. 96 h and

  10. 120 h of reperfusion.

  11. Morphometric quantification of affected tubular area.

  12. Mean cast number per field *p < 0.05 versus sham-operated rats.

Figure 6. Urinary Hsp72 levels in rats…
Figure 6. Urinary Hsp72 levels in rats which underwent to 30 min of ischemia with different periods of reperfusion

  1. Urinary Hsp72 levels assessed by ELISA.

  2. Relationship between urinary Hsp72 and % of tubular damaged area.

  3. Urinary Hp72 protein levels determined by Western blot.

  4. Relationship between Hsp72 and % of tubular injured area.

n = 3 per period of reperfusion, *p < 0.05 versus sham operated rats.
Figure 7. Urinary levels of Kim-1, NGAL…
Figure 7. Urinary levels of Kim-1, NGAL and IL-18 in rats which underwent different periods of ischemia and reperfusion (n = 6)

  1. A, B, C. Urinary levels of Kim-1, NGAL, and IL-18 in different degrees of renal injury induced by increasing periods of ischemia (10, 20, 30, 45 and 60 min), respectively.

  2. D, E, F. Urinary concentrations of Kim-1, NGAL and IL-18 after several times of reperfusion (3, 6, 9, 12, 18, 24, 48, 72, 96 and 120 h), respectively.

¤p < 0.05 versus 10 min, €p < 0.05 versus 20 min, £p < 0.05 versus 30 min and ¥p < 0.05 versus 45 min of ischemia group.
Figure 8. Urinary Hsp72 as a biomarker…
Figure 8. Urinary Hsp72 as a biomarker of renoprotection conferred by spironolactone in I/R rats (n = 5)

  1. Serum creatinine in I/R group without treatment (gray bar) and rats pre-treated with spironolactone (black bar),

  2. Creatinine clearance,

  3. Urinary Hsp72 assessed by ELISA and

  4. By WB analysis, the inset shows the individual analysis from five individual urines.

  5. Serum creatinine in I/R group and in rats pre-treated with lower doses of spironolactone (10, 5 and 2.5 mg/kg) and

  6. WB analysis of Hsp72 in rats pre-treated with lower doses of spironolactone, the superior inset shows the individual Hsp72 level from five different animals. *p < 0.05 versus I/R group.

Figure 9. Urinary Hsp72 levels as a…
Figure 9. Urinary Hsp72 levels as a biomarker of AKI in humans

  1. A, B. Hsp72 levels assessed by Western blot and ELISA, respectively, in five healthy kidney donors (▪) and nine patients that were diagnosed with AKI (▴).

  2. C. Serum creatinine from five patients with no evidence of AKI (○) or in five patients with AKI diagnosed with AKIN criteria (•).

  3. D. Urine output from five patients with no evidence of AKI (○) or in five patients with AKI diagnosed with AKIN criteria (•).

  4. E. Daily urinary Hsp72 levels from patients with diagnosed AKI.

  5. F. Daily urinary Hsp72 levels in patients without AKI.

The samples were collected during three days before and 5 days after AKI was diagnosed. *p < 0.05 versus 3 days before AKI.

