Background

Glomerular sieving coefficients (GSCs) of proteins have been measured extensively in animals but not humans. We have studied the proteinuria of Fanconi syndrome, a "knock-out" of renal tubular protein reabsorption, to estimate GSCs and detect potential contributors to development of renal failure.

Methods

Immunoassay of proteins and polypeptides in serum and urine of patients with early Dent's disease (mean GFR = 83 mL/min, range 60 to 101, N = 5), Lowe's syndrome (N = 3), and ADIF (N = 2) were used.

Results

Twenty-one proteins, ranging in mass from insulin (5.1 kD) and parathyroid hormone (PTH; 9.4 kD) to transferrin (78 kD) and intact IgG (160 kD), were present in Fanconi urine at> 6 to 1000-fold normal. A simple model assuming complete "knock-out" of the reuptake of each protein filtered normally by the glomerulus was applied to protein excretion by Dent's patients. GSCs were estimated for 12 plasma proteins, including albumin (7.7 0.9 10^-5) and IgG (4.2 0.28 10^-5; mean SEM). We calculated the albumin concentration in normal glomerular filtrate to be 3.5 0.41 mg/L (53 6.4 nmol/L), consistent with studies in rat and dog.

Conclusions

To our knowledge, this study provides the first estimates of human in vivo GSCs. Our model explains why tubular proteinuria of Fanconi syndrome includes proteins of mass of albumin and above as well as low-molecular-weight proteins, and further characterizes the endocytic pathway(s) believed defective in these syndromes. High urinary concentrations of potentially bioactive hormones such as PTH, insulin, IGF-1 and the chemokine monocyte chemoattractant protein-1 (MCP-1), were found; their presence in tubular fluid may contribute to the hypercalciuria, interstitial fibrosis, and the progressive renal failure of Fanconi syndromes.

METHODS

Patients

Clinical, laboratory, and molecular genetic features of the five affected male patients with Dent's disease whom we studied in detail have been described; these patients had relatively well-preserved creatinine clearances Table 1: Patients C/II/2, total CLCN5 deletion and F/II/1, W279X^9,12; a member of family 7.1/94, splice-site mutation with deletion of codons 132-241¹² and cases 4/96, R34X¹⁷ and 6/97, 1175-1176delGT; 346 amino acid deletion¹⁸. Each patient has a mutation in CLCN5 associated with loss of function of the chloride channel, tubular proteinuria typical of the disease, and a creatinine clearance> 60 mL/min. Creatinine clearance was either measured from a 24-hour collection of urine or calculated on the basis of serum creatinine, age, and weight¹⁹. A further three patients with a clinical, laboratory, and molecular genetic diagnosis of Dent's disease, but with creatinine clearances <45 mL/min, were also studied. The three patients with Lowe's syndrome have severe mental and growth retardation, visual impairment, and tubular proteinuria; two of the patients are brothers. The two patients with ADIF are father and son in a family described previously⁷. Creatinine clearances of patients studied are given in Table 1.

Table 1 - Estimated glomerular sieving coefficients (GSCs) of proteins and protein excretion by patients with Dent's disease, Lowe's syndrome and autosomal-dominant idiopathic Fanconi syndrome (ADIF).

Full table

Specimen collection

Midstream random specimens of urine following at least 18 hours of sexual inactivity were refrigerated immediately, frozen within four hours at -80°C for up to two days, and transferred to liquid nitrogen for up to one year before analysis. Serum specimens were obtained from two of the five patients with Dent's disease and stored in the same way.

