Detection of glomerular sialic acids in patients with diabetic nephropathy

A study on immunofluorescence of sialic acids in glomeruli from patients with diabetic nephropathy is described. Measurement of sialic acid in sera from 25 patients with diabetes mellitus was also performed. Renal biopsy specimens from 12 patients with diabetic nephropathy were stained with FITC-labeled antihuman IgG antiserum and rhodamine-labeled Triticum vulgaris (WGA) or Limulus polyphemus (LPA). These specimens were also stained with such reagents after treatment with neuraminidase, trypsin or citrate buffer. Both deposition of IgG and binding of WGA in the glomerular capillary walls were observed in all patients with diabetic nephropathy. The binding of WGA in the glomerular capillary walls in diabetic nephropathy was significantly increased compared with that in four normal renal tissues. However, the binding of LPA was hardly observed in the glomerular capillary walls of patients with diabetic nephropathy. The binding of WGA in the glomeruli was markedly decreased after treatment with neuraminidase although it was hardly decreased after treatment with trypsin or citrate buffer. The levels of sialic acid in sera from patients with diabetic nephropathy were markedly increased. It is suggested that accumulated substances in the glomerular capillary walls with an affinity for WGA are mainly composed of N-acetyl glucosamine and/or N-acetyl neuraminic acid in patients with diabetic nephropathy.     

Gouty kidney associated with membranous nephropathy: participation of renal tubular epithelial antigen

Histological studies were performed on 3 patients with gout and proteinuria measured at 1.0 g a day or more. Light microscopy revealed diffuse thickening of the glomerular capillary walls accompanied by spike formation and bubble-like appearance as well as tophaceous granuloma in the interstitium, tubular atrophy and benign nephrosclerosis. Immunofluorescence technique showed fine granular deposits of IgG and C3 along the glomerular capillary walls together with the renal tubular epithelial antigen (RTE) in 1 patient. Subepithelial dense deposits were also observed by electron microscopy. These findings suggest that the association of membranous nephropathy should be considered in patients with gout having moderate to severe proteinuria and that RTE may be involved in the pathogenesis of subepithelial deposits in gouty membranous nephropathy.     

Heparan sulfate proteoglycans are lost in patients with diabetic nephropathy.

The pathogenesis of diabetic nephropathy relative to the changes in the glomerular extracellular matrices was investigated. Renal tissues from 10 diabetic patients were immunostained with antibodies directed against heparan sulfate proteoglycans (HS-PGs), laminin, type IV collagen and fibronectin. Seven patients were nephrotic and had advanced glomerulosclerosis with nodular lesion, while the other 3 had no renal manifestations or minor glomerular tissue alterations. Controls included kidneys removed from patients with renal tumors and specimens obtained by renal biopsy from patients with IgA nephropathy. Relationships among proteinuria, intensity of fluorescence and glomerular changes were studied. In diabetes 3 patients with minor glomerular lesions were found to have no changes in various components of extracellular matrices. A marked reduction in the intensity of staining with anti-HS-PG antibodies was observed in renal specimens from patients with nodular glomerulosclerosis and proteinuria, while a mild decrease in the intensity of fluorescence was observed in tissues stained with antilaminin antibodies. An increase compared to normal control sample findings in type IV collagen and fibronectin was observed in the mesangium of sclerosing glomeruli. No loss of HS-PG was observed in patients with IgA nephropathy. These results indicate that glomerular extracellular matrix HS-PG is lost in association with diabetic nephropathy; this loss results in alteration of the charge-selective properties of glomerular capillaries. This alteration may, in part, be the cause of the proteinuria associated with diabetic nephropathy.     

Clinicopathological correlation of IgA nephropathy in children

22 patients with IgA nephropathy aged 7-16 years, 15 of whom were found by mass urine screening of school children, were divided into three groups based on the degree of their proteinuria at admission: group A (n = 6) below 0.5 g/day of urine protein, group B (n = 7) between 0.6 and 3.0, and group C (n = 9) above 3.0 g/day of urine protein. The degree of proteinuria seemed to be related to the severity of pathological changes of the glomerular basement membrane; most severe in group C, moderate in group B, and minimal in group A. IgA deposits in the mesangial area were found in all groups of patients, but those in the capillary walls were most frequently found in group C. In addition to electron-dense deposits in the mesangial area, which was found in all groups of patients, the subendothelial and subepithelial deposits were the most remarkable changes found in group C. During the clinical observation period, which was between 2.0 and 7.1 years, no patient belonging to group A progressed to groups B or C. 4 cases in group C developed chronic renal failure, but none in the other groups did. The amount of urine protein might be used as a valuable parameter of the pathological damage of the glomerulus in children with IgA nephropathy.     

