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,¶
Divisions of *Structural Biology and Biochemistry and Pathology, Hospital for Sick Children, Toronto, Canada; Departments of Pathobiology and 
Population Medicine, University of Guelph, Guelph, Canada; and ¶Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
Correspondence to Dr. Paul Thorner, Division of Pathology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, CANADA M5G 1X8. Phone: 416-813-5108; Fax: 416-813-5974;
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Abstract |
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Introduction |
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The pathogenesis of Alport syndrome has been linked to mutations in genes for type IV collagen. Type IV collagen is assembled from a family of distinct 
-chains designated 1 to 6, which are encoded by six different genes, COL4A1 to COL4A6, respectively (7). Over 300 mutations have been found in the COL4A5 gene in the X-linked form of Alport syndrome (4,8). Mutations have been identified in the COL4A3 and COL4A4 genes in patients with the autosomal recessive form (9,10). All these mutations result in an abnormal composition of type IV collagen -chains in the glomerular basement membrane (GBM); in most patients with Alport syndrome, the GBM contains the 1 and 2 chains rather than the 3, 4, and 5 chains of the normal GBM (11–14). As a consequence, there is progressive damage to the GBM, which ultrastructurally begins as bilaminar and progresses to multilaminar splitting and eventually involves all capillary loops. As the glomerular disease advances, there is increasing sclerosis of glomeruli, tubular atrophy, and fibrosis of the interstitium. Indicators of poor prognosis include massive proteinuria, an extensive multilaminar appearance of GBM, interstitial fibrosis, and obsolete glomeruli (3,4,15,16).
No curative therapy yet exists for Alport syndrome, and most treatments are aimed at slowing the progression of the disease. Beneficial effects have been reported for human Alport patients and in animal models using low-protein diets (17) and angiotensin-converting enzyme inhibitors (18,19). More recently, cyclosporine A was reported to be beneficial to patients with Alport syndrome in a small noncontrolled study (20,21). Massive proteinuria decreased or disappeared, whereas creatinine clearance remained stable throughout treatment. Changes in renal pathology were reported to be nonprogressive, but assessment was qualitative.
The availability of animal models for hereditary nephritis provides an opportunity to perform a controlled study to evaluate the benefits of cyclosporine A in the treatment of this disease. The renal disease occurring in a family of Samoyed dogs is a well-characterized model of the X-linked form of Alport syndrome, that results from a nonsense mutation in the COL4A5 gene and closely mimics the human form of the disease (22–25). In this model, the progression to end-stage renal disease occurs predictably, starting at 4 to 5 mo of age and resulting in terminal renal failure by 10 mo of age. The purpose of the present study was to investigate the effects of cyclosporine A on the progression to end-stage renal disease in this model and to determine the effects on renal pathology. We hypothesized that improvement in the clinical progression of the disease might correlate with changes at the tissue level, either glomerular or extraglomerular. The former would suggest developing specific therapies for Alport syndrome might be justified, whereas the latter would suggest nonspecific therapies might be equally beneficial (15).
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Materials and Methods |
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Clinical Biochemical Analysis
Blood for serum creatinine (µmol/L), total protein (g/L), and albumin (g/L) concentrations were collected once weekly by venipuncture. Routine urine dipstick and sediment analysis, urine Ponceau S protein (mg/L) and creatinine (mg/L) concentrations were determined routinely throughout the study. The half time plasma clearance of sodium sulfanilate (min) was used as a measure of GFR. Specific gravity was determined by refractometry. Urine protein/creatinine ratios were calculated. All testing was performed as described previously (23) apart from creatinine, which was done enzymatically (Randox Laboratories Canada Ltd., Mississauga, ON) on an automated chemistry analyzer (Hitachi 911; Boehringer Mannheim Corp, Indianapolis, IN). For urine electrophoresis, urine samples were first concentrated approximately 10x by ultrafiltration (15,000-dalton MW cutoff, Minicon Concentrator-B15; Amicon Canada Ltd, Oakville, Ontario). Concentrated urine samples were subjected to agarose gel electrophoresis with a barbital buffer (Titan Gel Serum Protein System; Helena Laboratories, Beaumont, Texas).
