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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 for6 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 AT1A to 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 6-12 in 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 (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. [ABSTRACT FROM AUTHOR]

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-1 sc) or the p38 inhibitor SB-239063 (15 mg·kg-1·day-1 sc) 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-1 in 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/mI, 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. [ABSTRACT FROM AUTHOR]

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, tubulointerstjtjal 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-I (Kim-1), and these were used to construct a sandwich Kim-1 ELISA. The assay range of this ELISA was 50 μg/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 1 were 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. [ABSTRACT FROM AUTHOR]