Dana Hoffmann

Comparative analysis of novel noninvasive renal biomarkers and metabonomic changes in a rat model of gentamicin nephrotoxicity.

Although early detection of toxicant induced kidney injury during drug development and chemical safety testing is still limited by the lack of sensitive and reliable biomarkers of nephrotoxicity, omics technologies have brought enormous opportunities for improved detection of toxicity and biomarker discovery. Thus, transcription profiling has led to the identification of several candidate kidney biomarkers such as kidney injury molecule (Kim-1), clusterin, lipocalin-2, and tissue inhibitor of metalloproteinase 1 (Timp-1), and metabonomic analysis of urine is increasingly used to indicate biochemical perturbations due to renal toxicity. This study was designed to assess the value of a combined (1)H-NMR and gas chromatography-mass spectrometry (GC-MS) metabonomics approach and a set of novel urinary protein markers for early detection of nephrotoxicity following treatment of male Wistar rats with gentamicin (60 and 120 mg/kg bw, s.c.) for 7 days. Time- and dose-dependent separation of gentamicin-treated animals from controls was observed by principal component analysis of (1)H-NMR and GC-MS data. The major metabolic alterations responsible for group separation were linked to the gut microflora, thus related to the pharmacology of the drug, and increased glucose in urine of gentamicin-treated animals, consistent with damage to the S(1) and S(2) proximal tubules, the primary sites for glucose reabsorption. Altered excretion of urinary protein biomarkers Kim-1 and lipocalin-2, but not Timp-1 and clusterin, was detected before marked changes in clinical chemistry parameters were evident. The early increase in urine, which correlated with enhanced gene and protein expression at the site of injury, provides further support for lipocalin-2 and Kim-1 as sensitive, noninvasive biomarkers of nephrotoxicity.

Expression, Circulation, and Excretion Profile of MicroRNA-21, -155, and -18a Following Acute Kidney Injury

MicroRNAs (miRNAs) are endogenous noncoding RNA molecules that are involved in post-transcriptional gene silencing. Using global miRNA expression profiling, we found miR-21, -155, and 18a to be highly upregulated in rat kidneys following tubular injury induced by ischemia/reperfusion (I/R) or gentamicin administration. Mir-21 and -155 also showed decreased expression patterns in blood and urinary supernatants in both models of kidney injury. Furthermore, urinary levels of miR-21 increased 1.2-fold in patients with clinical diagnosis of acute kidney injury (AKI) (n = 22) as compared with healthy volunteers (n = 25) (p < 0.05), and miR-155 decreased 1.5-fold in patients with AKI (p < 0.01). We identified 29 messenger RNA core targets of these 3 miRNAs using the context likelihood of relatedness algorithm and found these predicted gene targets to be highly enriched for genes associated with apoptosis or cell proliferation. Taken together, these results suggest that miRNA-21 and -155 could potentially serve as translational biomarkers for detection of AKI and may play a critical role in the pathogenesis of kidney injury and tissue repair process.

Evaluation of Putative Biomarkers of Nephrotoxicity after Exposure to Ochratoxin A In Vivo and In Vitro

The kidney is one of the main targets of xenobiotic-induced toxicity, but early detection of renal damage is difficult. Recently, several novel biomarkers of nephrotoxicity have been identified by transcription profiling, including kidney injury molecule-1 (Kim-1), lipocalin-2, tissue inhibitor of metalloproteinases-1 (Timp-1), clusterin, osteopontin (OPN), and vimentin, and suggested as sensitive endpoints for acute kidney injury in vivo. However, it is not known if these cellular marker molecules may also be useful to predict chronic nephrotoxicity or to detect nephrotoxic effects in vitro. In this study, a panel of new biomarkers of renal toxicity was assessed via quantitative real-time PCR, immunohistochemistry, and immunoblotting in rats treated with the nephrotoxin ochratoxin A (OTA) for up to 90 days and in rat proximal tubule cells (NRK-52E) treated with OTA in vitro. Repeated administration of OTA to male F344/N rats for 14, 28, or 90 days resulted in a dose- and time-dependent increase in the expression of Kim-1, Timp-1, lipocalin-2, OPN, clusterin, and vimentin. Changes in gene expression were found to correlate with the progressive histopathological alterations and preceded effects on traditional clinical parameters indicative of impaired kidney function. Induction of Kim-1 messenger RNA expression was the earliest and most prominent response observed, supporting the use of this marker as sensitive indicator of chronic kidney injury. In contrast, no significant increase in the expression of putative marker genes and proteins were evident in NRK-52E cells after exposure to OTA for up to 48 h, suggesting that they may not be suitable endpoints for sensitive detection of nephrotoxic effects in vitro.

