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Limited understanding of species differences in kidney transporters is a critical knowledge gap for prediction of drug-induced acute kidney injury, drug interaction, and pharmacokinetics in humans. Here, we report protein abundance data of 19 transporters in the kidney cortex across five species (human, monkey, dog, rat, and mouse). In general, the abundance of all of the 19 membrane transporters was higher in preclinical species compared with human except for multidrug resistance protein 1 (MDR1), organic cation transporter (OCT) 3, and OCTN1. In nonhuman primate, the total abundance of 12 transporters for which absolute data were available was 2.1-fold higher (P= 0.025) relative to human but the percentage of distribution of these transporters was identical in both species. Multidrug resistance-associated protein (MRP) 4, OCTN2, organic anion transporter (OAT) 2, sodium/potassium-transporting ATPase, MRP3, SGLT2, OAT1, MRP1, MDR1, and OCT2 were expressed differently with cross-species variabilities of 8.2-, 7.4-, 6.1-, 5.9-, 5.4-, 5.2-, 4.1-, 3.3-, and 2.8-fold, respectively. Sex differences were only significant in rodents and dog. High protein-protein correlation was observed in OAT1 versus MRP2/MRP4 as well as OCT2 versus MATE1 in human and monkey. The cross-species and sex-dependent protein abundance data are important for animal to human scaling of drug clearance as well as for mechanistic understanding of kidney physiology and derisking of kidney toxicity for new therapeutic candidates in drug development.

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)-β1to induce fibroblast proliferation and activates TGF-β1/pSMAD2 signaling. This study offers increased understanding of Fg expression and molecular interactions with TGF-β1in the progression to kidney fibrosis and, importantly, indicates that fibrinogenolytics like Ancrod present a treatment opportunity for a yet intractable disease.

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/brats. 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.

Introduction: There are significant rates of attrition in drug development. A number of compounds fail to progress past preclinical development due to limited tools that accurately monitor toxicity in preclinical studies and in the clinic. Research has focused on improving tools for the detection of organ-specific toxicity through the identification and characterization of biomarkers of toxicity.Areas covered: This article reviews what we know about emerging biomarkers in toxicology, with a focus on the 2012 Northeast Society of Toxicology meeting titled ‘Translational Biomarkers in Toxicology.' The areas covered in this meeting are summarized and include biomarkers of testicular injury and dysfunction, emerging biomarkers of kidney injury and translation of emerging biomarkers from preclinical species to human populations. The authors also provide a discussion about the biomarker qualification process and possible improvements to this process.Expert opinion: There is currently a gap between the scientific work in the development and qualification of novel biomarkers for nonclinical drug safety assessment and how these biomarkers are actually used in drug safety assessment. A clear and efficient path to regulatory acceptance is needed so that breakthroughs in the biomarker toolkit for nonclinical drug safety assessment can be utilized to aid in the drug development process.

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.

Acute kidney injury (AKI) may result from ischemia or by the use of nephrotoxic agents. The incidence of AKI is variable, depends on comorbidities, and ranges from 5 to 35% in all hospitalized patients. The mechanisms of kidney injury exist within a large network of signaling pathways driven by interplay of inflammatory cytokines/chemokines, reactive oxygen species (ROS), and apoptotic factors. The effects and progression of injury overlap extensively with the remarkable ability of the kidney to repair itself both by intrinsic and extrinsic mechanisms that involve specific cell receptors/ligands as well as possible paracrine influences. The fact that kidney injury is usually part of a generalized comorbid condition makes it all the more challenging in terms of assessment of severity. In this review, we attempt to analyze the mechanisms of ischemic injury and repair in acute and chronic kidney disease from the perspectives of both preclinical and human studies. WIREs Syst Biol Med2011 3 606–618 DOI: 10.1002/wsbm.133