References

    1. Bagshaw SM. Acute kidney injury: diagnosis and classification of AKI: AKIN or RIFLE. Nat Rev Nephrol. 2010;6:71–73.
    1. Coca SG, Parikh CR. Urinary biomarkers for acute kidney injury: perspectives on translation. Clin J Am Soc Nephrol. 2008;3:481–490.
    1. Csermely P, Soti C, Blatch GL. Chaperones as parts of cellular networks. Adv Exp Med Biol. 2007;594:55–63.
    1. Devarajan P. Update on mechanisms of ischemic acute kidney injury. J Am Soc Nephrol. 2006;17:1503–1520.
    1. Dieterle F, Perentes E, Cordier A, Roth DR, Verdes P, Grenet O, Pantano S, Moulin P, Wahl D, Mahl, et al. Urinary clusterin, cystatin C, beta2-microglobulin and total protein as markers to detect drug-induced kidney injury. Nat Biotechnol. 2010;28:463–469.
    1. Dieterle F, Sistare F, Goodsaid F, Papaluca M, Ozer JS, Webb CP, Baer W, Senagore A, Schipper MJ, Vonderscher J, et al. Renal biomarker qualification submission: a dialog between the FDA-EMEA and Predictive Safety Testing Consortium. Nat Biotechnol. 2010;28:455–462.
    1. Fan CY, Lee S, Cyr DM. Mechanisms for regulation of Hsp70 function by Hsp40 cell stress. Chaperones. 2003;8:309–316.
    1. Ferguson MA, Vaidya VS, Waikar SS, Collings FB, Sunderland KE, Gioules CJ, Bonventre JV. Urinary liver-type fatty acid-binding protein predicts adverse outcomes in acute kidney injury. Kidney Int. 2010;77:708–714.
    1. Friedewald JJ, Rabb H. Inflammatory cells in ischemic acute renal failure. Kidney Int. 2004;66:486–491.
    1. Han WK, Wagener G, Zhu Y, Wang S, Lee HT. Urinary biomarkers in the early detection of acute kidney injury after cardiac surgery. Clin J Am Soc Nephrol. 2009;4:873–882.
    1. Hernadez-Pando R, Pedraza-Chaverri J, Orozco-Estevez H, Silva-Serna P, Moreno I, Rondan-Zarate A, Elinos M, Correa-Rotter R, Larriva-Sahd J. Histological and subcellular distribution of 65 and 70 kD heat shock proteins in experimental nephrotoxic injury. Exp Toxicol Pathol. 1995;47:501–508.
    1. Kelly KJ. Stress response proteins and renal ischemia. Minerva Urol Nefrol. 2002;54:81–91.
    1. Kelly KJ. Acute renal failure: much more than a kidney disease. Semin Nephrol. 2006;26:105–113.
    1. Kelly KJ, Baird NR, Greene AL. Induction of stress response proteins and experimental renal ischemia/reperfusion. Kidney Int. 2001;59:1798–1802.
    1. Liangos O, Perianayagam MC, Vaidya VS, Han WK, Wald R, Tighiouart H, MacKinnon RW, Li L, Balakrishnan VS, Pereira BJ, et al. Urinary N-acetyl-beta-(D)-glucosaminidase activity and kidney injury molecule-1 level are associated with adverse outcomes in acute renal failure. J AmSoc Nephrol. 2007;18:904–912.
    1. Liano F, Pascual J. Epidemiology of acute renal failure: a prospective, multicenter, community-based study. Madrid Acute Renal Failure Study Group. Kidney Int. 1996;50:811–818.
    1. Lindquist S, Craig EA. The heat-shock proteins. Annu Rev Genet. 1988;22:631–677.
    1. Liu KD, Brakeman PR. Renal repair and recovery. Crit Care Med. 2008;36:S187–S192.
    1. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method Methods. 2001;25:402–408.
    1. Lopes JA, Fernandes P, Jorge S, Goncalves S, Alvarez A, Costa e S, Franca C, Prata MM. Acute kidney injury in intensive care unit patients: a comparison between the RIFLE and the acute kidney injury network classifications. Crit Care. 2008;12:R110.
    1. McIlroy DR, Wagener G, Lee HT. Neutrophil gelatinase-associated lipocalin and acute kidney injury after cardiac surgery: the effect of baseline renal function on diagnostic performance. Clin J Am Soc Nephrol. 2010;5:211–219.
    1. Mehta RL, Kellum JA, Shah SV, Molitoris BA, Ronco C, Warnock DG, Levin A. Acute kidney injury network: report of an initiative to improve outcomes in acute kidney injury. Crit Care. 2007;11:R31.
    1. Mejia-Vilet JM, Ramirez V, Cruz C, Uribe N, Gamba G, Bobadilla NA. Renal ischemia-reperfusion injury is prevented by the mineralocorticoid receptor blocker spironolactone. Am J Physiol Renal Physiol. 2007;293:F78–F86.
    1. Melnikov VY, Ecder T, Fantuzzi G, Siegmund B, Lucia MS, Dinarello CA, Schrier RW, Edelstein CL. Impaired IL-18 processing protects caspase-1-deficient mice from ischemic acute renal failure. J Clin Invest. 2001;107:1145–1152.
    1. Mishra J, Ma Q, Prada A, Mitsnefes M, Zahedi K, Yang J, Barasch J, Devarajan P. Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol. 2003;14:2534–2543.
    1. Mishra J, Dent C, Tarabishi R, Mitsnefes MM, Ma Q, Kelly C, Ruff SM, Zahedi K, Shao M, Bean J, et al. Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery. Lancet. 2005;365:1231–1238.