Clinicopathologic correlations in a series of 143 patients with IgA glomerulonephritis

In an unselected series of patients with IgA glomerulonephritis, old age, high blood pressure, and high urinary protein excretion at the time of renal biopsy were found to correlate with impaired renal function, whereas sex, estimated duration of the disease, or high serum IgA levels did not. The following clinical features were favorable prognostic signs: asymptomatic proteinuria, macroscopic hematuria, and isolated microscopic hematuria. The degree of diffuse mesangial alteration and the presence of segmental glomerular lesions correlated clearly with the subsequent clinical outcome. Vascular lesions, i.e. arteriosclerosis and renal vascular deposition of C3, were most often present in patients with severe glomerulopathy. The presence of electron-dense deposits in glomerular capillary walls was also an unfavorable prognostic finding. Renal biopsy findings of interstitial infiltrates of inflammatory cells and IgA distributed along glomerular capillary walls were usually associated with extrarenal manifestations of the disease.     

Specificity of IgA antibodies in sera from patients with IgA nephropathy.

A study was undertaken on the specificity of circulating IgA antibodies in patients with IgA nephropathy detected by immunofluorescence using avidin-biotin complexes. Renal biopsy specimens and serum samples were obtained from 33 patients with IgA nephropathy, 14 other glomerular diseases and 3 normal renal tissues. These renal specimens were treated with citrate buffer (pH 3.2), and then incubated with serum samples obtained from the same and other patients with IgA nephropathy, other glomerular diseases or healthy adults at 37 degrees C for 30 min. The specimens were incubated with biotin conjugated gout F(ab')2 anti-human IgA antiserum at 37 degrees C for 30 min, and then with fluorescein-labeled avidin at 37 degrees C for 30 min. It was found that IgA antibodies in the sera from patients with IgA nephropathy specifically combined with the autologous glomerular mesangial areas, but only 25.7% of them combined with allogeneic renal tissues of IgA nephropathy patients. Confirmatory findings were obtained using an automatic image analyzer. However, these IgA antibodies did not combine with the renal tissues from patients with other glomerular diseases or normal renal tissues. In parallel studies, in order to distinguish IgA nephropathy from other glomerular diseases before renal biopsy, the renal specimens from patients with IgA nephropathy were also incubated with serum samples obtained from 42 patients with proteinuria and/or hematuria before renal biopsy. It was demonstrated that the incidence of IgA binding in IgA nephropathy was significantly higher than that in other glomerular diseases prior to renal biopsy.(ABSTRACT TRUNCATED AT 250 WORDS)     

IgA affinity to ssDNA or endothelial cells and its deposition in glomerular capillary walls in IgA nephropathy

Autoantibodies to glomerular components may be pathogenetic in IgA nephropathy. We studied sera from 32 IgA nephropathy patients, 35 normal controls and 102 patients controls with other forms of glomerulonephritis for IgA isotype affinity to endothelial cells, denatured DNA (ssDNA), murine laminin, and cardiolipin measured by ELISA. Compared to the normal controls, patients with IgA nephropathy had significantly elevated IgA with affinity to endothelial cells (P = 0.01) and ssDNA (P = 0.04), but not to murine laminin or cardiolipin. Furthermore, serum IgA with affinity to ssDNA, but not to endothelial cells, was associated with the presence of IgA deposited in the glomerular capillary walls (P = 0.011), an indicator of poor prognosis in patients with IgA nephropathy. Therefore, IgA with affinity to ssDNA may represent an autoantibody with pathogenetic significance in IgA nephropathy.     

