Effect of human erythropoietin (hEPO) treatment on anemia in ICR-derived glomerulonephritis (ICGN) mice.

ICR-derived glomerulonephritis (ICGN) mice are a novel inbred strain with hereditary nephrotic syndrome and are thus considered a good animal model of human idiopathic nephrotic syndrome. In the present study, we investigated the effect to erythrocyte production by human erythropoietin (hEPO) treatment in ICGN mice during the early nephrotic stage. Erythrocyte count, hemoglobin concentration and hematocrit value in hEPO-treated (5 U/body/day, for 5 days) ICGN mice were recovered to the levels found in normal ICR mice. In addition, there was no correlation between plasma creatinine level, a marker of renal function, and erythrocyte count after hEPO treatment. Therefore, anemia in ICGN mice may be caused by decreased production of EPO in the kidney following progressive parenchymal damage.     

Development of nephrotic ICGN mice--the origin, reproductive ability, and incidence of glomerulonephritis

ICGN is a strain of spontaneous nephrotic mice with nonproliferative glomerular lesions. It was derived from an outbred Yok: ICR colony in our laboratory. The renal disease constantly occurred in animals of the first to the tenth generations (greater than 13.0%; 70 days of age). When affected males were mated with unaffected females, the incidence of the disease in their offspring was 38.8% (n = 49) at 70 days after birth. When both parents were affected, their offspring were all affected (n = 12). The disease evenly progressed in both sexes. It usually began 40 to 150 days after birth and death occurred within two months after onset. The animals usually showed sufficient reproductive ability as long as unaffected females were used for mating.     

Podocyte-secreted angiopoietin-like-4 mediates proteinuria in glucocorticoid-sensitive nephrotic syndrome

The main manifestations of nephrotic syndrome include proteinuria, hypoalbuminemia, edema, hyperlipidemia and lipiduria. Common causes of nephrotic syndrome are diabetic nephropathy, minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) and membranous nephropathy. Among the primary glomerular diseases, MCD is usually sensitive to glucocorticoid treatment, whereas the other diseases show variable responses. Despite the identification of key structural proteins in the glomerular capillary loop which may contribute to defects in ultrafiltration, many of the disease mechanisms of nephrotic syndrome remain unresolved. In this study, we show that the glomerular expression of angiopoietin-like-4 (Angptl4), a secreted glycoprotein, is glucocorticoid sensitive and is highly upregulated in the serum and in podocytes in experimental models of MCD and in the human disease. Podocyte-specific transgenic overexpression of Angptl4 (NPHS2-Angptl4) in rats induced nephrotic-range, and selective, proteinuria (over 500-fold increase in albuminuria), loss of glomerular basement membrane (GBM) charge and foot process effacement, whereas transgenic expression specifically in the adipose tissue (aP2-Angptl4) resulted in increased circulating Angptl4, but no proteinuria. Angptl4(-/-) mice that were injected with lipopolysaccharide (LPS) or nephritogenic antisera developed markedly less proteinuria than did control mice. Angptl4 secreted from podocytes in some forms of nephrotic syndrome lacks normal sialylation. When we fed the sialic acid precursor N-acetyl-D-mannosamine (ManNAc) to NPHS2-Angptl4 transgenic rats it increased the sialylation of Angptl4 and decreased albuminuria by more than 40%. These results suggest that podocyte-secreted Angptl4 has a key role in nephrotic syndrome.     

Advanced glomerulosclerosis is reversible in nephrotic mice

Advanced glomerulosclerosis, a common hallmark of chronic renal diseases (CRD) is believed to be irreversible, and it is thought that glomerular hyperfiltration and hypertrophy may participate in its pathogenesis. We demonstrate here that glomerulosclerosis is "reversible" in an animal model. We used nephrotic ICGN (nep/nep) mice which showed a rapid progression of glomerulosclerosis, accompanied by histological findings for glomerular hyperfiltration. It is known that ureter ligation reduces glomerular filtration in ligated kidneys. When ureter ligation was applied to our model, glomerulosclerosis (characterized by myofibroblast hyperplasia and over-accumulated matrix protein) weakened in conjunction with suppressed glomerular hypertrophy. During this process, glomerular myofibroblasts showed apoptotic cell death after unilateral ureter ligation (UUO) treatment. Our results suggest that inhibition of glomerular filtration in sclerotic tufts may cause glomerular remodeling through the modulation of molecular and cellular sclerogenesis.     