Ischemia/reperfusion (I/R) injury in the kidney is a major cause of acute kidney injury (AKI) in humans and is associated with significantly high mortality. To identify genes that modulate kidney injury and repair, we conducted genome-wide expression analysis in the rat kidneys after I/R and found that the mRNA levels of fibrinogen (Fg)α, Fgβ, and Fgγ chains significantly increase in the kidney and remain elevated throughout the regeneration process. Cellular characterization of Fgα and Fgγ chain immunoreactive proteins shows a predominant expression in renal tubular cells and the localization of immunoreactive Fgβ chain protein is primarily in the renal interstitium in healthy and regenerating kidney. We also show that urinary excretion of Fg is massively increased after kidney damage and is capable of distinguishing human patients with acute or chronic kidney injury (n = 25) from healthy volunteers (n = 25) with high sensitivity and specificity (area under the receiver operating characteristic of 0.98). Furthermore, we demonstrate that Fgβ-derived Bβ15-42peptide administration protects mice from I/R-induced kidney injury by aiding in epithelial cell proliferation and tissue repair. Given that kidney regeneration is a major determinant of outcome for patients with kidney damage, these results provide new opportunities for the use of Fg in diagnosis, prevention, and therapeutic interventions in kidney disease.

Ischemia/reperfusion (I/R) injury in the kidney is a major cause of acute kidney injury (AKI) in humans and is associated with significantly high mortality. To identify genes that modulate kidney injury and repair, we conducted genome-wide expression analysis in the rat kidneys after I/R and found that the mRNA levels of fibrinogen (Fg)α, Fgβ, and Fgγ chains significantly increase in the kidney and remain elevated throughout the regeneration process. Cellular characterization of Fgα and Fgγ chain immunoreactive proteins shows a predominant expression in renal tubular cells and the localization of immunoreactive Fgβ chain protein is primarily in the renal interstitium in healthy and regenerating kidney. We also show that urinary excretion of Fg is massively increased after kidney damage and is capable of distinguishing human patients with acute or chronic kidney injury (n = 25) from healthy volunteers (n = 25) with high sensitivity and specificity (area under the receiver operating characteristic of 0.98). Furthermore, we demonstrate that Fgβ-derived Bβ15-42 peptide administration protects mice from I/R-induced kidney injury by aiding in epithelial cell proliferation and tissue repair. Given that kidney regeneration is a major determinant of outcome for patients with kidney damage, these results provide new opportunities for the use of Fg in diagnosis, prevention, and therapeutic interventions in kidney disease.

Tubulointerstitial lesions are important in the progression of proteinuric renal disease. Tubular kidney injury molecule-1 (Kim-1) is induced in acute renal injury and reversible as a natural course. Kim-1 is also present in chronic renal damage; however, the dynamics of Kim-1 in chronic renal damage and effects of antiproteinuric treatment on Kim-1 are unknown. We studied Kim-1 in adriamycin nephrosis (AN) before and after renin-angiotensin system blockade. A renal biopsy was taken 6 wk after adriamycin injection to study renal damage and Kim-1 expression. Subsequently, ACE inhibition (ACEi; n= 23), angiotensin II antagonist (AT1A; n= 23), or vehicle (n= 10) was given for 6 wk; healthy rats served as controls (CON; n= 8). In AN, renal Kim-1 mRNA was induced 26-fold vs. CON at week 6, with further increase in vehicle to week 12(40-fold) but was reduced by ACEi and AT1Ato 10- and 12-fold vs. CON (P< 0.05 vs. week 6). Kim-1 protein was undetectable in CON; in AN, it was present in brush border of dilated tubules in areas with adjacent interstitial lesions. Renal Kim-1 protein levels increased from weeks 6–12in vehicle and decreased in ACEi- and AT1A-treated groups (P< 0.05). In vehicle, urinary Kim-1 was increased (P< 0.05 vs. CON), with a reduction by ACEi and AT1A(P< 0.05 vs. vehicle). Renal and urinary Kim-1 correlated with proteinuria and interstitial damage cross-sectionally. Reductions in proteinuria and renal Kim-1 correlated, which was not associated by corresponding changes in tubulointerstitial fibrosis. In conclusion, on longitudinal follow-up during antiproteinuric treatment increased renal Kim-1 expression is reversible in proportion to proteinuria reduction, likely reflecting reversibility of early tubular injury, supporting its potential as a biomarker for tubulointerstitial processes of damage and repair.

