Nephrotic Syndrome Induced by Dibasic Sodium Phosphate Injections for Twenty-eight Days in Rats

Abstract

Sprague-Dawley rats received once daily tail-vein injections of 360 mM dibasic sodium phosphate solution at 8 mL/kg for fourteen or twenty-eight days. Clinical examination revealed persistent proteinuria from three days after the first dosing and thereafter severe proteinuria from eight days or later in the phosphate-treated groups. Proteinuria developed without remission even after fourteen-day withdrawal in the fourteen-day dosed group. Phosphate-treated animals developed lipemia, hypercholesterolemia, anemia, higher serum fibrinogen levels, and lower serum albumin/globulin ratios on day 29. Renal weight increased significantly compared with control animals, and the kidneys appeared pale and enlarged with a rough surface. Histopathologically, glomerular changes consisted of mineralization in whole glomeruli, glomerular capillary dilatation, partial adhesion of glomerular tufts to Bowman’s capsule, and mesangiolysis. Ultrastructural lesions such as an increased number of microvilli, effacement of foot processes, and thickening of the glomerular basement membrane, and immunocytochemical changes in podocytes, mainly decreased podoplanin-positive cells and increased desmin expression, were also conspicuous in the phosphate-treated rats for twenty-eight days. Marked tubulointerstitial lesions were tubular regeneration and dilatation, protein casts, mineralization in the basement membrane, focal interstitial inflammation, and fibrosis in the cortex. These clinical and morphological changes were similar to features of human nephrotic syndrome.

Introduction

Dibasic sodium phosphate (Na₂HPO₄) has been used as an electrolyte replenisher and a buffer vehicle for injectable medicines in toxicity studies. However, we detected nephrotoxicity of dibasic sodium phosphate in a previous study in which a high-dose phosphate solution was given to Sprague-Dawley rats by daily bolus intravenous administration; urinalysis revealed proteinuria on day 3, and mineralization was found within the parietal epithelial cells and glomerular basement membrane (GBM) in the kidney with normal levels of serum calcium and inorganic phosphorus on day 9. Electron microscopy showed a small number of vacuoles in the cytoplasm of the podocytes after a single administration of phosphate solution, and effacement of foot processes, increased microvilli, and low-density lamellar structures within Bowman’s space, the GBM, and the mesangial matrix on day 4. Phosphorus and calcium were detected by x-ray microanalysis as fine particles admixed with lamellar structures (Tsuchiya et al. 2004; Tsuchiya et al. 2008). These results suggest that the onset of glomerular calcification is preceded by primary podocyte damage, and the pathogenesis of glomerular calcification could be considered to involve both transient excretion of high concentration phosphorus and dystrophic calcification based on cellular injury in the glomeruli.

The earliest changes detected, particularly foot-process loss together with increased proteinuria, were similar to the characteristics of minimal change disease in humans. The findings suggest that dibasic phosphate solution treatment conducted over a longer period may lead to more severe glomerular degeneration and subsequent tubular injury. The reversibility of glomerular function after phosphate-induced podocyte alteration is not clearly known. Therefore, we examined the progression of this alteration by repeat dose administration and assessed the reversibility of renal lesions after the drug withdrawal period.

Here we describe the pathological and biochemical changes caused by phosphate administration for twenty-eight days and their reversibility in rats.

Materials and Methods

Animals and Housing Conditions

All experimental procedures were conducted after approval of the study by the Institutional Animal Care and Use Committee of Shionogi Research Laboratories. Twenty male Sprague-Dawley rats (Jcl:SD) were obtained at five weeks of age from Clea Japan Inc. (Shiga, Japan) and acclimated for a week before treatment. We used only male animals because both male and female rats with phosphate treatment showed similar glomerular calcification in the preliminary two-week study (data not shown). The rats were divided into four groups of five rats each, for two control groups and two phosphate-treated groups, and were housed individually in plastic cages in an animal room kept under controlled conditions (temperature of 23°C ± 3°C, humidity of 50% ± 20%, lighting for twelve hours between 8:00 AM and 8:00 PM, ventilation ten times per hour). The rats were fed pelleted diet (CA-1, Clea Japan Inc., Tokyo) and provided water ad libitum via an automatic watering system after filtration through 30- and 3-μm-pore filters and exposure to ultraviolet radiation.