Performance of Novel Kidney Biomarkers in Preclinical Toxicity Studies

The kidney is one of the main targets of drug toxicity, but early detection of renal damage is often difficult. As part of the InnoMed PredTox project, a collaborative effort aimed at assessing the value of combining omics technologies with conventional toxicology methods for improved preclinical safety assessment, we evaluated the performance of a panel of novel kidney biomarkers in preclinical toxicity studies. Rats were treated with a reference nephrotoxin or one of several proprietary compounds that were dropped from drug development in part due to renal toxicity. Animals were dosed at two dose levels for 1, 3, and 14 days. Putative kidney markers, including kidney injury molecule-1 (Kim-1), lipocalin-2 (Lcn2), clusterin, and tissue inhibitor of metalloproteinases-1, were analyzed in kidney and urine using quantitative real-time PCR, ELISA, and immunohistochemistry. Changes in gene/protein expression generally correlated well with renal histopathological alterations and were frequently detected at earlier time points or at lower doses than the traditional clinical parameters blood urea nitrogen and serum creatinine. Urinary Kim-1 and clusterin reflected changes in gene/protein expression and histopathological alterations in the target organ in the absence of functional changes. This confirms clusterin and Kim-1 as early and sensitive, noninvasive markers of renal injury. Although Lcn2 did not appear to be specific for kidney toxicity, its rapid response to inflammation and tissue damage in general may suggest its utility in routine toxicity testing.

Integrated transcriptomic and proteomic evaluation of gentamicin nephrotoxicity in rats

Gentamicin is an aminoglycoside antibiotic, which induces renal tubular necrosis in rats. In the context of the European InnoMed PredTox project, transcriptomic and proteomic studies were performed to provide new insights into the molecular mechanisms of gentamicin-induced nephrotoxicity. Male Wistar rats were treated with 25 and 75 mg/kg/day subcutaneously for 1, 3 and 14 days. Histopathology observations showed mild tubular degeneration/necrosis and regeneration and moderate mononuclear cell infiltrate after long-term treatment. Transcriptomic data indicated a strong treatment-related gene expression modulation in kidney and blood cells at the high dose after 14 days of treatment, with the regulation of 463 and 3241 genes, respectively. Of note, the induction of NF-kappa B pathway via the p38 MAPK cascade in the kidney, together with the activation of T-cell receptor signaling in blood cells were suggestive of inflammatory processes in relation with the recruitment of mononuclear cells in the kidney. Proteomic results showed a regulation of 163 proteins in kidney at the high dose after 14 days of treatment. These protein modulations were suggestive of a mitochondrial dysfunction with impairment of cellular energy production, induction of oxidative stress, an effect on protein biosynthesis and on cellular assembly and organization. Proteomic results also provided clues for potential nephrotoxicity biomarkers such as AGAT and PRBP4 which were strongly modulated in the kidney. Transcriptomic and proteomic data turned out to be complementary and their integration gave a more comprehensive insight into the putative mode of nephrotoxicity of gentamicin which was in accordance with histopathological findings.

Assessment of candidate biomarkers of drug-induced hepatobiliary injury in preclinical toxicity studies.

This study was designed to assess the value of a set of potential markers for improved detection of liver injury in preclinical toxicity studies. Male Wistar rats were treated with drug candidates (BAY16, EMD335823, BI-3) that previously failed during development, in part due to hepatotoxicity, at two dose levels for 1, 3 and 14 days. Concentrations of lipocalin-2/NGAL and clusterin, which are frequently overexpressed and released from damaged tissues, and thiostatin, recently identified within PredTox as being elevated in urine in response to liver injury, were determined in rat urine and serum by ELISA. This was supplemented by confirmatory qRT-PCR and immunohistochemical analyses in the target organ. Serum paraoxonase-1 activity (PON1), which has been suggested as a marker of hepatotoxicity, was determined using a fluorometric assay. Clusterin and PON1 were not consistently altered in response to liver injury. In contrast, thiostatin and NGAL were increased in serum and urine of treated animals in a time- and dose-dependent manner. These changes correlated well with mRNA expression in the target organ and generally reflected the onset and degree of drug-induced liver injury. Receiver-operating characteristics (ROC) analyses supported serum thiostatin, but not NGAL, as a better indicator of drug-induced hepatobiliary injury than conventional clinical chemistry parameters, i.e. ALP, ALT and AST. Although thiostatin, an acute phase protein expressed in a range of tissues, may not be specific for liver injury, our results indicate that thiostatin may serve as a sensitive, minimally-invasive diagnostic marker of inflammation and tissue damage in preclinical safety assessment.