Significance of IgA deposits on the glomerular capillary walls in IgA nephropathy

Based on immunofluorescence findings, 232 patients with IgA nephropathy were classified into two groups; one consisted of 88 patients (38%) with IgA deposits in the glomerular capillary walls together with the mesangial deposits (capillary type), and the other consisted of 144 patients (62%) with deposits confined to the mesangium (mesangial type). Electron microscopic findings revealed dense deposits on the capillary walls (subepithelial, 50%; intramembranous, 65%; and subendothelial, 24%) in 37 of 46 patients with capillary type and six of 47 with mesangial type (P less than .001). Crescent formation observed in greater than or equal to 10% of glomeruli was more frequently found in patients with the capillary type (30/88, 34%) than those with the mesangial type (9/144, 6%) (P less than .01), especially higher in those with subepithelial deposits (15/26, 57%). The capillary type patients showed heavier proteinuria (1.7 +/- 0.2 g/d) than the mesangial type patients (0.6 +/- 0.1 g/d) (P less than .05). Thirteen of the 14 patients in an acute exacerbation phase, manifested by an abrupt increase in urinary protein and development of macroscopic hematuria, showed capillary type IgA deposits. The ratio of patients with normal renal function in the fifth year after apparent onset was lower in the capillary type (74.0%) than in the mesangial type patients (96.9%) (P less than .05). These findings suggest that capillary IgA deposition is closely related to clinical and histologic activities of IgA nephropathy and is considered to be an important factor responsible for the progression of the disease, possibly through crescent formation.     

Participation of complement components in glomerular deposition in IgA nephropathy

To clarify the role of complement components in glomerular deposition in IgA nephropathy, clinicopathological and immunohistological studies were performed on 299 patients (171 males and 128 females; age, 9-71 years). Glomerular IgA deposition with IgG and/or IgM was observed more frequently in patients with Clq and/or C4 than in those with only C3 deposition (P less than 0.001). Patients with glomerular deposition of Clq and/or C4 showed more severe proteinuria (1 g/24 hr less than), a lower glomerular filtration rate (GFR), a higher incidence of duplication of capillary walls and more severe proliferation of mesangial cells and an increase in mesangial matrix (P less than 0.05), as compared to those without both Clq and C4. Patients with glomerular C3 deposition had significantly lower serum CH50 levels at the time of renal biopsy (P less than 0.02) and a significantly higher incidence of sclerotic lesions (P less than 0.05). Patients with C3 deposition in the mesangium and peripheral capillaries had significantly higher serum IgA levels (P less than 0.02), a significantly higher incidence of adhesion (P less than 0.01), duplication and endocapillary proliferation (P less than 0.05) and a more severe increase in mesangial cells (P less than 0.01) than those with C3 deposition only in the mesangium. The above findings demonstrate that analysis of the complement system in glomeruli is important for the evaluation of glomerular damage, clinical findings and prognosis.     

Immunohistochemical analysis of extracellular components on the glomerular sclerosis in patients with glomerulonephritis and diabetic nephropathy

Immunofluorescence and immunoperoxidase staining were carried out to determine the correlations between the progression of glomerular sclerosis and changes in the amount and distribution of glomerular extracellular components, such as Type I, III, IV, V, VI collagen, laminin (LN) and fibronectin (FN) in patients with various types of glomerulonephritis and diabetic nephropathy. Six patients with IgA nephropathy, four patients with membrano-proliferative glomerulonephritis, four patients with rapidly progressive glomerulonephritis and six patients with diabetic nephropathy were examined. The intensity and distribution of Type IV collagen, LN and FN were similar between the glomeruli from normal individuals and patients with mild stages of glomerulonephritis and diabetic nephropathy. However, staining of Type I, III or V collagen was not observed in the glomeruli from normal individuals and such patients. In more advanced stages of glomerulonephritis and diabetic nephropathy, the amounts of Types IV and VI collagen, LN and FN were increased markedly in the mesangium, and their distribution extended along the glomerular capillary walls. The intensity of Type IV collagen, LN or FN in the nodular sclerotic lesions of glomeruli was decreased significantly in patients with glomerulonephritis and diabetic nephropathy. On the other hand, staining of Types I, III and V collagen was observed focally in the sclerotic or hyalinotic glomeruli and around such glomeruli in these patients. In light microscopic examinations, the patients who had marked staining of Type I, III or V collagen by immunofluorescence showed severe damage of the basement membrane in Bowman's capsules. It is concluded that hyperproduction and/or infiltration of interstitial collagens, i.e. Types I, III and V collagen, is closely linked to the progression of glomerular sclerosis and hyalinosis in patients with various types of glomerulonephritis and diabetic nephropathy.     

