High proteinuria selectivity index based upon IgM is a strong predictor of poor renal survival in glomerular diseases

Omran Bakoush, Ole Torffvit, Bengt Rippe and Jan Tencer

Department of Nephrology, Lund University Hospital, Lund, Sweden



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. The transport of large proteins across the glomerular capillary wall (GCW) may increase several fold in glomerular diseases. The occurrence of IgM in urine is a consequence of the presence of large defects or shunts in the GCW, whereas albuminuria is probably a result of an altered charge- and size-selectivity of the GCW. In order to examine whether patho-morphological differences influence the renal outcome in proteinuric glomerulopathies, we examined urinary excretion of IgM and albumin as prognostic markers of glomerular disease.

Methods. An observational study over a median of 41 (±3) months was conducted in 84 patients with biopsy-verified glomerular disease. The patients were subdivided into groups with low (<=0.002) and high (>0.002) proteinuria selectivity index based upon IgM (IgM-SI), and into groups with low (<=200 mg/mmol) and high (>200 mg/mmol) albumin creatinine index (ACI).

Results. In the high IgM-SI group, the median creatinine clearance (Ccr) decreased by 26%, and 62% of the patients decreased in Ccr by >5 ml/ min/year during the follow-up time. In comparison, the median Ccr decreased by 8% in the low IgM-SI group (P<0.001) and only 18% of the patients in this group deteriorated by >5 ml/min/year in the Ccr. Eleven (21%) of the 51 patients in the high IgM-SI group developed end-stage renal failure compared with none of the 33 patients in the low IgM-SI group. All the patients that progressed to uraemia had decreased Ccr (<60 ml/min) at entry into the study. However, among all these patients, only those with high IgM-SI, and none with low IgM-SI, developed end stage renal failure. The fall in Ccr did not differ significantly between the patients in high (12%) and low (16%) ACI groups.

Conclusion. The results of this study indicate that an increased IgM-SI value is a stronger predictor of clinical outcome in proteinuric glomerulopathies than baseline albuminuria. This finding may reflect different patho-histological mechanisms influencing renal survival in glomerular diseases.

Keywords: albuminuria; glomerulonephritis; IgM; macromolecular transport; proteinuria selectivity index



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Glomerular diseases, including primary glomerulonephritis (GN), are a major cause of end-stage renal failure [1,2]. However, whereas renal function might be impaired in many patients with GN, a significant percentage of these patients never show a deterioration of their glomerular filtration rate (GFR) [3]. Several factors, such as the type of the disease, gender and blood pressure may influence the clinical outcome. Furthermore, the degree and the type of proteinuria is important, both as a marker of the renal injury and as a factor involved in the pathophysiology of the disease [4]. As for today, there are few, if any, prognostic tools in proteinuric glomerulopathies, and therefore markers that could indicate prognosis in GN might be of great value in nephrological practice.

The permeability of the glomerular barrier to macromolecules is determined by the size- and charge-selectivity of the glomerular capillary wall (GCW), and by the size, shape and charge of the transported macromolecules [5,6]. In a ‘two-pore with a shunt’ model, based upon the two-pore theory of capillary size selectivity, hypothetical cylindrical pores, consisting of two pore populations, perforate the GCW [7]. The vast majority of the pores are ‘small pores’ of radius 29–31 Å vis-à-vis negatively charged rigid spherical proteins. The remaining pore population consists of a very small number of ‘large pores’ of radius 90–115 Å [8]. In addition to the two populations of pores, the intact GCW exhibits very sporadic physiological ‘membrane defects’ or ‘shunts’, large enough to allow the transport of very large proteins and red blood cells [8,9].

The small pores are essentially impermeable to macromolecules the size of albumin or larger. Such molecules are normally transported by convection across the large pores [9]. Very large proteins, such as IgM (molecular radius 120 Å), are able to pass the GCW only through the shunts. A repairing apparatus normally seals these shunts. Thus, increased transport of IgM indicates unsealing of these shunts and/or increased density of these defects in the GCW [10].