Immunoassays

The following methods were used for measurement of proteins and polypeptides in urine and serum (abbreviations as in Table 1): ₂m, IMx® analyzer (Abbott Diagnostics, Maidenhead, UK); RBP,²⁰; TSH, FSH and LH AxSym® analyzer (Abbott Diagnostics); ₁m, TTR and albumin, Array® analyzer (Beckman Coulter, High Wycombe, UK); kappa and lambda immunoglobulin light chains, ₁AG, DBP and ZAG, radial immunodiffusion²¹ using antibody from Dade-Behring (Milton Keynes, UK); ₂GI,²⁰; TRF, BNII® analyzer Dade-Behring (Milton Keynes, UK); SHBG and GH, Immulite® analyzer (DPC Corp., Los Angeles, CA, USA); IgG, modification of immunoassay for ₂GI with use of anti-IgG antibodies supplied by Dako (Ely, UK) and purified urinary IgG calibrant from Scipac (Sittingbourne, UK); IGF-1 and PTH (intact), Advantage® analyzer (Nicholls Institute, Newport, UK); MCP-1 (ELISA using matched antibody pair, R&D Systems, Abingdon, UK); insulin was measured on both the Access® (Beckman, High Wycombe, UK) and DELFIA® (Perkin-Elmer, Beaconsfield, UK) analyzers. Results of protein and hormone excretion are expressed per mmol creatinine determined by a kinetic Jaffé method.

Immunoblotting

To confirm the presence of intact IgG, urine samples were diluted in 62.5 mmol/L Tris chloride buffer, pH 6.8, and denatured by adding an equal volume of 10% sodium dodecyl sulfate (SDS), 20% glycerol, 0.003% bromophenol blue in the same buffer and heating at 40°C for 30 minutes. After electrophoresis on nonreducing 4 to 15% polyacrylamide gels (Bio-Rad, Hemel Hempstead, UK), proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 3% albumin and probed with anti-human gamma chain F(ab')₂ conjugated to horseradish peroxidase (HRP; Sigma, Poole, UK; Cat. No. A2290), diluted 1:60,000 in 0.1% bovine serum albumin (BSA), 500 mmol/L sodium chloride, 0.1% Tween 20, in 20 mmol/L Tris, pH 7.5, and visualized with ECL+® reagent (Amersham Pharmacia, Milton Keynes, UK).

RESULTS

Measurements of protein excretion in random urine samples obtained from five affected male patients with Dent's disease are shown in Table 1. The five patients chosen for study were early in the course of their disease with relatively preserved glomerular filtration rates (GFR; mean 83 mL/min, range 60 to 101). The results demonstrate greatly elevated excretion of proteins ranging in molecular weight from 11.6 to 160 kD. Increased excretion of IgG measured by ELISA was confirmed by immunoblotting, which showed that all material reacting with anti-Ig- antibody had the mobility expected of intact Ig on nonreducing SDS electrophoresis.

The excretion of both albumin and -1-microglobulin were evaluated in the five affected male patients with Dent's disease and relatively preserved glomerular filtration rates and three other Dent's patients with impaired GFR. There was no relationship between the amount of either protein excreted and GFR for the five patients in this group with GFR> 45 mL/min. However, increased excretion, not exceeding 2.6-fold, of either one or both these proteins was present in the three patients with GFR <45 mL/min (data not shown). Impaired GFR and associated glomerular disease was therefore unlikely to affect excretion of protein by the patients with Dent's disease shown in Table 1. Protein excretion by three patients with Lowe's syndrome and two patients with ADIF was similar to those with Dent's disease, except that excretion of LMW proteins by patients with Lowe's syndrome was even greater Table 1.

Calculation of glomerular sieving coefficients

Assuming that glomerular filtration is the only source of a protein in the urine of a patient with a Fanconi syndrome and that there are no losses from, or additions to, the filtrate, the glomerular filtrate concentration (GFC) of the protein is given by:

(Eq.1)

Random urine samples were used for measurements of protein excretion to minimize the effects of protein instability in urine and these are expressed as mg/mmol creatinine Table 1. The 24-hour excretion for individual proteins was then calculated based on the 24-hour creatinine excretion by each patient. The 24-hour volume of glomerular filtrate was calculated as the 24-hour creatinine clearance. When these measurements are substituted into equation 1, this becomes:

(Eq.2)

Then, by definition², we calculated:

(Eq.3)

Any errors introduced by these assumptions are considered in the Discussion section. The glomerular sieving coefficients (GSCs) were estimated using the above model in the five patients with Dent's disease Table 1, and Figure 1 shows the estimated GSC plotted against the molecular weight of each protein. Kappa and lambda immunoglobulin light chains, sex-hormone binding globulin and lysozyme could not be measured accurately in urine, either because of calibration bias or poor precision. Nevertheless, these four proteins were also present in at least tenfold increased concentrations over normal values (data not shown).

Figure 1.

Estimated glomerular sieving coefficients for 12 plasma proteins versus molecular weight. Data are from Table 1; mean ( SEM) of five determinations based on results from each of five patients with Dent's disease are shown. See Molecular size, charge and glomerular sieving for discussion of the anomalous behavior of ₁-acid glycoprotein (₁AG) and ₂-glycoprotein-I (₂GI). Abbreviations are: RBP, retinol-binding protein; ₂m, ₂-microglobulin; TSH, thyroid-stimulating hormone; ZAG, zinc-₂-globulin; DBP, vitamin D-binding protein; TTR, transthyretin; ALB, albumin; TRF, transferrin; IgG, immunoglobulin G.

Full figure and legend (15K)

When hormone and chemokine excretion were measured (Table 2 and Figure 2), greatly increased levels were found including those of several potentially bioactive proteins. Since the absolute concentrations of the hormones measured Table 2 and of TSH Table 1 were low, we looked for any effect of the analysis medium (urine vs. serum) on assay results. The apparent hormone levels were measured in the following mixtures using varying proportions of each component: serum and normal urine; serum and urine samples with high apparent levels of endogenous hormones and normal urine and urine samples with high apparent levels of endogenous hormone. This showed both over-recovery and under-recovery of up to 35% (IGF-1 and PTH, respectively) although generally not exceeding 20% (data not shown). Results in Table 2 are presented without adjustment for this apparent over-recovery and under-recovery. Insulin was measured by two assays based on different technologies and antibodies with almost identical results; measurements using the DELFIA® method only are given.

Figure 2.

Hormone and chemokine excretion in urine of patients with Dent's disease (); Lowe's syndrome (); autosomal-dominant idiopathic Fanconi syndrome (ADIF) (), and normal individuals (). Data are from Table 2. Excretion, in the following units, is expressed per mmol creatinine: parathyroid hormone, ng; insulin, pmol; IGF-1, nmol; prolactin, mIU; growth hormone, mIU; luteinizing hormone, mIU and MCP-1, ng.

Full figure and legend (24K)

Table 2 - Hormone and chemokine excretion in the urine of patients with Dent's disease, Lowe's syndrome and autosomal-dominant idiopathic Fanconi syndrome (ADIF).

Full table

DISCUSSION

Our results for the 12 proteins shown in Table 1 show that the excretion of most proteins involved in tubular proteinuria is largely a function of plasma concentration and molecular size Figure 1. These measurements permit the first estimates of the in vivo GSCs in humans and cover almost a 10⁵-fold range. The additional data of Table 2 and Figure 2 shows that several proteins of potentially high bioactivity are excreted in high concentration in the urine of patients with tubular proteinuria and, by implication, are present in tubular fluids at high concentration.

