Immunoreactive basement membrane antigens in normal human urine and serum.

Using a sheep antiserum to human glomerular basement membrane (GBM), studies of urine from healthy adults showed the presence of two cross-reactive antigens. These antigens were purified partially by preparative electrophoresis and electrofocusing, and separated on G-200; both appeared to be acidic, of high molecular weight, and carbohydrate rich. Their immunologic relationship to human GBM solubilized by several techniques was deduced from lines of identity with the native GBM digests in double diffusion analyses. These antigens will combine with homologous anti-GBM antibodies and block their fixation to human kidney sections, and will evoke heterologous anti-GBM antibody production in the rabbit. Fractionation studies of normal human serum indicated the presence of trace amounts of basement membrane antigens in the circulation. Although the serum antigens appear immunologically identical to the urinary antigens, the precise anatomic structures from which both are derived is not certain. Demonstration of immunoreactive basement membrane antigens in the circulation provides a plausible source of immunogen for the potential development of anti-GBM antibody-mediated glomerulonephritis as well as a clue to a mode for reestablishment of tolerance in such an autoimmune disorder.

Glomerulonephritis mediated by antibody to glomerular basement membrane. Immunological, clinical, and histopathological characteristics.

A prospective study was undertaken to establish the incidence of glomerular basement membrane (GBM) antibody-mediated glomerulonephritis and its histopathological characteristics in a clinical group of patients presenting with renal disease. Biopsies from 43 of 409 consecutive patients technically satisfactory for direct immunofluorescent (IF) examination had diffuse and generalized linear localization of host immunoglobulin (Ig); two other badly scarred kidneys tested negative to IF although GBM antibodies were eluted. Confirmatory evidence of GBM antibody-mediated disease in these patients came from whole kidney or biopsy elutions (15 patients), serologic assays for circulating GBM antibodies by indirect IF (9 of 38 patients), radioimmunoassay (26 of 34), and hemagglutination (31 of 32). Although sera were not tested from six patients, circulating antibodies were demonstrated by some test in 36 of 39 of the remainder. Histologically, half of the patients had minor and nonspecific glomerular abnormalities or mild focal proliferative glomerulonephritis. More severely involved kidneys had focal necrotizing (17%), rapidly progressive (7%), and chronic, usually sclerosing, glomerulonephritis (27%). Clinical courses of these patients comparably were quite variable, ranging from indolent microhematuria and/or gross hematuric bouts to progressive renal failure; nephrotic syndrome was observed in 11 patients. GBM antibody-mediated glomerulonephritis may be a relatively mild disease with apparently stable renal function, although 16 patients have experienced functional deterioration, and 11 have progressed to dialysis, renal transplantation, or death.

Observations on the mechanisms of glucosuria during glucose loads in normal and nondiabetic subjects.

The study of 14 normal young men by glucose titration procedures has defined the magnitude of splay in this population, differing from previously reported data in its unexpected deviation from the line of theoretic unity high on the titration curve. Compared to these normal subjects, a group of glucosuric men could be divided into two subclasses, those with normal maximal rate of glucose reabsorption (Tm(G)) and those with subnormal Tm(G), both with comparably abnormal splay. Most consistent glucosurics fall into the latter group. Nephritic patients studied were not such a homogeneous group in terms of age and sex, but did manifest an abnormal splay during their titration curves in most cases. They also demonstrated a greater than normal reabsorptive rate of glucose per unit measured glomerular filtration rate. It is concluded that renal glucosuria must be defined not only in terms of the concept of Tm(G) but also by deviation of the glucose titration curve expressing an unusual degree of splay. The latter is presumed, as has been suggested by others, to be a characteristic of nonhomogeneity of glucose handling units in the kidney. This seems subject to exaggeration in the adaptations which accompany chronic renal disease.

Characterization of human anti-glomerular basement membrane antibodies eluted from glomerulonephritic kidneys.