With those considerations in mind, we examined the prognostic implications of baseline albuminuria, calculated as albumin creatinine index (ACI), and proteinuria selectivity index based upon IgM (IgM-SI) in patients with non-diabetic, proteinuric glomerulopathies. The results of this study indicate that high IgM-SI, and not the degree of albuminuria per se, predicts the progress of the decline in renal function in the studied glomerular diseases.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Subjects
The patients in the present study were participants in a large investigation of all forms of glomerular disease currently being conducted at the Department of Nephrology, University Hospital, Lund, Sweden. The morphological diagnoses were in all cases established by evaluation by light microscopy and immunofluorescent staining of representative percutanous renal biopsy specimens. All patients gave written consent to participate in the study.

Patients investigated between July 1993 and December 1997 with diagnoses of mesangial proliferative glomerulonephritis, IgA nephropathy, membranous glomerulonephritis, minimal change nephropathy or nephrosclerosis secondary to hypertension were invited to participate in the present study. Exclusion criteria were: advanced renal failure on admission (serum creatinine >400 µmol/l); diabetic nephropathy; systemic diseases; or ACI <20 mg/mmol. All together, 98 patients commenced the study. During the follow-up, five patients were excluded because of death due to non-renal causes and nine patients refused further controls.

The 84 patients (56 males, 28 females) that fulfilled the study criteria were followed at several outpatient nephrology clinics in southern Sweden at intervals of 6–12 months (narrower when needed). The end point of the study was the last planned visit during the year 1999 or start of renal replacement therapy. None of the patients had any restrictions concerning protein diet during the follow-up. Causes of renal function impairment, other than the primary renal disease, were excluded.

The patients were subdivided according to baseline levels into groups with low (<=0.002) and high (>0.002) IgM-SI, and low (<=200 mg/mmol) and high (>200 mg/mmol) ACI. The levels of IgM-SI and ACI used to make the subdivisions were based on our previous observation [11].

The median follow-up time was 39 months in the high IgM-SI group and 43 months in the low IgM-SI group. The reason for this difference was the fact that several patients in the high IgM-SI group started renal replacement therapy before the end of 1999.

The number, age, gender and baseline data of the patients in the two IgM-SI groups are presented in Table 1Go and the histological diagnoses are presented in Table 2Go. Patients with diagnoses of IgA nephropathy, proliferative glomerulonephritis and membranous glomerulonephritis were represented in both groups, while all patients with minimal change nephropathy appeared in the low IgM-SI group, and eight out of nine patients with nephrosclerosis were inherent in the high IgM-SI group (Table 2Go).


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Table 1. Number of patients, gender and medians (ranges in parentheses) of age, mean arterial blood pressure, serum creatinine and creatinine clearance in 84 patients with glomerular diseases, subdivided into 33 patients with low IgM-SI and 51 patients with high IgM-SI

 

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Table 2. Number of patients with different diagnosis (84 patients in total), subdivided into a low IgM-SI group (33 patients) and a high IgM-SI group (51 patients)

 
Blood pressure was measured using a mercury sphygmomanometer with the patient in the supine position. The diastolic blood pressure was measured at Korotkoff phase V. Mean blood pressure was calculated by adding one third of the pulse pressure to diastolic blood pressure. At the start of the study, the blood samples and the first voided urine specimens were obtained in the morning of the day the kidney biopsy was performed. Portions of 30 ml of urine were collected in polyethene vessels (Kebo AB, Sweden). The samples were, after addition of 1 ml of a preservation solution, kept frozen at -20°C until assayed. The preservation solution, described earlier, contained benzamidinium chloride, EDTA, sodium azide and Tris base representing inhibitors of serine- and metalloproteinases, an antimicrobial agent and a buffer substance, respectively. This solution has previously been demonstrated to result in stable levels of proteins in frozen urine samples [12,13].

Serum and urine creatinine was determined enzymatically using a Kodak Ektachem 700 XR-C system. Serum and urine albumin, and serum IgM were determined by immunoturbidimetry [14] using a Cobas Mira S system (Roche Inc., Stockholm, Sweden) and monospecific rabbit antisera obtained from Dako (Copenhagen, Denmark). Urine IgM was measured by an ELISA technique presented previously [11]. The degree of albuminuria was presented as ACI. We use the urine protein to creatinine ratio measured in a spot urine sample since, in our own experience and that of many others, it is a reliable estimate of the degree of proteinuria and is highly correlated to 24 h urinary protein excretion. Furthermore, it helps to avoid errors caused by problems with reliable urine sampling in an outpatient population [4,1517].