Calculation of glomerular sieving coefficients

Several assumptions are made in the calculation of the GSCs and errors in these will affect the final results differently. The effect of instability of proteins in urine was minimized by the use of rapidly processed random urine specimens. Conversely, any impact of disease on glomerular permeability might be expected to increase the estimated GSC, at least for less freely filtered molecules. No evidence was found for further increases in the excretion of ₁m and albumin until the creatinine clearance of patients with Dent's disease fell to <45 mL/min; measurements were specifically made early in the progression of renal impairment. Residual tubular protein uptake in Dent's disease, that is, an incomplete knock-out of tubular protein reabsorption, cannot be wholly excluded because the exact pathway(s) have not been defined. However the results for ₂m demonstrated a recovery in urine of 91 14% of all ₂m filtered by the glomerulus; recovery is calculated as (GFC/plasma protein concentration) where GFC is found from equation 2 in the Results section using the data from Table 1. Since ₂m is probably freely filtered², this finding suggests that most tubular reuptake, at least for this protein, is disrupted; given the known instability of this protein in urine, the true recovery of ₂m is probably even higher³. The three diseases of diverse molecular pathology all appear to show similar increases in the excretion of each protein Table 1 and therefore the simplest explanation is that there is a common loss of function of a single final pathway. The higher excretion of the low molecular weight proteins ₂m and RBP in the unselected patients with Lowe's syndrome may be related to lower glomerular filtration in these Table 1, compared with the Dent's patients who were chosen for study because of relatively preserved GFR. Indeed, increased RBP excretion is found in patients with a variety of tubular and non-tubular diseases when GFR is reduced by more than 70%²². The approach used in Table 1 to estimate GSC cannot be used for the hormones and chemokines shown in Table 2, because of the wide physiological fluctuations in their plasma levels.

Comparison between human and animal results

Measurements of the glomerular filtrate concentration of albumin by micropuncture in rats and dogs have yielded a range from <1 to 50 mg/L. The reasons for this variability have been discussed in detail². The approach here, using equation 2 (Results section), gives a value of 3.5 0.41 mg/L (536.4 nmol/L) in humans, for this important pathophysiological measurement. There is controversy about the contribution of transcellular reabsorption to retrieve albumin from the glomerular filtrate^23,24,25. It is not known how the molecular defect in the syndromes we have studied might affect any transcellular reabsorption, and some residual tubular reabsorption would mean an underestimation of the reported albumin GSC here. Nevertheless, even a twofold underestimate of true albumin filtration predicts a glomerular fluid concentration of albumin of only 7.0 mg/L (106 nmol/L), which is still toward the lower end of that determined by invasive animal studies.

Molecular size, charge, and glomerular sieving

Not unexpectedly, Figure 1 shows several deviations from a monotonic curve. Excretion of ₁-acid glycoprotein and ₂-glycoprotein I appear anomalously low and high respectively Figure 1. Alpha₁-acid glycoprotein has an unusually low isoelectric point, pI = 2.7,²⁶ whereas in contrast the pI of ₂GI is unusually high, pI = 6.4 to 8.2 for nine major isoforms in urine^4,5. The isoelectric points of most of the other proteins studied, except for IgG, are all in the range from about 4.0 to 6.0²⁶. An effect of extremes of charge on GSC is therefore apparent for those proteins with GSCs that neither approach one nor are very low (Table 1 and Figure 1). This is consistent with studies of the glomerular filtration of dextrans in humans, which suggest that fixed, negatively charged components of the capillary wall relatively hinder filtration of more negatively charged proteins²⁷. Tables 1 and 2 show that before development of marked renal failure, the commonly used LMW protein markers (₂m, RBP, and ₁m) account for about one half of the proteinuria in the Fanconi syndrome, albumin for some 30% and the remainder comprises a diverse group of proteins.

Implications for specificity of the endocytic pathway

Receptor-mediated endocytosis is believed to underlie protein transport from the proximal tubular lumen and defects of this pathway have been studied in Dent's disease [abstract; Norden et al, J Am Soc Nephrol (in press)]^13,14. The exact structure of the receptor(s) is unclear, although both megalin and cubilin are almost certainly involved^{13,14,28,29,30,31}. In vitro receptor-binding studies of a variety of proteins to megalin and cubilin are entirely consistent with our results (abstract; Christensen, J Am Soc Nephrol 11:49A, 2000)^{4,28,30,32,33}. However, the putative receptor(s) for several important potential ligands described here, remain to be identified: These include thyroid-stimulating hormone, immunoglobulin G and monocyte chemoattractant protein-1. Although our findings do not delineate which pathways are defective in Dent's disease, they suggest that the normal endocytic pathway can process an even wider array of ligands than described so far.