Renal Morphometry
Renal biopsies were taken at taken at various timepoints ranging from 1 to 10 mo of age. All 31 dogs underwent renal biopsies: three times for 8 dogs, twice for 7 dogs, and once for the remaining dogs. Dogs were given general anesthesia and biopsies were obtained by laparotomy using a disposable single-action biopsy 18-gauge needle (E-Z Core; Products Group International Inc., Lyons, CO). Samples were fixed in 4% formaldehyde/1% glutaraldehyde, post-fixed in 2% osmium tetroxide, dehydrated in graded acetone, and embedded in Epon. Thick (1-µm) sections were stained with toluidine blue, and blocks containing glomeruli were selected for ultrastructural examination. Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a JEM1230 (JEOL, Tokyo, Japan). Images of capillary loops were captured electronically at a magnification of x12000 and transferred to a morphometric analysis program (Scion Image, Frederick, MD). A total of 351 images were analyzed in a blinded fashion. The length of GBM with multilaminar splitting was expressed as a percentage of the total length of GBM examined. Tangentially cut portions of the GBM were not included in the measurements. The proportions of normal, segmentally obsolete, and globally obsolete glomeruli were determined from toluidine blue-stained thick sections. The interstitial area was measured adapting the approach of Kashtan et al. (15). Instead of a grid-counting system, images of cortex were captured electronically at a magnification of x20 using an Olympus light microscope and transferred to Scion Image for direct measurement. A total of 52 images were analyzed in a blinded fashion. The interstitial area was obtained by subtracting the area of the tubules, glomeruli, and vessels from the total cortical area, and expressed as a fraction of interstitial area over total cortical area.
Statistical Analyses
Data were expressed as individual values observed in each dog. Relationships between age, dog status and treatment, and body weight, serum concentrations of creatinine and albumin, sodium sulfanilate half-time, percent split GBM, and proportion of obsolete glomeruli were assessed using least squares linear regression. Differences in the slopes of regression lines were assessed by the F test. Where indicated, the Wilcoxon rank sum test was used to assess treatment differences (Statistix 7; Analytical Software, Tallahassee, FL). Differences with P 
0.05 were considered significant.
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Results |
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Urinary protein/creatinine ratios were determined because it is difficult to do 24-h urine collections in animals. Cyclosporine A treatment had no significant effect on the urinary protein/creatinine ratio in normal or affected dogs up to 4 mo of age, but ratios were significantly increased as a result of cyclosporine A treatment in both normal and affected dogs older than 4 mo (Table 1). On average for all groups of dogs, ratios doubled with cyclosporine A treatment. In normal dogs, cyclosporine A treatment decreased the urine specific gravity, but the effect was only significant in older dogs (Table 2). In contrast, treatment of affected dogs was associated with significantly improved concentrating ability in both age groups. Urine agarose gel electrophoresis showed that albumin comprised between 38 to 46% of the protein present in affected dogs, regardless of whether the animals received cyclosporine or not (results not shown).
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1/2 of sodium sulfanilate are shown in Figure 3. Cyclosporine A treatment had no effect on the sodium sulfanilate half-times in normal dogs; thus the data from treated and untreated normal dogs were pooled. Between 3 and 4 mo of age, the half-time clearance of sodium sulfanilate began to increase in affected dogs, both treated and untreated. However, the increases were significantly lower in treated affected dogs (P = 0.02).
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Discussion |
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In the present study, affected and normal dogs treated with cyclosporine A were compared with untreated dogs. Cyclosporine A treatment of affected dogs had significantly beneficial effects on weight gain, renal function, and renal morphometry. Weight gain, urine-specific gravity, serum creatinine concentration/body weight ratio, and GFR were all significantly improved. In fact, weight gain and serum creatinine concentration/body weight ratio were indistinguishable from normal dogs. Although all treated and untreated affected dogs eventually attained 100% splitting of GBM, the onset and rate of progression of multilaminar splitting were significantly delayed in treated dogs. Additionally, cyclosporine A treatment was significantly associated with decreased glomerulosclerosis. In no instances, however, were the dogs cured of their disease; the relentless progression to renal failure was only delayed, not halted.
