Lysyl oxidase (LOX), an extracellular enzyme, plays a key role in the post-translational modification of collagens and elastin, catalyzing inter- and intra-crosslinking reactions. Because the crosslinked extracellular matrices (ECMs) are highly resistant to degradative enzymes, it is considered that the over-expression of LOX may cause severe fibrotic degeneration. In the present study, we addressed the role of LOX-mediated crosslinking in chronic renal tubulointerstitial fibrosis using an animal model of hereditary nephrotic syndrome, the Institute of Cancer Research (ICR)-derived glomerulonephritis (ICGN) mouse. Ribonuclease protection assay (RPA) revealed that LOX mRNA expression was up-regulated in the kidneys of ICGN mice as compared with control ICR mice. High-level expression of LOX and transforming growth factor (TGF)-beta1 (an up-regulator of LOX) mRNA was detected in tubular epithelial cells of ICGN mouse kidneys by in situ hybridization. Type-I and -III collagens, major substrates for LOX, were accumulated in tubulointerstitium of ICGN mouse kidneys. The present findings imply that TGF-beta1 up-regulates the production of LOX in tubular epithelial cells of ICGN mouse kidneys, and the excessive LOX acts on interstitial collagens and catalyzes crosslinking reactions. As a result, the highly crosslinked collagens induce an irreversible progression of chronic renal tubulointerstitial fibrosis in the kidneys of ICGN mice.

ABSTRACT. The ICR-derived glomerulonephritis (ICGN) mouse, a novel inbred mouse strain with a hereditary nephrotic syndrome, develops severe anemia associated with chronic renal failure. To reveal the pathogenic mechanism of anemia in ICGN mice, we subcutaneously administered recombinant human erythropoietin (rhEPO; 5 IU/mouse/day) or saline for 5 days to ICGN mice. In terminal-stage ICGN mice with severe anemia, rhEPO significantly increased hematocrit (Ht), red blood cells (RBC) and hemoglobin levels. Endogenous EPO levels in peripheral blood were reduced by rhEPO injection. No histopathological changes in bone marrow and kidneys w ere induced by rhEPO injection. Insufficiency of EPO may cause anemia in ICGN mice. KEY WORDS: anemia associated with chronic renal failure, erythropoietin, hereditary nephrotic mouse (ICGN).J. Vet. Med. Sci. 66 (7): [883][884][885][886] 2004 The Institute of Cancer Research (ICR)-derived glomerulonephritis (ICGN) mouse, an inbred mouse strain with a hereditary nephrotic syndrome, is considered to be a good model of human idiopathic nephrotic syndrome [9]. Homozygous ICGN mice show the typical symptoms of nephrosis [10][11][12] and exhibit severe glomerular and tubulointestitial fibrosis [14]. Age-dependent increases in apoptotic cell and proliferating cell densities were also observed in the kidneys of ICGN mice [15]. Renal deterioration eventually leads to death as a result of chronic renal disorder (CRD). Moreover, ICGN mice develop a marked anemia with the deterioration of renal function [16]. Anemia associated with chronic renal failure is a major subsequent symptom that appears in the earlier stages of renal disease. It is a normochromic and normocytic anemia with hypoplastic bone marrow. The anemia associated with chronic renal failure is initially mild and inconsequential, however, ultimately it directly affects quality of life [3,13].Twenty week-age homozygous ICGN mice were prepared by mating homozygous males (nep/nep) and heterozygous females (nep/-), and age-and sex-matched ICR mice, were purchased from Clea Japan (Tokyo, Japan) as healthy controls. They received humane care as outlined in the "Guide for the Care and Use of Laboratory Animals" (Kyoto University Animal Care Committee according to NIH No. 86-23; revised 1999). Peripheral blood samples were obtained from the cervical vein under ether anesthesia and used for hematological and biochemical analyses. Hematological analyses were performed using an automatic counter (K-4500; Sysmex Co., Kobe, Japan), as were serum biochemical analyses (Fuji Drichem 3500V; Fuji Film, Tokyo, Japan), according to the manufacturer's instructions.Based on serum creatinine (sCre) levels, ICGN mice were categorized into three groups (latent, progressing and terminal) [14][15][16]. Briefly, ICGN mice with less than 0.34 mg/dl of sCre (mean plus SD of healthy ICR mice: 0.27 plus 0.07) were categorized as being in the latent stage. ICGN mice with 0.35-0.54 mg/dl (twice the mean of ICR mice) and those with more than 0.55 mg/dl were class...

ABSTRACT. Anemia is a major secondary symptom in chronic renal disorder (CRD), but the precise cause of insufficient production of erythropoietin (EPO) remains unclear owing to the controversial localization of EPO-producing cells in the kidneys. The ICR-derived glomerulonephritis (ICGN) mouse, a new hereditary nephrotic mouse, is an appropriate model of anemia associated with CRD. By using an amplified in situ hybridization technique, we detected and counted the renal EPO-producing cells under both normoxic and hypoxic conditions. The expression levels of renal EPO mRNA were quantified and oxygen gradients were also assessed immunohistochemically. Amplified in situ hybridization clarified that EPO-producing cells were peritubular interstitial cells in the middle region of renal cortex in both ICR and ICGN mice. Hypoxia (7% O 2 ) induced low oxygen tension in proximal tubular epithelial cells of renal cortex, and increased the expression of EPO mRNA and the number of EPO-producing cells in both ICR and ICGN mice. However, hypoxia did not increase the serum EPO levels in ICGN mice. The ICGN mouse is a good model for anemia associated with CRD, and the suppression of EPO protein production in the renal EPO-producing cells is considered to be a potential cause of anemia associated with CRD. KEY WORDS: amplified in situ hybridization, anemia with chronic renal disorder (CRD), erythropoietin (EPO)-producing cell, hereditary nephrotic ICR-derived glomerulonephritis (ICGN) mouse, kidney.J. Vet. Med. Sci. 67(9): 891-899, 2005 Anemia associated with chronic renal disorder (CRD) is a major secondary symptom that appears in the early stages of renal disease. This anemia is initially mild; however, ultimately, severe anemia directly affects the quality of life (QOL) [25,37]. Anemia associated with CRD is normochromic and normocytic, and, based on case studies, insufficient serum levels of erythropoietin (EPO) are considered to be a major cause [6,25,37].EPO, a 30-34 kDa glycoprotein, is classified as part of the hematopoietic cytokine family [5,14,24], controls the erythropoiesis in the bone marrow, and regulates the proliferation, differentiation and survival of erythroid progenitor cells through EPO receptor (EPOR)-mediated signal transduction [5,12,14,24]. The kidney is a principal organ of EPO production, accounting for more than 90% under normal conditions, with extra-renal sources estimated to produce than 10% [3,26]. Due to its strong erythropoietic effect, recombinant human EPO (rhEPO) is generally used as a therapeutic agent for anemia associated with CRD [7,19]. However, the precise cause of the insufficient serum levels of EPO in anemia associated with CRD remains unclear, primarily owing to the lack of knowledge of the identity of the EPO-producing cells and how they are regulated in the kidneys.Studies on anemia associated with CRD have been mainly accomplished by using artificial models of acute renal failure, i.e. drug-induced, antibody-induced and/or nephrectomized animal models. Compared with these exper...