Renal thrombotic microangiopathy in a genetic model of hypertension in mice

Our goal was to develop a model of accelerated hypertension with renal microangiopathy. Transgenic mice that are hypertensive because of overexpression of human renin (R+ mice) and human angiotensin (A+ mice) genes were studied. To increase arterial pressure to levels comparable to those that may be seen in malignant hypertension, high salt was added to the diet and/or the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methylester (L-NAME), was added to the drinking water. Renal lesions, decline in renal function, and proteinuria developed within 10 weeks in R+/A+ mice given both L-NAME and a high-salt diet, and within 24 weeks in mice given either L-NAME or a high-salt diet. Renal morphology showed features of severe thrombotic microangiopathy, with extensive vascular and glomerular lesions in all R+/A+ mice on high salt, L-NAME, or high salt plus L-NAME. Vascular lesions included fibrin thrombi and onion skinning of the vessel walls, whereas glomerular lesions included segmental sclerosis, mesangiolysis, fibrin thrombi within glomerular capillaries, and double-contour formation of glomerular capillary walls. Renal morphology was normal in control mice fed high salt and/or L-NAME. No R+/A+ mice fed a normal diet developed vascular lesions, whereas a few mice developed mild focal glomerular lesions. In summary, these studies characterize vascular and glomerular lesions in R+/A+ mice fed high salt, L-NAME, or both high salt and L-NAME, and provide a murine model of malignant hypertension with renal thrombotic microangiopathy.     

Deficiency of the tensin2 gene in the ICGN mouse: an animal model for congenital nephrotic syndrome

The ICGN mouse is a model for nephrotic syndrome (NS) which presents with proteinuria, hyperlipidemia, and edema. In this study we attempted to identify the gene(s) responsible for NS. By analyzing albuminuria in 160 (ICGN × MSM)F₁ × ICGN backcross progenies, we found that NS in the ICGN mouse is caused by more than one gene. We then performed a quantitative trait locus (QTL) analysis and detected a QTL with a very high LOD score peak in the telomeric region of Chr 15. By analyzing the nucleotide sequence of 22 genes located close to the QTL, we found that the tensin2 gene of the ICGN mouse possessed an 8-nucleotide deletion mutation in exon 18, leading to a frameshift and giving rise to a terminal codon at a premature position. Analyses of in situ hybridization and immunohistochemistry revealed that tensin2 was expressed in podocytes and tubular epithelial cells in normal mice but not in the ICGN mouse. These data raise the possibility that a mutation of the tensin2 gene is responsible for NS of the ICGN mouse and tensin2 is a prerequisite for the normal kidney function.     

A new mouse strain manifesting high proteinuria and kidney glomerular defect.

A mutant strain of mice manifesting high proteinuria, wasting syndrome, and kidney glomerular defect was established from the F5 offspring of an interstrain cross of CBA/Nga and RFM/Nga mice. Affected mice had high levels of proteinuria after 40 days of age. The body weight of about 22.6% of affected mice decreased rapidly and they died between 3 and 5 months of age. We learned that this abnormality is controlled by two pairs of autosomal recessive genes; the mutant strain of mice is designated FGS/Nga. The mutant strain has been characterized by high proteinuria and renal lesions with focal sclerosis of glomeruli and tubular atrophy with interstitial nephritis in the kidney resembling the human disease. The FGS/Nga mouse strain is a potential animal model for studying kidney glomerular defect in humans.     

Nephrotic Syndrome with Heart Disease: A Reappraisal

The evidence that heart failure alone may cause a nephrotic syndrome is inconclusive. Mercurial diuretics, which have also been implicated as a cause of the nephrotic syndrome, had been given in 23 of the 24 well-documented cases.Two cases of heart disease and nephrotic syndrome are described. Glomerular lesions were minimal on light microscopy, but thickening of the glomerular tuft basement membrane and partial fusion of the epithelial cell foot processes were apparent on elecronmicroscopy. The response to prednisone was such as to justify a trial of corticosteroid therapy in such cases despite the presence of cardiac disease.     