Eluates from glomerulonephritic kidneys of nine patients with anti-glomerular basement membrane (anti-GBM)-mediated nephritis were studied to define their antigenic specificity and content of kidney-fixing antibodies. Five of these patients had Goodpasture's syndrome with pulmonary and renal involvement clinically; four patients did not. All had in vivo fixation of IgG in the characteristic linear pattern by direct immunofluorescence, and eluted IgG fixed to normal human kidney sections. Eluates from kidneys of patients with Goodpasture's syndrome fixed more frequently to homologous nonglomerular renal and extrarenal antigenic sites and to heterologous GBM than did non-Goodpasture eluates over a hundredfold range of antibody concentrations; both could be blocked by prior absorption with soluble GBM antigens. By radial immunodiffusion and precipitation tests the content of IgG in the eluates was measured to range from 2 to 20% of the total protein eluted. By paired label isotopic fixation studies with some of the eluates the per cent of IgG that was kidney fixing ranged from 0.6 to 23.4%. Although the in vivo fixation studies with radiolabeled eluates failed to indicate significant fixation to monkey lung, the observations define quantitative as well as qualitative differences between anti-GBM antibody populations mediating the Good-pasture syndrome compared to those causing glomerulonephritis without lung involvement.

Specificities of antibodies eluted from human cadaveric renal allografts. Multiple mechanisms of renal allograft injury.

The purpose of the present experiments was to evaluate the role of circulating antibodies in the rejection of human renal allografts and to study the apparent target(s) for antibody binding. Eluates obtained from surgical biopsy and nephrectomy specimens of rejecting, cadaveric human renal allografts were tested for antibodies directed to structural antigens of normal kidney and for cytotoxic antibody activity against mononuclear cell populations. By indirect immunofluorescence 23 of 35 eluates contained immunoglobulin that bound to normal kidney. Staining was in smooth muscle only in 10 patients, in smooth muscle and other structures such as tubular basement membranes, proximal cells, or brush border in 9 patients, and in structures other than smooth muscle in 4 patients. All 16 eluates tested contained antibodies cytotoxic for cells derived from a panel of normal volunteers. Six were cytotoxic to T cells and 10 to B cell and monocyte-enriched preparations. Absorption of eluates with pooled buffy coat cells, platelet concentrates and packed, cultured B cells removed antibodies reactive with vascular wall smooth muscle and endothelium, but not antibodies to tubular basement membranes, proximal or distal tubular cells, brush border, or other structures of kidney sections. Two of five eluates containing antikidney antibodies were found to bind to rat kidneys in vivo. These results suggest that circulating antibodies participate in cadaveric renal allograft destruction and demonstrate that they can be recovered directly from the allograft. Moreover, the data indicate that there are different antibody populations involved: some clearly directed to allo-specific differences and others that are apparently kidney-specific.

Effect of hemodialysis on left ventricular function. Dissociation of changes in filling volume and in contractile state.

Prior studies of the effect of hemodialysis on left ventricular function have not distinguished between the removal of uremic toxins and the change in cardiac filling volume. To separate these effects, left ventricular function was examined by serial echocardiography in five stable hemodialysis patients before and after three different dialysis procedures: (a) hemodialysis with volume Loss, (b) ultrafiltration (volume loss only), and (c) hemodialysis without volume loss. The patients were similarly studied under control conditions and after increased (5 degrees of head-down tilt for 90 min) and decreased (lower body negative pressure) cardiac filling volume. After hemodialysis with volume loss, end-diastolic volume (EDV) decreased from 167 to 128 ml (P less than 0.001) and end-systolic volume (ESV) decreased from 97 to 51 ml (P less than 0.001) without a change in stroke volume (SV). Ejection fraction increased from 42 to 52% (P less than 0.001) and mean velocity of circumferential fiber shortening (VCF) increased from 0.61 to 1.04 circumferences (circ)/s (P less than 0.001). After ultrafiltration, EDV decreased from 167 ml to 124 ml (P less than 0.001) and SV from 73 ml to 39 ml (P less than 0.001), without significant changes in ESV or VCF. In contrast to the maneuvers in which volume loss occurred, after hemodialysis without volume loss ESV decreased from 95 to 66 ml (P less than 0.001) and SV increased from 74 ml to 97 ml (P less than 0.001) without changes in EDV. EF increased from 44 to 59% (P less than 0.001) and VCF increased from 0.64 to 1.26 circ/s (P less than 0.001). Ventricular function curves plotted from data obtained under conditions of altered cardiac filling volume before and after the three dialysis maneuvers demonstrate that ultrafiltration produced a pure Frank-Starling effect, while hemodialysis with or without volume loss produced a shift in the ventricular function curves, which demonstrated an increase in the contractile state of the left ventricle. The changes in left ventricular function produced by regular hemodialysis are the combined effects of a decrease in EDV and an increase in the contractile state of the left ventricle.