Calculations
IgM-SI was calculated according to the formula [11,18]:


Ccr was calculated using the Cockcroft and Gault formula [19], where:


ACI (mg/mmol) was defined as ratio of urine albumin (mg/l) to urine creatinine (mmol/l).

Statistical methods
The data in the tables are expressed as medians followed by ranges. Statistical comparison between the patient groups was performed with the non-parametric Mann–Whitney test. P<0.01 was selected as the level of significance. Correlation was tested using Spearman's correlation coefficient. The statistical package for social science (SPSS, version 10) was used.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The clinical and laboratory data of the patients in the IgM-SI groups are shown in Tables 1Go and 3Go. The individual levels of serum creatinine in the low and high IgM-SI group during the follow-up time are shown in Figure 3Go.


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Table 3. Percentage of creatinine clearance decline and number of patients started on dialysis treatment during follow-up time in 84 patients with glomerular diseases, subdivided in 33 patients with low IgM-SI and 51 patients with high IgM-SI

 


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Fig 3. The course of individual values of serum creatinine in patients with low and high IgM-SI during follow-up.

 
The patients in the low IgM-SI group had a higher degree of albuminuria (411 mg/mmol) than the patients in the high IgM-SI group (151 mg/mmol) (P<0.001).

The median Ccr decreased by 26% (0–90%) in patients with high IgM-SI compared with 8% (0–62%) in patients with low IgM-SI (P<0.001). Furthermore, 32 out of the 51 patients in the high IgM-SI group, compared with six out of 33 patients in the low IgM-SI group, experienced deterioration in Ccr by>5 ml/min/year during the follow-up time (Figure 1Go). There was no difference in the decrease of Ccr between the low (16%) and the high (12%) ACI groups, although high ACI group had significantly lower initial Ccr (56 ml/min) than the low ACI group (86 ml/min) (Figure 2Go).



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Fig 1. Decrease in creatinine clearance (ml/min/year) during the follow-up time in patients subdivided into groups with low and high values of IgM-SI.

 


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Fig 2. Decrease in creatinine clearance (ml/min/year) during the follow-up time in patients subdivided into groups with low and high values of ACI.

 
Eleven patients (eight males, three females) developed end-stage renal failure during the follow-up. All of these 11 patients were in the high IgM-SI group. However, since the initial GFR was lower in the high IgM-SI group compared with the initial GFR in the low IgM-SI group, the groups were also compared after excluding of all patients with initial GFR >=60 ml/min (Table 4Go). Among the 29 patients with initial Ccr <60 ml/min, the initial Ccr levels were 31 and 38 ml/min in the low and high IgM-SI groups, respectively. The patients with high IgM-SI decreased in Ccr by 54% compared with a 0% decrease in the patients with low IgM-SI (P=0.009). Furthermore, no patients with low IgM-SI developed uraemia during the follow-up time, despite the decreased Ccr at entry into the study (Table 4Go).


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Table 4. Number of patients, gender, age and medians (ranges in parentheses) of mean arterial blood pressure, serum creatinine and creatinine clearance, and ACI in the 29 patients with initial GFR <60 ml/min, in low and high IgM-SI groups

 
Because of the spotty distribution of patients with minimal change nephropathy and nephrosclerosis in the IgM-groups (Table 2Go), a comparison between the high and the low IgM-SI groups was performed among the 67 patients with diagnoses other than these two. Also in this comparison, the prior findings were confirmed and the differences found to be statistically significant. Also in this cohort, the patients with low IgM-SI had a lesser extent of Ccr decline (8%) than the patients with high IgM-SI (21%) during the follow-up time (P=0.004). Furthermore, also in this selected group, patients with low IgM-SI had higher initial ACI (346 mg/mmol) than patients with high IgM-SI (151 mg/mmol).