Abnormal distribution of mesangium-specific laminin in glomeruli of patients with idiopathic membranous nephropathy

Using a monoclonal antibody, the distribution of the mesangium-specific laminin that binds only to the mesangium in normal kidney, was studied immunohistochemically in renal tissues obtained from patients with different glomerulonephritides including idiopathic membranous nephropathy. In minimal change nephrotic syndrome and IgA nephropathy, the mesangium-specific laminin staining was restricted to the mesangium, as is observed in the normal kidney. However, in patients with idiopathic membranous nephropathy, the monoclonal antibody against the mesangium-specific laminin reacted with the glomerular peripheral capillary walls including subepithelial protrusions or 'spikes', as well as with the mesangium. These results suggest that the quality of laminin produced in response to the challenge of immune deposits in the peripheral capillary walls may be different from that in the rest of the glomerular basement membrane in idiopathic membranous nephropathy.     

Combined glomerular deposition of polymeric rat IgA and IgG aggravates renal inflammation

BACKGROUND: IgA nephropathy (IgAN) is characterized by deposition in the glomerular mesangium of IgA together with C3, C5b-9, and properdin. IgG deposition as a risk factor in IgAN was recently confirmed by a long-term follow-up of patients with IgAN. We previously reported on an acute model of IgA-mediated glomerular inflammation in Wistar rats. METHODS: To investigate the effect of the combination of IgA and IgG on glomerular injury, Wistar rats were injected with a minimum dose of rat IgG in the presence or absence of a subnephritogenic dose of polymeric rat IgA. Subsequently, glomerular complement activation, influx of inflammatory cells, proteinuria, and hematuria were assessed. RESULTS: Administration of IgG to the rats resulted in maximal proteinuria of 20.3 +/- 12.1 mg/24 h on day 2 and an absence of overt glomerular inflammation. Administration of polymeric rat IgA antibodies to rats resulted in hematuria with a moderate mesangial complement deposition. In the combination group, however, glomerular deposition of C5b-9 was dramatically increased. This was accompanied by increased proteinuria as compared with rats receiving IgA or IgG antibody injections alone on day 7. Microhematuria occurred in rats receiving either polymeric rat IgA or IgG alone or the combination. While both rat IgG and polymeric IgA induced minor mesangial cell (MC) proliferation and MC lysis, the combination resulted in a pronounced, significant increased percentage of aneurysm formation on day 7 after injection. CONCLUSIONS: We conclude that in this model of IgA-induced glomerulopathy, a selective, complement-dependent glomerular inflammation is induced in Wistar rats by glomerular codeposition of rat isotypic monoclonal antibodies.     

Impaired solubilization of glomerular immune deposits by sera from patients with IgA nephropathy

A study of the solubilization of glomerular immune deposits by sera from patients with IgA nephropathy is described. Renal biopsy specimens were obtained from patients with IgA nephropathy and other glomerular diseases. These specimens were incubated with fresh and heated sera from the same patients and healthy adults at 37 degrees C for one hour in plastic tubes. The sections were stained with fluorescein isothiocyanate (FITC)-labeled heavy chain specific anti-human IgA antiserum and then examined with a fluorescent microscope. It was shown that the solubilization of glomerular immune deposits by sera from patients with IgA nephropathy was significantly less than that by sera from healthy adults. It is possible that impaired solubilization of immune complexes in vivo could lead to the accumulation of glomerular immune deposits in patients with IgA nephropathy.     

A clinicopathologic study of thrombotic microangiopathy in the setting of IgA nephropathy