Test Materials

Na₂HPO₄·12H₂O (Kanto Chemical Co., Inc. Tokyo, Japan) was diluted with physiological saline to produce 360 mM Na₂HPO₄ solutions (409.6 mg/kg/day Na₂HPO₄). On the basis of our previous study (Tsuchiya et al. 2004), we selected 360 mM Na₂HPO₄ solution to induce stable glomerular calcification. The Na₂HPO₄ solution was adjusted to pH 5.5–6.5 using 1 N sulfuric acid, and its osmolarity was 748 mOsm/kg.

Experimental Design

The rats were administered 360 mM Na₂HPO₄ at 4 mL/kg/min for two minutes via the tail vein through a polyethylene catheter connected to a plastic syringe mounted on an infusion pump (STC-525, Terumo Corp., Tokyo). Two phosphate-treated groups consisting of five male rats each were dosed once daily for fourteen (the withdrawal group) or twenty-eight days (the twenty-eight-day group) with dibasic sodium phosphate solution at 360 mM per day, respectively. Animals dosed for fourteen days were followed by a treatment-free period of fourteen days. The control animals received physiological saline in the same way. Clinical signs and body weights were observed and measured each day. All rats were sacrificed on day 29.

Urinalysis

All rats were kept in individual metabolic cages and deprived of food and water, and urine was collected four hours after dosing on days 1, 3, 5, 8, 16, 19, 22, and 26. Protein, color, pH, glucose, ketone bodies, urobilinogen, bilirubin, and occult blood were measured with Clinitek 200 Plus (Bayer Corporation Diagnostics Division, Elkhart, IN, USA) for qualitative analysis. The measurement range of proteinuria was as follows: 0 was negative, 15 mg/dL was ± (minimal), 30 mg/dL was + (mild), 100 mg/dL was 2+ (moderate), 300 mg/dL was 3+, and 1000 mg/dl was 4+ (severe). All rats were also assessed for urinary volume on days 16, 19, 22, and 26 in all groups.

Blood Chemistry and Hematology

Immediately prior to necropsy, blood was collected from each animal via the posterior vena cava under sodium pentobarbital anesthesia and placed in a vacuum blood-collecting tube containing heparin sodium. Plasma was analyzed for total protein, albumin, creatinine, urea nitrogen, total cholesterol, triglyceride, inorganic phosphorus, calcium, sodium, potassium, and chloride using an Automatic Analyzer 7170 (Hitachi, Tokyo, Japan), and albumin/globulin ratio (A/G ratio) was calculated as albumin/(total protein-albumin). Whole blood treated with EDTA-2K was subjected to analysis using ADIVA 120 Hematology System (Siemens Healthcare Diagnostics Inc., IL, USA). Plasma was obtained from blood treated with 3.8% sodium citrate solution and subjected to analysis using an Automated Blood Coagulation Analyzer CA-6000 (Sysmex Corporation, Kobe, Japan). The following items were examined: white blood cell count (WBC); red blood cell count (RBC); hemoglobin concentration; packed cell volume; mean corpuscular volume; mean corpuscular hemoglobin; mean corpuscular hemoglobin concentration; platelet count; leukocyte count for neutrophil, lymphocyte, monocyte, eosinophil, basophil and large unstained cells; prothrombin time; activated partial thromboplastin time; and fibrinogen concentration.

Microscopy

All animals were euthanized by exsanguinations by cutting both the abdominal aorta and vena cava under deep anesthesia with pentobarbital sodium, and were then necropsied. The kidneys were removed and weighed together (PM400, Mettler-Toledo, Greifensee, Switzerland). The following organs were microscopically examined: kidneys, liver, lung, heart, spleen, urinary bladder, brain, thyroid, parathyroid, pituitary, adrenal glands, testes, spinal cord, stomach, small intestine, large intestine, and femur (including bone marrow). These organs were fixed in 10% neutral buffered formalin, processed routinely, and embedded in paraffin. Paraffin sections were prepared and stained with hematoxylin and eosin (H&E). The kidney sections were also stained by the von Kossa, periodic acid-Schiff (PAS) reagent, periodic acid-methenamine-silver (PAM) stain, and Masson’s trichrome stain.

For transmission electron microscopic observation, renal cortical tissues from two animals per group were fixed in 3% glutaraldehyde immediately and 2% osmic acid and embedded in epoxy resin. Ultrathin sections were double-stained with uranyl acetate and lead citrate and examined under an electron microscope (JEM-1010, JEOL Ltd., Tokyo, Japan).