In vitro fertilization and microinsemination with round spermatids for propagation of nephrotic genes in mice

The ICGN is an inbred strain of mice with a hereditary nephrotic syndrome of an unknown cause. The disease progresses to renal failure, resulting in the deterioration of the systemic condition and in a drastic reduction of reproductive capacity. The present study was undertaken to determine if in vitro fertilization (IVF) and microinsemination using round spermatids could ameliorate the reduced fertility of ICGN mice. Mature oocytes (9.4 +/- 1.1 per mouse) were obtained from PMSG/hCG-primed females 2 to 5 mo of age. When spermatozoa from males aged 3 to 4 mo was used for IVF, a high fertilization rate (82.6%) was achieved and a high rate of embryos developed into blastocysts (92.6%). When spermatozoa from males aged 5 to 7 mo was used, the rates of fertilization and development to blastocysts were significantly lower (63.9 and 47.1%, respectively; P < 0.001). However, the production rate of offspring after embryo transfer was satisfactory irrespective of the age of males (59.1%). When males older than 9 mo were used, no fertilization was achieved due to the very poor motility of the spermatozoa. Microinsemination techniques with round spermatids (electrofusion and intracytoplasmic injection) resulted in the production of normal offspring from the older males, including one azoospermic case. These findings indicate that a conventional IVF technique and microinsemination using round spermatids can be used for propagating mutant genes which cause poor reproductive ability in mice.     

Transgenic mouse model of kidney disease: insertional inactivation of ubiquitously expressed gene leads to nephrotic syndrome.

Transgenic mouse strains carrying proviruses were generated by exposing mouse embryos to a recombinant retrovirus. Animals carrying a single provirus were intercrossed to derive mice homozygous for a given proviral insertion. Adult mice homozygous for the Mpv17 integration developed nephrotic syndrome and chronic renal failure. Histologically, affected kidneys showed progressive glomerular sclerosis. Similar lesions are seen in patients with progressive renal function deterioration. A probe to DNA sequences flanking the provirus detected a 1.7 kb RNA ubiquitously expressed during embryogenesis and in adults with high levels in kidney, brain, and heart. This RNA was not detected in tissues of homozygous animals, suggesting that the provirus interferes with RNA expression. Sequence analysis of the cDNA revealed that the gene encodes a 176 amino acid peptide containing hydrophobic regions, suggesting membrane association of the putative protein. The Mpv17 mutant is a potentially useful experimental system for studying mechanisms leading to renal disorders in man.     

Sphingolipid activator proteins in a human hereditary renal disease with deposition of disialogangliosides

Summary Congenital nephrotic syndrome of the Finnish type is a recessively inherited renal disease with glomerular deposits of the disialogangliosideO-acetyl-GD3. Sphingolipid activator proteins (saposins) stimulate the degradation of glycosphingolipids by lysosomal enzymes, and defects in saposins cause accumulation of substrate lipids in the affected tissues in lysosomal storage diseases. Here we report a study of the role of saposins in the accumulation ofO-acetyl-GD3 in kidneys of congenital nephrotic syndrome patients. At the mRNA level, the expression of saposin precursor in diseased kidneys appeared normal, and the nucleotide sequence analysis of cDNA clones did not reveal abnormalities in the prosaposin gene. Immunohistologically, saposins were localized mainly to the epithelial cells of the distal renal tubules or to the parietal epithelial cells of glomeruli. In the nephrotic syndrome kidneys, the staining pattern was highly granular and appeared mostly in the apical part of the epithelial lining, unlike the control kidneys. These results show that a major site of ganglioside metabolism is located in the distal nephron. Furthermore, these results suggest that saposins are not directly involved in the metabolism of the terminal sialic acids of disialogangliosides in the nephrotic syndrome kidneys.     