Chronic transplant dysfunction is characterized by renal function decline and proteinuria. Kidney injury molecule (KIM)-1, a transmembrane tubular protein with unknown function, is undetectable in normal kidneys, but markedly induced after injury. Urinary KIM-1 excretion has been quantified as biomarker of renal damage. We prospectively studied whether urinary KIM-1 predicts graft loss, independent of renal function and proteinuria.

Kidney injury molecule-1 (Kim-1) is associated with ischemic and proteinuric tubular injury; however, whether dysregulation of the renin-angiotensin system (RAS) can also induce Kim-1 is unknown. We studied Kim-1 expression in homozygous Ren2 rats, characterized by renal damage through excessive RAS activation. We also investigated whether antifibrotic treatment (RAS blockade or p38 MAP kinase inhibition) would affect Kim-1 expression. At 7 wk of age, homozygous Ren2 rats received a nonhypotensive dose of candesartan (0.05 mg·kg−1·day−1sc) or the p38 inhibitor SB-239063 (15 mg·kg−1·day−1sc) for 4 wk; untreated Ren2 and Sprague-Dawley (SD) rats served as controls. Kim-1 mRNA and protein expression were determined by quantitative PCR and immunohistochemistry, respectively, and related to markers of prefibrotic renal damage. Urinary Kim-1 was measured in 8-wk-old Ren2 and SD rats with and without angiotensin-converting enzyme inhibition (ramipril, 1 mg·kg−1·day−1in drinking water for 4 wk). Untreated Ren2 rats showed a >20-fold increase in renal Kim-1 mRNA (expressed as Kim-1-to-GAPDH ratio): 75.5 ± 43.6 vs. 3.1 ± 1.0 in SD rats (P< 0.01). Candesartan and SB-239063 strongly reduced Kim-1 mRNA: 3.1 ± 1.5 (P< 0.01) and 9.8 ± 4.2 (P< 0.05), respectively. Kim-1 protein expression in damaged tubules paralleled mRNA expression. Kim-1 expression correlated with renal osteopontin, α-smooth muscle actin, and collagen III expression and with tubulointerstitial fibrosis. Damaged tubular segments expressing activated p38 also expressed Kim-1. Urinary Kim-1 was increased in Ren2 vs. SD (458 ± 70 vs. 27 ± 2 pg/ml, P< 0.01) rats and abolished in Ren2 rats treated with ramipril (33 ± 5 pg/ml, P< 0.01). Kim-1 is associated with development of RAS-mediated renal damage. Antifibrotic treatment through RAS blockade or p38 MAP kinase inhibition reduced Kim-1 in the homozygous Ren2 model.

Recent studies from our laboratory have shown that the mineralocorticoid receptor (MR) blockade with spironolactone (Sp) prevented renal dysfunction and reduced renal injury in both acute and chronic cyclosporine (CsA) nephrotoxicity. This study was designed to evaluate whether Sp administration reduces functional and structural renal damage associated in the setting of preexisting chronic CsA nephrotoxicity. Twenty eight male Wistar rats were fed a low-sodium diet. Fourteen received vehicle (V) and the others were treated with CsA (15 mg/kg sc). After 18 days one half of each group received Sp (20 mg/kg po) for the subsequent 18 days. Creatinine clearance, arteriolopathy, tubulointerstitial fibrosis, arteriolar thickening, glomerular diameter, apoptosis index and TGF-β, procaspase-3, and kidney injury molecule 1 (Kim-1) mRNA levels as well as Kim-1 shedding in urine were evaluated. Sp reduced the progression of renal dysfunction and tubulointerstitial fibrosis in preexisting chronic CsA nephrotoxicity. There was a significant reduction of arteriolar thickening in the CsA+Sp group that was associated with greater glomerular diameter and reduction of apoptosis index. These renoprotective effects were associated with reduction of TGF-β, procaspase-3, and Kim-1 mRNA levels as well as Kim-1 shedding into the urine. In conclusion, MR blockade with Sp prevented the progression of renal injury in preexisting chronic CsA nephropathy. These results suggest that Sp may reduce CsA-induced established nephrotoxicity in patients.