Renal cortical tissues from two animals per group were also fixed by 2.5% glutaraldehyde and were processed for scanning electron microscopy employing a ligand osmium tetroxide and 1% tannic acid solution during secondary fixation followed by ethanol dehydration. The samples were critical point dried using an HCP-2 critical point dryer (Hitachi Koki Co., Tokyo, Japan) and coated with palladium-gold using an SC500A Sputter Coater (EMscope Co., Kent, UK) before being viewed with scanning electron microscope (S-800, Hitachi High-Tech Fielding Corporation, Tokyo, Japan).

Immunohistochemistry

To assess the proliferating activity and functional changes of the glomerulus, kidney sections were subjected to indirect immunoperoxidase staining using the following primary antibodies: proliferating cell nuclear antigen (PCNA, 1:200, Dako Corporation, Carpinteria, CA, USA); podoplanin (specific markers for podocytes and parietal epithelial cells, 1:1600, P1995, Sigma, St. Louis, MO, USA); and desmin (1:100, M0760, Dako Corporation). Four-micron–thick sections of formalin-fixed, paraffin-embedded kidney were deparaf-finized, rehydrated and then incubated with pepsin (Dako Corporation) at 37°C for twenty minutes or boiled in citric acid (10 mmol; pH 6.0) for ten minutes to unmask antigens. The sections were immersed in PBS (pH 7.4) containing 3% H₂O₂ to inactivate endogenous peroxidase. Equilibration buffer was applied to the sections for thirty minutes at room temperature. Immunoperoxidase staining was performed in accordance with the N-Histofine Simple Stain Rat MAX PO kit (Nichirei Biosciences Inc., Tokyo, Japan) or Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA), and the staining was visualized with diaminobenzidine. They were then counterstained with Mayer’s hematoxylin, dehydrated, and mounted.

Semiquantitative Analysis of the Glomerulus

Semiquantitative analysis was performed for the results of immunohistochemical examination by assessing the amount of podoplanin or desmin expression in glomeruli and counting the number of PCNA-positive cells per field at 400X magnification. Fifty to 60 glomeruli in two sections were randomly chosen and examined (n = 3 per group). The numbers of PCNA-positive cells per glomerulus were counted (Gross et al. 2004). The extent of glomerular expression of podoplanin was also evaluated. Scores were assigned to individual glomeruli in each section as follows: 0, no expression; 1, expression in few glomeruli; 2, up to 50% of glomeruli with positive cells; 3, 50%–75% of glomeruli with positive cells; 4, positive cells all over the glomeruli (Gross et al. 2004; Macconi et al. 2006). For analysis of desmin immunohistochemistry, the capillary tuft was divided into four quarters, and the following scoring system was used: 0, no expression; 1, desmin-positive cells in 25% of the capillary tuft; 2, desmin-positive cells in 50% of the capillary tuft; 3, desmin-positive cells in 75% of the capillary tuft; and 4, desmin-positive cells in 100% of the capillary tuft. The final score per section was then calculated as the weighted mean as follows: S_desmin= [(0×N₀) + (1×N₁) + (2×N₂) + (3×N₃) + (4×N₄)]/[N₀+N₁+N₂+N₃+N₄], where Ni (i = 0 to 4) is the number of glomeruli in each category (Gross et al. 2004; Macconi et al. 2006).

Statistical Analysis

All continuous data are expressed as means ± standard deviation (SD). Statistical significance was analyzed by Aspin-Welch’s t test for body and kidney weights, blood chemistry, and hematology, the cumulative chi-square test was used for urinalysis, and the Mann-Whitney test was used for immunohistochemistry compared with each control group.

Results

Clinical Observations and Biochemical Analysis

Table 1 shows laboratory data in the control and phosphate-treated rats. In all animals, neither abnormal clinical signs nor body weight changes were observed. Increased urinary protein excretion was first detected on day 3 in six out of ten rats. On day 8 or later, all phosphate-treated rats showed consecutive moderate to severe proteinuria. Even fourteen days after cessation of treatment (day of dissection), rats in the fourteen-day dosed group continued to excrete urinary protein (Table 1). Urinary volume from phosphate-treated rats tended to be larger compared with control animals. No abnormalities were observed for any other items.