Requirement for class II phosphoinositide 3-kinase C2alpha in maintenance of glomerular structure and function

An early lesion in many kidney diseases is damage to podocytes, which are critical components of the glomerular filtration barrier. A number of proteins are essential for podocyte filtration function, but the signaling events contributing to development of nephrotic syndrome are not well defined. Here we show that class II phosphoinositide 3-kinase C2α (PI3KC2α) is expressed in podocytes and plays a critical role in maintaining normal renal homeostasis. PI3KC2α-deficient mice developed chronic renal failure and exhibited a range of kidney lesions, including glomerular crescent formation and renal tubule defects in early disease, which progressed to diffuse mesangial sclerosis, with reduced podocytes, widespread effacement of foot processes, and modest proteinuria. These findings were associated with altered expression of nephrin, synaptopodin, WT-1, and desmin, indicating that PI3KC2α deficiency specifically impacts podocyte morphology and function. Deposition of glomerular IgA was observed in knockout mice; importantly, however, the development of severe glomerulonephropathy preceded IgA production, indicating that nephropathy was not directly IgA mediated. PI3KC2α deficiency did not affect immune responses, and bone marrow transplantation studies also indicated that the glomerulonephropathy was not the direct consequence of an immune-mediated disease. Thus, PI3KC2α is critical for maintenance of normal glomerular structure and function by supporting normal podocyte function.     

Early glomerular filtration defect and severe renal disease in podocin-deficient mice

Podocytes are specialized epithelial cells covering the basement membrane of the glomerulus in the kidney. The molecular mechanisms underlying the role of podocytes in glomerular filtration are still largely unknown. We generated podocin-deficient (Nphs2-/-) mice to investigate the function of podocin, a protein expressed at the insertion of the slit diaphragm in podocytes and defective in a subset of patients with steroid-resistant nephrotic syndrome and focal and segmental glomerulosclerosis. Nphs2-/- mice developed proteinuria during the antenatal period and died a few days after birth from renal failure caused by massive mesangial sclerosis. Electron microscopy revealed the extensive fusion of podocyte foot processes and the lack of a slit diaphragm in the remaining foot process junctions. Using real-time PCR and immunolabeling, we showed that the expression of other slit diaphragm components was modified in Nphs2-/- kidneys: the expression of the nephrin gene was downregulated, whereas that of the ZO1 and CD2AP genes appeared to be upregulated. Interestingly, the progression of the renal disease, as well as the presence or absence of renal vascular lesions, depends on the genetic background. Our data demonstrate the crucial role of podocin in the establishment of the glomerular filtration barrier and provide a suitable model for mapping and identifying modifier genes involved in glomerular diseases caused by podocyte injuries.     

The struggle for energy in podocytes leads to nephrotic syndrome

Podocytes are terminally differentiated post-mitotic cells similar to neurons, and their damage leads to nephrotic syndrome, which is characterized by massive proteinuria associated with generalized edema. A recent functional genetic approach has identified the pathological relevance of several mutated proteins in glomerular podocytes to the mechanism of proteinuria in hereditary nephrotic syndrome. In contrast, the pathophysiology of acquired primary nephrotic syndrome, including minimal change disease, is still largely unknown. We recently demonstrated the possible linkage of an energy-consuming process in glomerular podocytes to the mechanism of proteinuria. Puromycin aminonucleoside nephrosis, a rat model of minimal change disease, revealed the activation of the unfolded protein response (UPR) in glomerular podocytes to be a cause of proteinuria. The pretreatment of puromycin aminonucleoside rat podocytes and cultured podocytes with the mammalian target of rapamycin (mTOR) inhibitor everolimus further revealed that mTOR complex 1 consumed energy, which was followed by UPR activation. In this paper, we will review nutritional transporters to summarize the energy uptake process in podocytes and review the involvement of the UPR in the pathogenesis of glomerular diseases. We will also present additional data that reveal how mTOR complex 1 acts upstream of the UPR. Finally, we will discuss the potential significance of targeting the energy metabolism of podocytes to develop new therapeutic interventions for acquired nephrotic syndrome.     

Immune complex glomerulonephritis in baboons (Papio cynocephalus) with indwelling intravascular catheters

Baboons with long term, indwelling, intravascular catheters developed clinical signs of renal and hepatic impairment. These included proteinuria and hypoalbuminemia without edema, and albumin to globulin ratios were reversed. Serum IgM, IgG, rheumatoid factor, and liver enzyme concentrations were above normal. Immunofluorescent staining of renal glomerular capillary loops was positive for IgG, IgM, B1c, and C4. Major microscopic lesions were membranoproliferative glomerulonephritis, chronic active hepatitis, degenerative arthritis, and chronic sialoadenitis. Electron microscopy of renal glomeruli demonstrated dense deposits in a variety of locations, mesangial cell interpositioning, and foot process fusion. These alterations, found in conjunction with the isolation of Staphylococcus aureus from the blood of affected baboons as well as the intravascular catheters, suggested that chronic bacterial infection was important in the pathogenesis of this disease.     