Acute kidney injury is a common condition and is associated with a high mortality rate. It has been recognised that routinely used measures of renal function, such as levels of blood urea nitrogen and serum creatinine, increase significantly only after substantial kidney injury occurs and then with a time delay. Insensitivity of such tests delays the diagnosis in humans, making it particularly challenging to administer putative therapeutic agents in a timely fashion. Furthermore, this insensitivity affects the evaluation of toxicity in preclinical studies by allowing drug candidates, which have low, but nevertheless important, nephrotoxic side effects in animals, to pass the preclinical safety criteria only to be found to be clinically nephrotoxic with great human costs. This review presents the current status of sensitive and specific biomarkers to detect preclinical and clinical renal injury and summarises the techniques used to quantitate these biomarkers in biological fluids.

Kim-1, a recently discovered membrane protein, is undetectable in normal kidneys but markedly induced in proximal tubules after ischemic and toxic injury. The function of Kim-1 is unclear, but it is implicated in damage/repair processes. The Kim-1 ectodomain is cleaved by metalloproteinases and detectable in urine. We studied Kim-1 in a nontoxic, nonischemic, model of tubulointerstitial damage caused by acute proteinuria. Uninephrectomized (NX) rats received daily (ip) injections of 2 g BSA (NX+BSA, n= 12) or saline (NX, n= 6) for 3 wk. Kidneys were stained for various damage markers by immunohistochemistry (IHC). Kim-1 mRNA (RT-PCR, in situ hybridization), protein (IHC, Western blotting), and urinary Kim-1 (Luminex) were determined. Spatial relations between Kim-1 and other damage markers were studied by double labeling IHC. NX+BSA rats developed massive proteinuria (1,217 ± 313 vs. 18 ± 2 mg/day in NX, P< 0.001) and significant renal damage. Kim-1 mRNA was upregulated eightfold in NX+BSA (ratio Kim-1/β-actin, 4.08 ± 2.56 vs. 0.52 ± 0.64 in NX, P< 0.001) and localized to damaged tubules. Kim-1 protein expression was markedly induced in NX+BSA (2.46 ± 1.19 vs. 0.39 ± 0.10% staining/field in NX, P< 0.001). Urinary Kim-1 was significantly elevated in NX+BSA (921 ± 592 vs. 87 ± 164 pg/ml in NX, P< 0.001) and correlated with tissue Kim-1 expression (r= 0.66, P=0.02). Kim-1 protein was found at the apical membrane of dilated nephrons. Kim-1 expression was limited to areas with inflammation (MØ), fibrosis (α-smooth muscle actin), and tubular damage (osteopontin), and only occasionally with tubular dedifferentiation (vimentin). These results implicate involvement of Kim-1 in the pathogenesis of proteinuria-induced renal damage/repair. Urinary Kim-1 levels may serve as a marker of proteinuria-induced renal damage.