A semi-quantitative evaluation of histological lesions showed a significantly higher percentage of global glomerulosclerosis in patients with high IgM-SI. A median of 18% (0–80%) of the glomerulae in the patients with high IgM-SI were affected by global glomerulosclerosis, compared with a median of 3% (0–91%) in patients with low IgM-SI (P<0.001). Diffuse interstitial fibrosis was seen in 49% of the patients in the high IgM-SI group. In this group, only 16% of the patients had normal interstitium. Interstitial fibrosis occurred in 12% of the biopsies in the low IgM-SI, and 45% of the patients had normal interstitium in the light microscopy examination of their biopsies. The difference between the groups, in regard to the appearance of interstitial fibrosis, was highly significant (P<0.001). In the high and low ACI groups, the percentage of patients with diffuse interstitial fibrosis was 39 and 30%, respectively (non-significant, ns), and the percentage of glomerulae affected by global glomerulosclerosis was 7 and 15% respectively (ns).

There were no statistically significant differences between the IgM-SI groups concerning age, or the initial mean arterial blood pressure. However, there was a tendency towards higher blood pressure levels during the follow-up time in the high IgM-SI group. The percentage of males was higher in the high IgM-SI group (38 out of 51 patients) compared with the low IgM-SI group (18 out of 33 patients).

Immunosuppressive therapy was administered to 12 patients in the high IgM-SI group (Table 5Go). The patients had diagnoses of proliferative glomerulonephritis (six patients), IgA nephropathy (one) and membranous glomerulonephritis (five). Immunosuppressive treatment was given to 18 patients in the low IgM-SI group during follow-up time (Table 5Go). The patients had diagnoses of minimal change nephropathy (seven patients), proliferative glomerulonephritis (five) and membranous glomerulonephritis (six). ACE inhibitors were prescribed to 20 patients (60%) in the low IgM-SI group and 37 (71%) in the high IgM-SI group for a median of 38±4 months.


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Table 5. Number of patients with different diagnoses in low and high IgM-SI groups, who received immunossupression treatment during the follow-up, subdivided in three treatment modalities: A, steroids only; B, combination of steroids and cyclosporine A; and C, Ponticelli regimen with cyclophosphamide and steroids

 
In the high IgM-SI group, Ccr declined by 33% (0–90%) in patients treated with ACE inhibitors compared with 19% (0–89%) in patients without such treatment (ns). Patients treated with immunosuppressive drugs had a 26% (0–90%) decline in Ccr compared with 29% (0–89%) decrease in patients without immunosuppressive treatment (ns). There was no difference in the initial GFR between those treated and those not treated with ACE inhibitors (a median of 67 and 60 ml/min, respectively; ns) as well as between those treated or not with immunosuppression (71 and 56 ml/min, respectively; ns).

Among determinants of the glomerular disease progression; age, sex, blood pressure, creatinine clearance, histological diagnosis, degree of fibrosis and glomerulosclerosis, type of treatment, degree of albuminuria and IgM-SI, only the IgM-SI, glomerulosclerosis and interstitial fibrosis were correlated with Ccr decline (r=0.44, 0.33 and 0.35, respectively; P<0.01). Stepwise multiple regression analysis showed that the high IgM-SI is the major predictor of Ccr decline (P=0.001).



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The findings of this study indicate that the decline of renal function in proteinuric glomerulopathies is predicted by IgM-SI independently of the GFR and the degree of albuminuria.

Clinical and experimental data indicate that glomerular proteinuria enhances the progression of renal impairment in glomerular diseases by the formation of tubulointerstitial fibrosis [4]. However, several recent reports suggest that it is not albumin per se that might cause these sequelae, but rather other factors associated with the enhanced urinary leakage of plasma proteins, e.g. complement factors or protein-bound inflammatory cytokines such as TGF-ß [2022].

According to the ‘two-pore with a shunt’ theory, macromolecules of the size of albumin or larger are normally transported through ‘large pores’ of the GCW [8]. Since the population of these pores is, under normal conditions, relatively small, the transport of albumin across the GCW is usually low. Very large proteins, such as IgM, are able to pass the GCW only through the extremely rare shunts [7,10].

A low value of SI reflects a condition in which the functional nephrons only allow passage into the urine of relatively small quantities of high molecular weight proteins in comparison to albumin. Thus, ‘selective proteinuria’, which means low SI value, is usually a consequence of a charge-selective defect rather than a size-selective disorder, irrespective of the histological picture. In such conditions, loss of the charge-selectivity of the glomerular filter increases the ‘effective’ small pore radius from ~30 to ~45 Å and enables albumin to escape in large amounts through the small pores. Proteins larger than albumin are still unable to pass through the small pore pathway. This situation is conceivably the case in minimal change nephropathy, but also in some other glomerular disease [6]. Once the glomerular disease produces alterations in the size-selective properties of the GCW, the proteins found in the final urine contain increased amounts of large proteins such as IgG and IgM. However, whereas the increased urinary excretion of IgG reflects increased density of ‘large pores’ in the GCW, the occurrence of IgM in the final urine reflects a markedly increased population of highly unselective pathways, i.e. ‘shunts’.