Plasma cholesterol and triglyceride were significantly higher (up to 4.8-fold) and the A/G ratio was significantly lowered by 60% because of low plasma albumin level. The number of RBC, packed cell volume, and hemoglobin level decreased by 70% to 80% compared with control groups. Platelet count was up to 1.4-fold higher and fibrinogen was elevated up to 2.3-fold. There were no changes in inorganic phosphorus and calcium level in the phosphate-treated groups. Plasma creatinine and urea nitrogen in phosphate-treated animals tended to be higher than in the controls.

Necropsy Findings

Treatment-related higher absolute and relative kidney weights were observed in the phosphate-treated rats (Table 2). The kidneys from all phosphate-treated rats were pale in color, markedly enlarged, and had a rough surface (Figure 1).

Light Microscopy

No significant changes were observed in the saline-administered control animals. In the phosphate-treated rats, the glomeruli showed severe calcium deposition in the GBM of the vascular tufts and Bowman’s capsule, parietal epithelial cells, and mesangial matrix. Dilatation of the capillary lumen by mineralization and thickening of the basement membrane were also observed in the glomeruli. Glomerular spaces were slightly enlarged, and expanded glomerular tufts partially adhered to the wall of Bowman’s capsule. Parietal epithelial cells were hypertrophic but accompanied by mineralization in some glomeruli. A number of glomeruli showed mild mesangiolysis, and a few glomeruli displayed notable focal mesangial sclerosis. Desquamated podocytes with giant or multiple nuclei appeared occasionally in the Bowman’s space (Figure 2).

In the twenty-eight-day group, proximal tubules showed marked nuclear enlargement suggesting regenerative changes, increased mitosis, dilatation with occasional protein casts, and mineralization of the tubular basement membrane. The damage of proximal tubules was characterized by scattered single cell death, detached necrotic epithelial cells in the lumen, cytoplasmic blebbing at the luminal surface, cytoplasmic vacuolization, and granular degeneration. Hyaline droplets were observed less frequently in the tubular epithelial cells. A patchy interstitial cellular infiltrate consisting predominantly of mononuclear cells was noted, and fibrosis was also observed in the cortex by Masson’s trichrome stain (Figure 2). Focal tubular regeneration and hyaline casts were also seen in the medulla of one animal from the twenty-eight-day group. The animals receiving phosphate solution per day for fourteen days followed by a fourteen-day withdrawal period showed no evidence of recovery. On the contrary, even after the withdrawal period, the lesions showed more deterioration compared to the morphological changes during the fourteen days of administration, with focal infiltration of mononuclear cells and mild fibrosis.

Electron Microscopy

Transmission electron microscopy showed high deposition of lamellar structures in the GBM, mesangial area, and podocytes, capillary dilatation, and thickening of the GBM in the phosphate-treated rats. The cell processes of the podocytes often fused with each other, the number of microvilli increased, and the Bowman’s space was filled with large amounts of debris (Figure 3). Scanning electron microscopic examination demonstrated effacement of foot processes and marked increase of microvilli of the podocytes (Figure 4).

Immunohistochemistry

There were no significant differences in the number of PCNA-positive glomerular cells between treated and control animals. PCNA expression markedly increased in enlarged nuclei of the renal tubules in the cortex.

In the control rats, podoplanin was expressed in a linear pattern on the cell membranes of podocytes and the parietal epithelial cells of the Bowman’s capsule. The number of podoplanin-positive podocytes and parietal epithelial cells in each glomerulus decreased in both phosphate-treated groups, and decreased podoplanin expression was significant in podocytes of the twenty-eight-day group (Figures 5 and 7).

Conversely, anti-desmin antibody reacted mainly with the mesangial cells and vascular smooth muscle cells, and very weak positive staining was observed in the podocytes of the control kidney. Desmin expression extended over half of the glomerular tuft and tended to increase in the phosphate-treated groups compared to the control groups (Figures 6 and 8).

Findings for Other Organs/Tissues

The spleen was enlarged in all rats in the twenty-eight-day group. Histopathological examination revealed focal mineralization in the alveolar septum of the lung, small artery wall in the heart, endothelium of the aortic arch, and interstitium of the thyroid gland and the parathyroid gland in the twenty-eight-day group. The spleen was congestive and showed excessive extra-medullary hematopoiesis. In the withdrawal group, mineralization was also observed in the thyroid gland. There were no lesions in any other organs including the bone in phosphate-treated animals.