Nephrotic syndrome and subepithelial deposits in a mouse model of immune-mediated anti-podocyte glomerulonephritis

Subepithelial immune complex deposition in glomerular disease causes local inflammation and proteinuria by podocyte disruption. A rat model of membranous nephropathy, the passive Heymann nephritis, suggests that Abs against specific podocyte Ags cause subepithelial deposit formation and podocyte foot process disruption. In this study, we present a mouse model in which a polyclonal sheep anti-mouse podocyte Ab caused subepithelial immune complex formation. Mice developed a nephrotic syndrome with severe edema, proteinuria, hypoalbuminemia, and elevated cholesterol and triglycerides. Development of proteinuria was biphasic: an initial protein loss was followed by a second massive increase of protein loss beginning at approximately day 10. By histology, podocytes were swollen. Electron microscopy revealed 60-80% podocyte foot process effacement and subepithelial deposits, but no disruption of the glomerular basement membrane. Nephrin and synaptopodin staining was severely disrupted, and podocyte number was reduced in anti-podocyte serum-treated mice, indicating severe podocyte damage. Immunohistochemistry detected the injected anti-podocyte Ab exclusively along the glomerular filtration barrier. Immunoelectron microscopy localized the Ab to podocyte foot processes and the glomerular basement membrane. Similarly, immunohistochemistry localized mouse IgG to the subepithelial space. The third complement component (C3) was detected in a linear staining pattern along the glomerular basement membrane and in the mesangial hinge region. However, C3-deficient mice were not protected from podocyte damage, indicating a complement-independent mechanism. Twenty proteins were identified as possible Ags to the sheep anti-podocyte serum by mass spectrometry. Together, these data establish a reproducible model of immune-mediated podocyte injury in mice with subepithelial immune complex formation.     

CC chemokine ligand 5/RANTES chemokine antagonists aggravate glomerulonephritis despite reduction of glomerular leukocyte infiltration

The chemokine CC chemokine ligand (CCL)5/RANTES as well as its respective receptor CCR5 mediate leukocyte infiltration during inflammation and are up-regulated early during the course of glomerulonephritis (GN). We tested the effects of the two CCL5/RANTES blocking analogs, Met-RANTES and amino-oxypentane-RANTES, on the course of horse apoferritin (HAF)-induced GN. HAF-injected control mice had proliferative GN with mesangial immune complex deposits of IgG and HAF. Daily i.p. injections of Met-RANTES or amino-oxypentane-RANTES markedly reduced glomerular cell proliferation and glomerular macrophage infiltration, which is usually associated with less glomerular injury and proteinuria in HAF-GN. Surprisingly, however, HAF-GN mice treated with both analogs showed worse disease with mesangiolysis, capillary obstruction, and nephrotic range albuminuria. These findings were associated with an enhancing effect of the CCL5/RANTES analogs on the macrophage activation state, characterized by a distinct morphology and increased inducible NO synthetase expression in vitro and in vivo, but a reduced uptake of apoptotic cells in vivo. The humoral response and the Th1/Th2 balance in HAF-GN and mesangial cell proliferation in vitro were not affected by the CCL5/RANTES analogs. We conclude that, despite blocking local leukocyte recruitment, chemokine analogs can aggravate some specific disease models, most likely due to interactions with systemic immune reactions, including the removal of apoptotic cells and inducible NO synthetase expression.     

Nephrotic Syndrome in Monkey Infected with Human Quartan Malaria

A splenectomized aotus monkey infected with human quartan malaria (Plasmodium malariae) developed oedema and proteinuria. Histological examination revealed a generalized diffuse glomerulonephritis and immunofluorescent staining showed granular deposits of IgM in the glomeruli. The pathological picture resembled that shown by human patients with the quartan malaria nephrotic syndrome.