Evidence suggesting persistence of nephritogenic immunopathologic mechanisms in patients receiving renal allografts.

Direct immunofluorescent (IF) examinations and elutions were performed on native kidneys and allografts of 24 patients undergoing renal transplantation. Immunoglobulins (Ig) were detected by IF on native kidneys of 12 of the 24; 11 of the 12 later had Ig localized to allograft glomeruli by direct IF. In addition, three other patients also developed Ig deposition on allograft glomeruli, although direct IF of native kidneys was negative. Elution studies indicated: (a) that linear Ig deposition on allograft glomeruli was the result of antiglomerular basement membrane (GBM) antibodies, (b) Ig localizing to allograft glomeruli in many of these patients was the result of persistent immunopathogenetic mechanisms existing at the time of allograft placement, and (c) occasionally, kidneys negative for Ig localization by direct IF contain elutable nephritogenic antibodies.

Immunopathologic findings in idiopathic renal hematuria.

Eighty patients had idiopathic renal hematuria and normal renal function. Renal biopsy showed minimal changes or focal glomerulonephritis in two thirds of the patients. The remainder exhibited diffuse proliferative glomerulonephritis and included nine patients with segmental glomerular sclerosis. Electron microscopy disclosed alterations of the capillary loop in 23 biopsy specimens and electron-dense deposits in 11. Immunofluorescent microscopy identified glomerular-bound immunoglobulins, C3, or fibrinogen in 58% in a generalized distribution. IgG was the immunoglobulin seen most commonly. IgA and IgM were present in 14 and 13 biopsy specimens, respectively. These findings suggest that idiopathic renal hematuria is a clinical syndrome rather than a single disease with varying underlying renal findings. Both immunologic and nonimmunologic mechanisms may be involved, and the prognosis is favorable even in patients followed up for 11 years.

Characterization of B cell antibodies in kidney transplant recipients.

Antibodies against B lymphocytes were found in the serum of the majority of 59 kidney transplant recipients and of 22 eluates obtained from kidney allografts undergoing rejection. To characterize these B cell lymphocytotoxins we have used a mouse monoclonal anti-DR antibody (L227) that inhibits cytotoxicity of antibodies against HLA-DR antigens and a chicken serum against human Ia-like antigens that also inhibits antibodies against DR-related supertypic determinants and other Class II histocompatibility antigens. Three types of B cell cytotoxins were defined: antibodies against HLA-DR, antibodies against Ia-like antigens other than DR, and antibodies against non-Ia-related B cell antigens. Before transplantation, B cell antibodies were detected in about a third of the patients studied. They were inhibited by monoclonal anti-DR more often in recipients who ultimately rejected a kidney allograft (67%) than in those in whom the graft was successful (44%, P less than 0.03). After transplantation, antibodies inhibited by L227 were found in 56% of the patients with functioning grafts and in 94% of the recipients whose grafts had been removed because of rejection (P less than 0.001). B cell antibodies inhibited by monoclonal anti-DR were found in the majority of kidney eluates. However, although 85% of the B cell reactions of kidney eluates were blocked by this antibody, only 55% of the B cell reactions of sera obtained from the same recipients after nephrectomy were similarly inhibited. Thus it appears that antibodies against HLA-DR were bound and concentrated in the transplanted organ and other B cell antibodies were not. These results indicate that anti-DR antibodies blocked by the monoclonal antibody L227 are the most common type of B cell lymphocytotoxins formed in kidney transplant recipients. Their role in kidney allografts undergoing rejection, where they are bound in high concentration, needs to be determined.

Primary tubulointerstitial nephritis caused by antibodies to proximal tubular antigens.