In GN, activation of inflammatory mediator systems by immunological mechanism [23] may cause damage to the GCW, resulting in the appearance of shunts in the GCW, allowing urinary escape of high molecular weight plasma proteins including complement factors, membrane attack complexes and protein-bound inflammatory cytokines, which mediate tubulointerstitial fibrosis and progression of glomerular diseases. Recently, varying degrees of ultrastructural defects in glomerular basement membranes, measuring 15–200 nm in diameter, were revealed in nephrotic patients by transmission electron microscopy using a tissue negative staining method. These ultrastructural defects were not seen in the normal renal tissue [24]. Thus, proteins the size of IgM could make a sensitive marker of such defects, which might explain the correlation between the GFR decline and IgM-SI in glomerular diseases.

Patients with high IgM-SI have a significantly high degree of fibrosis and global glomerulosclerosis, whereas the degree of albuminuria does not correlate to these histopathological renal biopsy findings. Thus, high IgM-SI reflects severe glomerular damage and development of global glomerulosclerosis and fibrosis. Bazzi et al. found significant correlation between IgG-SI and the degree of fibrosis, but not with the degree of glomerulosclerosis [25]. IgG is smaller than IgM, and high IgG-SI indicates an increased number of large pores while IgM-SI indicates an increased shunt pathway and severity of the glomerular lesion. However, both IgG-SI and IgM-SI correlate to the development of tubulointerstitial fibrosis, further indicating that high molecular weight proteins are more damaging to the tubules than albumin per se.

We believe that IgM-SI predicts renal outcome in patients with glomerular diseases, irrespective of the glomerular histopathological findings. A biopsy specimen represents only a small number of glomerulae, whereas determination of SI has the advantage of reflecting the overall permeability of the active nephrons. Thus, proteinuria with high IgM-SI reflects a severe glomerular injury in the majority of the active nephrons.

Isolated albuminuria may represent alterations in either the charge- or size-selective properties of the GCW, or both. These alterations do not necessarily correlate to gross damage of the GCW, which, in turn, could explain the lack of correlation between albuminuria and the progress of the renal function impairment or development of interstitial fibrosis.

Improved blood pressure control, treatment with ACE inhibitors, and immunosuppressive therapy are considered to be important factors in slowing the progression of renal diseases. In the present study, the initial mean arterial blood pressure and the percentage of patients on ACE inhibitors during the follow-up time did not differ between the low and the high IgM-SI group. The tendency towards increasing blood pressure in the high IgM-SI group during the follow-up time could be explained by the poor renal survival in this group and is not sufficient to explain the poor renal prognosis in this group. Furthermore, treatment with immunosuppressive therapy or ACE inhibitors does not slow the deterioration of kidney function in patients with high IgM-SI. Regression analysis showed that high IgM-SI is a better predictor of the progression of the glomerular diseases studied than that of degree of albuminuria, initial GFR, and degree of fibrosis or other known determinants of disease progression such as gender, blood pressure or treatment.

The findings of the present study indicate that it is possible to predict the rate of progression in renal disease in patients with non-diabetic glomerulopathies by the type of proteinuria rather than the degree of albuminuria. Urine IgM assay is a simple, easy technique and can be performed in all laboratories. We suggest the use of a selectivity index based upon IgM as a part of the investigation programme in all patients with suspect glomerular diseases.



   Acknowledgments
 
We wish to thank the physicians and the nurses at the renal clinics in southern Sweden for assisting in collecting patients’ data. Many thanks to Åsa Pettersson for help with the analysis of urine IgM. This study was supported by grants from the Swedish Medical Research Council.



   Notes
 
Correspondence and offprint requests to: Omran Bakoush, Department of Nephrology, Lund University Hospital, S-221 85 Lund, Sweden. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 29.11.00
Revision received 19. 2.01.