BACKGROUND: IgA nephropathy is the most common glomerulonephritis in the world. Thrombotic microangiopathy occurs in a number of clinical settings, including but not limited to thrombotic thrombocytopenic purpura/hemolytic uremic syndrome, malignant hypertension, anti-phospholipid antibody syndrome and radiation nephropathy. Renovascular complications, such as thrombotic microangiopathy, in the setting of IgA nephropathy may be overlooked and their significance as a concomitant histologic finding is unclear. METHODS: We conducted a clinicopathologic study to understand the possible relationship between IgA nephropathy and a concurrent thrombotic microangiopathy injury process. We identified 10 patients with an established diagnosis of IgA nephropathy and concurrent findings of thrombotic microangiopathy based on their renal biopsies. RESULTS: Six patients presented with malignant hypertension, while three others had severe hypertension (> or = 100 mmHg, diastolic). Five patients had nephrotic-range proteinuria. Seven patients had occasional arteriolar thrombi identified by light microscopy and prominent glomerular subendothelial space widening by electron microscopy, while three patients demonstrated only ultrastructural features of thrombotic microangiopathy. Other possible etiologic causes of thrombotic microangiopathy were not identified with the available clinical information. CONCLUSION: Our study suggests that a thrombotic microangiopathy injury, when present, is usually found in advanced stages of IgA nephropathy and can be associated with severe proteinuria. Although other possible causes of thrombotic microangiopathy, such as anti-phospholipid antibody syndrome, were excluded in only two patients, the thrombotic microangiopathy injury process may be a cause or a consequence of the severe hypertension encountered in most of the patients which, in turn, may be a consequence of the disease progression of IgA nephropathy.     

Correlations among expression of glomerular intercellular adhesion molecule 1 (ICAM-1), levels of serum soluble ICAM-1, and renal histopathology in patients with IgA nephropathy.

The purpose of the present study was to evaluate the correlations among expression of intercellular adhesion molecule 1 (ICAM-1) in glomeruli, levels of soluble ICAM-1 (sICAM-1) in sera, and renal injuries in patients with IgA nephropathy. The levels of sICAM-1 in sera from 27 patients with IgA nephropathy and 7 healthy controls were measured by the human soluble ICAM-1 immunoassay. The expression of ICAM-1 in glomeruli was detected by indirect immunofluorescence. We observed marked expression of ICAM-1 in glomerular capillary walls and mesangial areas in patients with advanced-stage, but not in those with mild IgA nephropathy. Since the histopathological changes in the advanced stage of this disease were characterized by diffuse mesangial cell proliferation and tubulointerstitial injury, the expression of ICAM-1 in the glomeruli may be of value in evaluating the degree of renal lesions in patients with IgA nephropathy. However, there was no significant change in the levels of serum sICAM-1 among mild-stage and advanced-stage patients and healthy controls. It appears that the measurement of serum sICAM-1 is not useful in evaluating the degree of renal injuries in patients with IgA nephropathy.     

Immunohistochemical analysis of extracellular components in the glomerular sclerosis of patients with glomerulonephritis

Immunofluorescence and immunoperoxidase staining was carried out to determine correlations between the progression of glomerular sclerosis and changes in the amount or distribution of glomerular extracellular components, including type I, III, IV, and VI collagens, laminin and fibronectin, in patients with IgA nephropathy, membranoproliferative glomerulonephritis and rapidly progressive glomerulonephritis. Staining of type I and III collagens was not observed in glomeruli from normal individuals or patients with mild glomerulonephritis. In the advanced stages of glomerulonephritis, the staining of type IV and VI collagens, laminin and fibronectin was marked in the glomerular mesangium, and the distribution of fibronectin extended to the glomerular capillary walls in the sclerotic lesions of glomeruli. However, the staining intensity of type IV collagen, laminin and fibronectin was gradually decreased during the progression of glomerular sclerosis. On the other hand, the staining of type I and III collagens was observed focally in sclerotic or hyalinotic glomeruli and around such glomeruli in those patients. Light microscopic examination revealed that patients who showed marked staining of type I and III collagens by immunofluorescence had severe damage of Bowman's capsules. These results suggest that the hyperproduction and/or invasion of interstitial collagens, i.e., types I and III, are closely linked to the progression of glomerular sclerosis and hyalinosis in patients with various types of glomerulonephritis.     

Ultrastructural alterations of glomerular anionic sites in IgA nephropathy

The ultrastructural alterations of glomerular anionic sites were studied in biopsy specimens from 34 patients with IgA nephropathy using polyethyleneimine (PEI). Prominent common findings in the glomeruli of the patients were few PEI particles in electron dense deposits in the mesangial and subepithelial area and marked reduction in glomerular anionic sites covered with deposits. The anionic sites of the glomerular basement membrane (GBM) and epithelial cell surface coat (ESC) appeared unaltered in the patients with hematuria and/or mild proteinuria. But in patients with proteinuria in the nephrotic range, focally discrete loss of anionic sites in the lamina rara externa (LRE) was seen and the number of anionic sites of the ESC were decreased with retraction of the foot processes. The anionic sites of the lamina rara interna showed much less change in these patients. Subepithelial deposits were often seen concomitantly with focal loss of anionic sites in the LRE at the site of the deposits, but subendothelial deposits had little influence on the anionic sites of the neighboring GBM. The anionic sites of GBM that showed focal thinning with small GBM projections were appreciably decreased in number, but those in split GBM were not decreased. These results suggest that either loss of the negative charge on the glomerular capillary wall associated with subepithelial immune deposition or morphological changes of the GBM contribute to the progression of proteinuria in IgA nephropathy.     