Acute oliguric renal failure requiring hemodialysis developed in a 59-year-old man who had myasthenia gravis. A renal biopsy on day 21 was diagnostic of tubulointerstitial nephritis. Despite regular hemodialysis, the patient died of complications 17 weeks after the onset of oliguria. At post-mortem examination, a thymoma was found, and renal histopathology indicated tubulointerstitial nephritis and concomitant generalized epimembranous and intramembranous electron-dense deposits of glomerular capillary walls. By direct immunofluorescence, immunoglobulins and C'3 were visualized in peritubular granular deposits around proximal tubules, but not on glomeruli. The renal acid-eluate contained immunoglobulins that bound to proximal tubule brush border, intracellular cytoplasmic granules, and a granular antigen probably associated with basement membrane of proximal tubule cells of normal human kidney and the patient's kidney, whereas the patient's serum apparently contained antibodies only to proximal tubule brush border. The renal eluate did not bind to normal or the patient's glomeruli, and partial elution of the patient's kidney did not expose new binding sites for the eluate. The data indicate a unique instance of tubulointerstitial nephritis caused by antibodies to multiple proximal tubule antigens apparently forming immune complexes in situ.

Interstitial nephritis caused by methicillin. Studies in a case complicating staphylococcal sepsis with acute glomerulonephritis.

A 16-year-old student was admitted with acute, oliguric renal failure complicating staphylococcal sepsis. During treatment with methicillin drug hypersensitivity was suspected, and antibiotic was changed to vancomycin; by day 19 hemodialysis was discontinued. Renal biopsy showed two pathologic processes: acute exudative glomerulonephritis and widespread tubulointerstitial nephritis. In addition to glomerular immunoglobulin and C'3 deposits, interstitial and focal tubular basement membrane deposits of IgG were seen. Antiserum to DPO (methicillin) haptens localized apparently to the same tubular sites, as did fluorescein-conjugated antibodies from the patient's serum. The data suggest that interstitial nephritis was caused by serum antibodies to methicillin which bound to sites in renal tubules to which methicillin also had fixed. The acute tubulointerstitial nephritis complicated acute oliguric glomerulonephritis of staphylococcal sepsis.

Cryoimmunoglobulinaemia in patients with primary renal disease and systemic lupus erythematosus. I. IgG- and DNA-binding assessed by co-precipitation.

Sera were tested for cryoglobulin precipitates from 206 consecutive patients with renal disease, ninety-eight normals and sixteen patients with systemic lupus erythematosus (SLE) without evident renal disease. Cryoprecipitates were detected in 17% of test subjects overall and 2% of normals; the incidence was highest in patients with SLE, regardless of detectable renal disease. Cryoprecipitates usually were comprised of IgG and IgM or IgG, IgM, and IgA in thirty-six out of forty-two instances, although a single immunoglobulin class was detected in five patients. Co-precipitation experiments showed IgG-binding by virtually all sera forming cryoprecipitates; isolated cryoprecipitates bound radiolabelled homologous IgG, and Fc fragment and sometimes IgG subclass proteins preferentially. Freshly forming cryoprecipitates sometimes co-precipitated DNA, whereas all isolated cryoprecipitates co-precipitated DNA from dilute solutions. The data are compatible with the current hypothesis that cryoimmunoprecipitates are immune complexes that are insoluble in vitro in the cold, that they usually comprise mixed immunoglobulins with anti-IgG activity, and may contain a mixture of antigens and antibodies.

IgA-associated glomerulonephritis.

In summary, IgA-associated glomerulonephritis is an interesting clinical problem. The immunohistochemical identification of renal IgA deposits is the sine qua non of its diagnosis, although most of the patients reported have had hematuric syndromes, particularly recurrent gross hematuria. The importance of this immunopathologic entity devolves from the crucial use of special stains to identify IgA, the enigmatic role of IgA, the usual mesangioapthic expression of histologic response, and the ill-defined relationship of this clinical problem to nephropathies associated with systemic diseases that also have glomerular IgA deposits. Although still unproven, it is likely that the usual instance of IgA-associated glomerulonephritis is due to deposition of circulating immune complexes containing IgA. The nature of the exciting antigen(s), quantitative measures and characteristics of such complexes, and the role of mediating systems, including coagulation, have not yet been elucidated.