Discussion

Dibasic sodium phosphate solution administered by bolus injection for twenty-eight days induced severe glomerular lesions in Sprague-Dawley rats. The induced glomerular changes were not only irreversible but also progressive, because clinical parameters and morphological changes in the animals that received phosphate solution per day for fourteen days continued to deteriorate even after a fourteen-day withdrawal period. The clinical and morphological characteristics of the renal changes induced were very similar to those of chronic nephrotic syndrome.

The histopathological changes also suggested severe structural and functional damage of glomeruli, as well as secondary damage of the tubular epithelium caused by severe proteinuria. Among the morphological changes of the glomerular components, the formation of numerous microvilli of podocytes was strong evidence of severe proteinuria (Olson 2007). Nuclear enlargement in the proximal tubular epithelial cells might reflect increased proliferating activity of the tubular epithelium or regenerative changes after destruction, although single cell necrosis was observed less frequently in the proximal tubules, and a relatively small number of detached necrotic epithelial cells were scattered in the lumen of the lower renal tubules.

The relationship between tubulointerstitial nephritis and proteinuria has been characterized for experimental nephrosis in rats (Robertson 1998). It has been proposed that various substances may directly cause tubular damages; for example, growth factors and other substances may cause up-regulation of cytokines/chemokines or the complement may be activated. Several in vitro studies have demonstrated that urinary proteins can stimulate proximal tubular cells to synthesize chemokines: monocyte chemoattractant protein-1 (MCP-1), Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES), and fractalkine, which recruit monocytes, and T-cells and interleukin-8, which attract neutrophils (Abbate et al. 2006; Eddy 2004). Albumin might activate the signal and transducer activator of transcription (STAT) 1 and STAT 5 pathways, which are important pathways from cytokine and growth factor receptors, in tubular cells (Nakajima et al. 2004), and also act as a carrier for other inflammatory mediators such as lipids (Eddy 2004). These findings suggest that a similar sequence of events may occur in vivo, providing a plausible mechanistic link between proteinuria and interstitial inflammation. In rats with overload proteinuria, up-regulation of MCP-1 and osteo-pontin and increased NF-κB activity in tubular epithelial cells were associated with an interstitial inflammatory reaction (Eddy 2004). Brunskill (2004) suggested that albumin may be acting as a cytokine by producing profibrotic substances and matrix proteins. Macrophages may also regulate matrix accumulation, primarily by producing fibrosis-promoting growth factors (e.g., TGF-β), vasoactive products (e.g., endothelin-1, angiotensin II), and products that impair matrix degradation (Eddy 2004). Although a series of reports has indicated a link between proteinuria and mononuclear cell accumulation into the interstitium in experimental models and patients, the true mechanism of tubular damage by proteinuria has not been fully explained for an in vivo experimental system (Abbate et al. 2006). The present system using rats may provide a useful tool for further clarification of tubular damage involved in proteinuria and other factors such as hypertension or lipidemia.

Human nephrotic syndrome is associated with a spectrum of primary and secondary glomerular diseases. The more common causes of nephrotic syndrome are minimal change disease (MCD), glomerulonephritis, and focal segmental glomerulosclerosis. As animal models of human nephrotic syndrome, aminonucleoside puromycin (PAN), adriamycin (doxorubicin), experimental histamine H₂-receptor antagonists, and 5-lipoxygenase inhibitor have been repeatedly reported to induce proteinuria and glomerular lesions. The induced nephrotoxicity has some features in common with human MCD or focal and segmental glomerular sclerosis (FSGS) (Bertani et al. 1982; Eddy and Michael 1988; Morley et al. 1997; Read et al. 1995; Robertson 1998; van der Vijgh et al. 1987). After seven to twenty-one days of treatment with these agents, electron microscopy revealed damage to podocytes and effacement of foot processes associated with proteinuria and functional changes, including increases in plasma cholesterol, triglycerides, urea, and creatinine and loss of plasma albumin. Tubular dilatation, casts with interstitial mononuclear cell infiltration, and fibrosis were revealed in rats administered 5-lipoxygenase inhibitors for fourteen or twenty-one days following intracardiac ADR injection or intraperitoneal PAN injection. Severe tubulointerstitial lesions have been reported over twelve weeks after injection in most experimental models. The Wistar rat strain has been frequently used as an experimental nephrosis model. Morphologic injury and nephrotic syndrome were more apparent in the Wistar-Munich strain than Sprague-Dawley rats in PAN nephrosis (Anderson et al. 1988). PAN or ADR could induce nephrosis in rats by central venous, intracardiac, or single intravenous or intraperitoneal injection, and other agents could lead to nephrosis with intravenous or repeated oral administration. The phosphate-treated nephrotic syndrome model differs from these experimental models; there was continuous proteinuria from days 3 to 28 and the occurrence of tubulointerstitial lesions in twenty-eight days with the Sprague-Dawley rat and a simple technique of administration by intravenous injection via the tail vein. Although experiments have demonstrated a high mortality rate and decreased body weight in the PAN nephrosis model, and hepatic toxicity and cardiotoxicity in rats by ADR administration (Anderson et al. 1988; Mettler et al. 1977; Rangan et al. 2001), the phosphate nephropathy model showed a lower mortality rate, no body weight suppression, and no fatal impact on other organs.