    1. Molinas SM, Rosso M, Wayllace NZ, Pagotto MA, Pisani GB, Monasterolo LA, Trumper L. Heat shock protein 70 induction and its urinary excretion in a model of acetaminophen nephrotoxicity. Pediatr Nephrol. 2010;25:1245–1253.
    1. Mueller T, Bidmon B, Pichler P, Arbeiter K, Ruffingshofer D, VanWhy SK, Aufricht C. Urinary heat shock protein-72 excretion in clinical and experimental renal ischemia. Pediatr Nephrol. 2003;18:97–99.
    1. Negishi K, Noiri E, Doi K, Maeda-Mamiya R, Sugaya T, Portilla D, Fujita T. Monitoring of urinary L-type fatty acid-binding protein predicts histological severity of acute kidney injury. Am J Pathol. 2009;174:1154–1159.
    1. Nejat M, Pickering JW, Walker RJ, Endre ZH. Rapid detection of acute kidney injury by plasma cystatin C in the intensive care unit. Nephrol Dial Transplant. 2010;25:3283–3289.
    1. Parikh CR, Abraham E, Ancukiewicz M, Edelstein CL. Urine IL-18 is an early diagnostic marker for acute kidney injury and predicts mortality in the intensive care unit. J Am Soc Nephrol. 2005;16:3046–3052.
    1. Parikh CR, Mishra J, Thiessen-Philbrook H, Dursun B, Ma Q, Kelly C, Dent C, Devarajan P, Edelstein CL. Urinary IL-18 is an early predictive biomarker of acute kidney injury after cardiac surgery. Kidney Int. 2006;70:199–203.
    1. Price PM, Safirstein RL, Megyesi J. The cell cycle and acute kidney injury. Kidney Int. 2009;76:604–613.
    1. Ramirez V, Trujillo J, Valdes R, Uribe N, Cruz C, Gamba G, Bobadilla NA. Adrenalectomy prevents renal ischemia-reperfusion injury. Am J Physiol Renal Physiol. 2009;297:F932–F942.
    1. Ritossa FA. New puffing pattern induced by temperature shoch and DNP in Drosophila. Experientia. 1962;18:571–573.
    1. Taman M, Liu Y, Tolbert E, Dworkin LD. Increase urinary hepatocyte growth factor excretion in human acute renal failure. Clin Nephrol. 1997;48:241–245.
    1. Tsutsumi S, Neckers L. Extracellular heat shock protein 90: a role for a molecular chaperone in cell motility and cancer metastasis. Cancer Sci. 2007;98:1536–1539.
    1. Turman MA, Rosenfeld SL. Heat shock protein 70 overexpression protects LLC-PK1 tubular cells from heat shock but not hypoxia. Kidney Int. 1999;55:189–197.
    1. Uchida K, Gotoh A. Measurement of cystatin-C and creatinine in urine. Clin Chim Acta. 2002;323:121–128.
    1. Vaidya VS, Ramirez V, Ichimura T, Bobadilla NA, Bonventre JV. Urinary kidney injury molecule-1: a sensitive quantitative biomarker for early detection of kidney tubular injury. Am J Physiol Renal Physiol. 2006;290:F517–F529.
    1. Vaidya VS, Waikar SS, Ferguson MA, Collings FB, Sunderland K, Gioules C, Bradwin G, Matsouaka R, Betensky RA, Curhan GC, et al. Urinary biomarkers for sensitive and specific detection of acute kidney injury in humans. Clin Transl Sci. 2008;1:200–208.
    1. Vaidya VS, Ford GM, Waikar SS, Wang Y, Clement MB, Ramirez V, Glaab WE, Troth SP, Sistare FD, Prozialeck WC, et al. A rapid urine test for early detection of kidney injury. Kidney Int. 2009;76:108–114.
    1. Vaidya VS, Ozer JS, Dieterle F, Collings FB, Ramirez V, Troth S, Muniappa N, Thudium D, Gerhold D, Holder DJ, Bobadilla NA, Marrer E, et al. Kidney injury molecule-1 outperforms traditional biomarkers of kidney injury in preclinical biomarker qualification studies. Nat Biotechnol. 2010;28:478–485.
    1. Wagener G, Jan M, Kim M, Mori K, Barasch JM, Sladen RN, Lee HT. Association between increases in urinary neutrophil gelatinase-associated lipocalin and acute renal dysfunction after adult cardiac surgery. Anesthesiology. 2006;105:485–491.
    1. Waikar SS, Curhan GC, Wald R, McCarthy EP, Chertow GM. Declining mortality in patients with acute renal failure, 1988 to 2002. J Am Soc Nephrol. 2006;17:1143–1150.
    1. Wu I, Parikh CR. Screening for kidney diseases: older measures versus novel biomarkers. Clin J Am Soc Nephrol. 2008;3:1895–1901.
    1. Yamamoto T, Noiri E, Ono Y, Doi K, Negishi K, Kamijo A, Kimura K, Fujita T, Kinukawa T, Taniguchi H, et al. Renal L-type fatty acid-binding protein in acute ischemic injury. J Am Soc Nephrol. 2007;18:2894–2902.
    1. Yu Y, Jin H, Holder D, Ozer JS, Villarreal S, Shughrue P, Shi S, Figueroa DJ, Clouse H, Su M, et al. Urinary biomarkers trefoil factor 3 and albumin enable early detection of kidney tubular injury. Nat Biotechnol. 2010;28:470–477.
    1. Zhipeng W, Li L, Qibing M, Linna L, Yuhua R, Rong Z. Increased expression of heat shock protein (HSP)72 in a human proximal tubular cell line (HK-2) with gentamicin-induced injury. J Toxicol Sci. 2006;31:61–70.

Source: PubMed

Sponsors en medewerkers

Medische omstandigheden

Geneesmiddelinterventies

3
Abonneren