Prolonged membranous lupus nephritis with change of anti-ssDNA antibody titer and repeated renal relapse

We report a case of a 44-year-old woman with nephrotic syndrome who underwent renal biopsy three times. On each occasion, light microscopy showed membranous nephropathy with mild to moderate thickening of the glomerular capillary walls. Immunofluorescence microscopy showed predominant deposition of immunoglobulin (Ig) G, IgG1, IgG2, IgG3, and IgG4; C3; and C1q along the glomerular capillary walls and deposition of IgM and IgA in some parts of the walls. Electron microscopy revealed the accumulation of electron-dense deposits in the mesangium and the subepithelial area of the glomerular basement membrane. Virus-like particles were detected in the subendothelial cells in all three biopsy specimens. A definitive diagnosis of systemic lupus erythematosus (SLE) was made at the time of the second admission, when she was 31 years old. A diagnosis of membranous lupus nephritis was then made on the basis of the pathological and clinical findings. A change in anti-single-stranded (ss)DNA antibody titers was of particular interest in this patient. Occasional small increases in anti-double-stranded (ds)DNA antibody were found, but increased anti-ssDNA antibody titers occurred before there was any elevation of urinary protein during renal relapse, and a sustained increase in the titers was shown subsequently. Hypocomplementemia occurred in parallel with the increase of anti-ssDNA antibody. Immunosuppressive therapy with steroid promptly eliminated anti-dsDNA antibody, but anti-ssDNA antibody remained positive. The patient had normocomplementemia and proteinuria was absent. Later, anti-ssDNA antibody decreased. Renal function has remained in the normal range for 20 years.     

Glomerular deposition and serum levels of complement control proteins in patients with IgA nephropathy

We examined complement control proteins focusing on the role of modulating the complement activation in IgA nephropathy. Glomerular C4-binding protein (C4-bp) deposits were found in 60% of patients with IgA nephropathy, while C4 deposits were found in 30%. Glomerular deposits of beta 1H globulin (beta 1H) were found in 85% of patients with IgA nephropathy. The frequency of glomerular deposits of C4-bp tended to be higher in the group with deposits of various immunoglobulin types than in the group with deposits of IgA alone, and it increased parallel with the progression of glomerular histologic changes. The serum C4-bp level was higher in IgA nephropathy patients. No significant correlation was found between the serum level of C4-bp or beta 1H and the extent or distribution of tissue deposits of these complement control proteins. These results suggest that glomerular immune deposits in IgA nephropathy may be attributable to the activation not only of the alternative pathway but also of the classical pathway, the latter of which may take place locally in glomeruli with advanced histologic change or various immunoglobulin deposits in IgA nephropathy.     

Specific binding of circulating IgA antibodies in patients with IgA nephropathy

Detection of circulating IgA antibodies which are specific in patients with IgA nephropathy is described. Freeze and thawed extracts of pharyngeal cells obtained from patients with IgA nephropathy, other glomerular diseases, and healthy adults were cultured with fibroblasts such as Vero or Hel cells at 37 degrees C for 2 weeks. Serum samples were obtained from these patients and healthy adults. The cultured fibroblasts were fixed on slide glasses, and then incubated with the serum samples from the same or other patients with IgA nephropathy. The cells were stained with FITC-labeled heavy-chain specific anti-human IgA antiserum and then examined with a fluorescent microscope. It was demonstrated that the IgA antibodies in sera obtained from patients with IgA nephropathy or HSP nephritis were bound with the nuclear regions of such fibroblasts. It was suggested that IgA antibodies in sera could be bound with some antigenic substances which were transferred from pharyngeal cells of patients with IgA nephropathy to fibroblasts in vitro.