Pathophysiological role of tubular fluid

Progressive renal failure is a very common feature of the Fanconi syndrome^6,7. There is evidence from animal studies that bioactivity of tubular fluid may contribute to the progression of renal disease³⁴, and our results demonstrate that a number of potentially bioactive proteins are excreted in large amounts by patients with Dent's disease, Lowe's syndrome, and ADIF. The high levels of excretion in urine imply that tubular fluid distal to the earliest part of the proximal tubule is perfused with high concentrations of these potentially bioactive proteins. Recent studies indicate that parathyroid hormone receptors are expressed on apical surfaces of tubular epithelium and that activation of these apical receptors by PTH down-regulates the type IIa NaPi cotransporter³⁵. This might lead to phosphaturia, enhanced 1-hydroxylation and either increased or inappropriately elevated serum 1,25(OH)₂-vitamin D levels leading to enhanced intestinal calcium absorption and thus explaining the hypercalciuria of Dent's disease¹³. Elevated urinary PTH levels have also recently been reported in a mouse model of Dent's disease¹³. It is notable that the levels we have found in the urine of patients with Dent's disease, expressed as a multiple of normal, are frequently higher than those reported in that mouse model Table 2.

Monocyte chemoattractant protein-1 in tubular fluid may promote tubulointerstitial fibrosis³⁶ and the potential tubular fluid concentrations calculated from the results of Table 1 are well within the range of those known to have biological activity. Increased urinary MCP-1 is found in patients with active renal vasculitis and persistence of this increase is associated with progression to end-stage renal failure (abstract; Tam, J Am Soc Nephrol 11:99A, 2000). The relative contributions of any local renal production and glomerular filtration to the amount excreted in these conditions are unclear. An important role for MCP-1 in macrophage-mediated tubular injury role has been found by the study mice deficient in this chemokine³⁶. Similarly, bioactive IGF-1, which has been shown by micropuncture studies to occur in the tubular fluids of nephrotic rats³⁴, might contribute to the tubulointerstitial lesions of patients with the renal Fanconi syndrome.

Progressive renal failure has been described as a feature of most forms of the Fanconi syndrome^6,7. The explanation for this renal failure may lie in progressive tubular and interstitial damage caused by the high levels of potentially bioactive hormones and MCP-1 identified (Table 2 and Figure 2).

Our findings provide a unifying quantitative explanation for the excretion of large amounts of higher molecular weight proteins in addition to the more freely filtered LMW proteins in patients with the renal Fanconi syndrome. Results of glomerular sieving coefficients estimated here are both self-consistent (Table 1 and Figure 1) and consistent with much animal work. Further molecular studies are needed to account for the versatility and efficiency of the proximal tubular endocytic uptake system and to assess the pathophysiological consequences of the probable high hormone and chemokine content of tubular fluids in the Fanconi syndrome.

References

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Acknowledgments

This study was supported by grants from the Sir Jules Thorn Charitable Fund (AGWN), NIH award DK46838 (SJS), and the MRC and Wellcome Trust (RVT). Part of this work was presented at the 33^rd meeting of the American Society of Nephrology, October 2000. We thank Professor C.N. Hales for helpful discussion and the staff in the Department of Clinical Biochemistry, Addenbrooke's Hospital for immunoassays. Scipac Ltd. (Sittingbourne, UK) kindly donated purified human urinary proteins. Dr. D. Teale, Guildford University, performed initial insulin measurements. MCP-1 ELISA was performed by Mr. J.-S. Sanders at the Hammersmith Hospital.