Many strains of rats develop age-related chronic progressive nephropathy (CPN) (Gray 1977; Peter et al. 1986; Solleveld and Boorman 1986). The Sprague-Dawley strain is considered one of the most susceptible to CPN, which becomes apparent by twenty weeks of age. Seely et al. have reported that glomerular hypertrophy or hyperfiltration have been postulated as the possible mechanism for glomerular damage and subsequent nephron loss as well as the spectrum of renal lesions of CPN (Seely et al. 2002). The basic pathogenetic mechanism has not been definitively determined, as CPN is a complex disorder; however, the essential lesions are very similar to proteinuria-induced nephropathy (Peter et al. 1986). CPN has been confounded with lesions of renal toxicity in young rats, and questions have been raised about the validity of the rat as a model for renal disease in humans. In the present study, the phosphate-induced nephropathy had similar morphological changes to early change in CPN in rats; however, tubulointerstitial lesions occurred by just ten weeks of age, and the extent of the lesion appeared not in individual nephrons but throughout the cortex.

Immunohistochemical examination revealed decreased podoplanin-positive podocytes and a tendency toward increased desmin expression. Podoplanin is a mucinlike substance expressed on the surface of rat podocytes that contributes to the negative charge (Matsui et al. 1998). In kidneys of adult rats, the expression of podoplanin shows an exclusively linear pattern on cell membranes of podocytes and the parietal epithelium of Bowman’s capsule (Breiteneder-Geleff et al. 1997). The decreased number of podoplanin-positive podocytes in the phosphate-treated rats suggests impairment of podocyte function. The up-regulation of desmin expression may play a critical role in the morphological changes of podocytes in response to injury (Zou et al. 2006).

Along with experimental data suggesting GBM defects, we detected protein overload, glomerular hyperfiltration, anemia, hypoalbuminemia, and hyperlipidemia in this study. The decrease in red cell parameters should be attributable to secondary effects of renal failure, because deficiency of erythropoiesis factor or decreased transferrin from renal disorder has been suggested to cause anemia, and no direct effect of dibasic sodium phosphate on the destruction of erythrocytes was recognized. The extramedullary hematopoiesis that increased in the spleen may have been a result of anemia in the twenty-eight-day group. Hypoalbuminemia has been suggested to play a key role in the pathogenesis of nephrotic hyperlipidemia (Bernard 1988). Hypercholesterolemia is one of the major features of nephritic syndrome in human and is, in part, caused by acquired low-density lipoprotein receptor deficiency (Kim et al. 2007). Furthermore, induction of hyperlipidemia was reported to accelerate glomerular and interstitial damage (Attman et al. 1997). Higher platelet counts and serum fibrinogen levels are also common laboratory findings in nephrotic syndrome owing to compensative coagulation mechanism (Cucuianu et al. 1991). Moreover, higher serum fibrinogen level might be caused by enhanced protein production function of the liver.

Early changes by fourteen-day injections detected in the kidney were noted in the glomeruli, in which podocytes showed effacement of foot processes and mineralization without any biochemical abnormality. In the more advanced lesions, the degree of foot process loss became more obvious, and changes in the glomeruli and renal tubules were visible by light microscopy. The morphological changes increased in severity with time, and symptoms similar to human nephrotic syndrome occurred. We conclude that this nephritic model can contribute to elucidating the mechanism of irreversible progress in renal injury and should serve as a useful tool for developing therapeutic agents to treat nephrotic syndrome.

Investigations are now under way to characterize the exacerbation process of renal lesions produced by high-dose dibasic phosphate solution and to search for ways to induce remission in chronic nephropathy.