Sensitive and specific biomarkers are needed to detect early kidney injury. The objective of the present work was to develop a sensitive quantitative urinary test to identify renal injury in the rodent to facilitate early assessment of pathophysiological influences and drug toxicity. Two mouse monoclonal antibodies were made against the purified ectodomain of kidney injury molecule-1 (Kim-1), and these were used to construct a sandwich Kim-1 ELISA. The assay range of this ELISA was 50 pg/ml to 5 ng/ml, with inter- and intra-assay variability of <10%. Urine samples were collected from rats treated with one of three doses of cisplatin (2.5, 5, or 7.5 mg/kg). At one day after each of the doses, there was an approximately three- to fivefold increase in the urine Kim-1 ectodomain, whereas other routinely used biomarkers measured in this study [plasma creatinine, blood urea nitrogen (BUN), urinary N-acetyl-β-glucosaminidase (NAG), glycosuria, proteinuria] lacked the sensitivity to show any sign of renal damage at this time point. When rats were subjected to increasing periods (10, 20, 30, or 45 min) of bilateral ischemia, there was an increasing amount of urinary Kim-1 detected. After only 10 min of bilateral ischemia, Kim-1 levels on day 1were 10-fold higher (5 ng/ml) than control levels, whereas plasma creatinine and BUN were not increased and there was no glycosuria, increased proteinuria, or increased urinary NAG levels. Thus urinary Kim-1 levels serve as a noninvasive, rapid, sensitive, reproducible, and potentially high-throughput method to detect early kidney injury in pathophysiological studies and in preclinical drug development studies for risk-benefit profiling of pharmaceutical agents.

Kidney fibrosis constitutes the shared final pathway of nearly all chronic nephropathies, but biomarkers for the non-invasive assessment of kidney fibrosis are currently not available. To address this, we characterize five candidate biomarkers of kidney fibrosis: Cadherin-11 (CDH11), Sparc-related modular calcium binding protein-2 (SMOC2), Pigment epithelium-derived factor (PEDF), Matrix-gla protein, and Thrombospondin-2. Gene expression profiles in single-cell and single-nucleus RNA-sequencing (sc/snRNA-seq) datasets from rodent models of fibrosis and human chronic kidney disease (CKD) were explored, and Luminex-based assays for each biomarker were developed. Plasma and urine biomarker levels were measured using independent prospective cohorts of CKD: the Boston Kidney Biopsy Cohort, a cohort of individuals with biopsy-confirmed semiquantitative assessment of kidney fibrosis, and the Seattle Kidney Study, a cohort of patients with common forms of CKD. Ordinal logistic regression and Cox proportional hazards regression models were used to test associations of biomarkers with interstitial fibrosis and tubular atrophy and progression to end-stage kidney disease and death, respectively. Sc/snRNA-seq data confirmed cell-specific expression of biomarker genes in fibroblasts. After multivariable adjustment, higher levels of plasma CDH11, SMOC2, and PEDF and urinary CDH11 and PEDF were significantly associated with increasing severity of interstitial fibrosis and tubular atrophy in the Boston Kidney Biopsy Cohort. In both cohorts, higher levels of plasma and urinary SMOC2 and urinary CDH11 were independently associated with progression to end-stage kidney disease. Higher levels of urinary PEDF associated with end-stage kidney disease in the Seattle Kidney Study, with a similar signal in the Boston Kidney Biopsy Cohort, although the latter narrowly missed statistical significance. Thus, we identified CDH11, SMOC2, and PEDF as promising non-invasive biomarkers of kidney fibrosis.

Kidney toxicity due to drugs and chemicals poses a significant health burden for patients and a financial risk for pharmaceutical companies. However, currently no sensitive and high‐throughput in vitro method exists for predictive nephrotoxicity assessment. Primary human proximal tubular epithelial cells (HPTECs) possess characteristics of differentiated epithelial cells, making them a desirable model to use in in vitro screening systems. Additionally, heme oxygenase 1 (HO‐1) protein expression is upregulated as a protective mechanism during kidney toxicant‐induced oxidative stress or inflammation in HPTECs and can therefore be used as a biomarker for nephrotoxicity. In this article, we describe two different methods to screen for HO‐1 increase: A homogeneous time resolved fluorescence (HTRF) assay and an immunofluorescence assay. The latter provides lower throughput but higher sensitivity due to the combination of two readouts, HO‐1 intensity and cell number. The methods described in the protocol are amendable for other cell types as well. © 2016 by John Wiley & Sons, Inc.