Fibrinogen Excretion in the Urine and Immunoreactivity in the Kidney Serves as a Translational Biomarker for Acute Kidney Injury

Fibrinogen (Fg) is significantly up-regulated in the kidney after acute kidney injury (AKI). We evaluated the performance of Fg as a biomarker for early detection of AKI. In rats and mice with kidney tubular damage induced by ischemia/reperfusion (I/R) or cisplatin administration, respectively; kidney tissue and urinary Fg increased significantly and correlated with histopathological injury, urinary kidney injury molecule-1 (KIM-1) and N-acetyl glucosaminidase (NAG) corresponding to the progression and regression of injury temporally. In a longitudinal follow-up of 31 patients who underwent surgical repair of abdominal aortic aneurysm, urinary Fg increased earlier than SCr in patients who developed postoperative AKI (AUC-ROC = 0.72). Furthermore, in a cohort of patients with biopsy-proven AKI (n = 53), Fg immunoreactivity in the tubules and interstitium increased remarkably and was able to distinguish patients with AKI from those without AKI (n = 59). These results suggest that immunoreactivity of Fg in the kidney, as well as urinary excretion of Fg, serves as a sensitive and early diagnostic translational biomarker for detection of AKI.

Pharmacological and genetic depletion of fibrinogen protects from kidney fibrosis

Fibrinogen (Fg) has been implicated in the pathogenesis of several fibrotic disorders by acting as a profibrotic ligand for a variety of cellular surface receptors and by modulating the provisional fibrin matrix formed after injury. We demonstrated increased renal Fg expression after unilateral ureteral obstruction and folic acid (FA) nephropathy in mice, respectively. Urinary Fg excretion was also increased in FA nephropathy. Using in vitro and in vivo approaches, our results suggested that IL-6 mediates STAT3 activation in kidney fibrosis and that phosphorylated (p)STAT3 binds to Fgα, Fgβ, and Fgγ promoters in the kidney to regulate their transcription. Genetically modified Fg heterozygous mice (∼75% of normal plasma Fg levels) exhibited only 3% kidney interstitial fibrosis and tubular atrophy after FA nephropathy compared with 24% for wild-type mice. Fibrinogenolysis through Ancrod administration after FA reduced interstitial fibrosis more than threefold compared with vehicle-treated control mice. Mechanistically, we show that Fg acts synergistically with transforming growth factor (TGF)-β1 to induce fibroblast proliferation and activates TGF-β1/pSMAD2 signaling. This study offers increased understanding of Fg expression and molecular interactions with TGF-β1 in the progression to kidney fibrosis and, importantly, indicates that fibrinogenolytics like Ancrod present a treatment opportunity for a yet intractable disease.

Urinary biomarkers track the progression of nephropathy in hypertensive and obese rats

AIMS To determine whether urinary biomarkers of acute kidney injury can be used to monitor the progression of chronic kidney injury in a rat model of hypertension and obesity. MATERIALS & METHODS A suite of novel urinary biomarkers were used to track the progression of kidney damage in SHROB and SHR-lean rats. RESULTS Urinary albumin, NAG, clusterin, osteopontin, RPA-1 and fibrinogen levels were significantly elevated over time and were closely associated with the severity of histopathologically determined nephropathy in both SHROB and SHR-lean rats. CONCLUSION Urinary biomarkers, such as albumin, fibrinogen, NAG, clusterin, RPA-1 and osteopontin, may serve as useful tools to track the progression of chronic kidney disease associated with hypertension and obesity.

Impaired renal function and development in Belgrade rats

Belgrade rats carry a disabling mutation in the iron transporter divalent metal transporter 1 (DMT1). Although DMT1 plays a major role in intestinal iron absorption, the transporter is also highly expressed in the kidney, where its function remains unknown. The goal of this study was to characterize renal physiology of Belgrade rats. Male Belgrade rats died prematurely with ∼50% survival at 20 wk of age. Necropsy results indicated marked glomerular nephritis and chronic end-stage renal disease. By 15 wk of age, Belgrade rats displayed altered renal morphology associated with sclerosis and fibrosis. Creatinine clearance was significantly lower compared with heterozygote littermates. Urinary biomarkers of kidney injury, including albumin, fibrinogen, and kidney injury molecule-1, were significantly elevated. Pilot morphological studies suggest that nephrogenesis is delayed in Belgrade rat pups due to their low iron status and fetal growth restriction. Such defects in renal development most likely underlie the compromised renal metabolism observed in adult b/b rats. Belgrade rat kidney nonheme iron levels were not different from controls but urinary iron and transferrin levels were higher. These results further implicate an important role for the transporter in kidney function not only in iron reabsorption but also in glomerular filtration of the serum protein.