One of the most striking results in human Alport patients receiving cyclosporine A was a marked reduction or elimination of proteinuria (20,21). Protein balance in the present study was assessed by determining serum protein concentrations and the urinary protein/creatinine ratio. The latter is useful in dogs because the ratio is highly correlated with 24-h protein loss in the urine, and timed urine collections are logistically difficult in animals. Serum albumin levels were reduced in affected dogs compared with normal dogs, implying these animals had persistent proteinuria. The levels were comparable to normal dogs until about 2 mo, which corresponds with the onset of proteinuria in affected animals. Furthermore, treatment of affected dogs was associated with increased urinary protein/creatinine ratios. The increased ratios could theoretically result in part from decreased urinary creatinine, except that muscle mass was increased in treated dogs and GFR improved, hence urinary creatinine would be unlikely to decrease. The increased ratio would then indicate increased or at least persistent proteinuria, with cyclosporine A. Urine protein loss was still elevated after urine protein concentrations were adjusted for a standardized urine creatinine concentration (data not shown), supporting our conclusion that a reduced creatinine did not account for the higher ratios. Additional studies, likely utilizing timed urine collections, would be needed to resolve the magnitude of the urine protein loss. We can nevertheless conclude that, unlike the protein-sparing effect of cyclosporine A treatment demonstrated in human patients with Alport syndrome, for this canine model of Alport, cyclosporine A treatment does not have any protein-sparing effect. This lack of protein sparing is not related to preexisting GBM damage; dogs were started on treatment at 1 mo of age, when morphologic evidence for GBM damage is just starting. Urine electrophoresis confirmed that the amount of albumin spilled in the urine is comparable in treated and untreated affected dogs, indicating that the selective permeability of the GBM is not restored by cyclosporine A in our animal model and that the benefits of treatment come from other mechanisms.
One advantage of an animal model over human-based studies is the ability to follow sequential histologic changes. We hypothesized that if cyclosporine A reduces or eliminates proteinuria in humans and slows the decline of renal function in affected dogs and humans, this effect might be at the glomerular level and may manifest as improved glomerular histology. We had previously found that multilaminar splitting of the GBM begins around 1 mo of age and progresses steadily until virtually 100% is affected by about 4 mo of age (22,27). In the present study, affected dogs receiving cyclosporine A showed a significant 1-mo delay in the onset of multilaminar splitting but then developed this change at about the same rate as untreated affected dogs. This suggests that at least part of the beneficial effect of cyclosporine A treatment was associated with a delay in glomerular deterioration. We did not find, however, that cyclosporine A halted progression of renal pathologic changes, as was found in the human study (21). In that study, no morphometric assessment of biopsies was performed, so progression of changes at the ultrastructural level may have gone undetected. Alternatively, the rate of ultrastructural change in human Alport syndrome is likely considerably slower than that in our canine model.
The measurement of the total number of obsolete glomeruli was based on work by Kashtan et al. (15) in which this parameter was found to correlate with a decline in GFR and renal outcome. In both human and canine Alport syndrome, as glomeruli become progressively damaged, they develop segmental and global sclerosis. In untreated affected dogs, by 3 mo of age, segmental sclerosis was well-established and there was global sclerosis in a minority of glomeruli, but this increased such that almost all glomeruli are globally sclerotic by the time the dog reaches end-stage renal disease. Treatment with cyclosporine A had the dramatic effect of reducing the extent of global sclerosis to almost zero over the same time period. Sclerosis still progressed in treated animals, but it remained predominately segmental for the duration of the study. The functional and morphometric data support that cyclosporine A treatment is associated with a delay in glomerular deterioration. In patients receiving cyclosporine A, detailed counts of obsolete glomeruli were not performed, but two of eight patients were reported to show segmental sclerosis on biopsy, which was nonprogressive over 7 to 10 yr. Of note, all the patients in the cyclosporine A study were under 25 yr of age (21). Kashtan et al. (15) found that global sclerosis was present in about 50% of patients by age 25, suggesting the possibility that the patients in the cyclosporine A study may have been affected with a milder form of Alport syndrome.
The later stages of renal damage common to most glomerular diseases include tubular atrophy and interstitial fibrosis. In Alport syndrome, there is a strong correlation between increased cortical interstitial volume and deterioration in renal function (15). An increase was rarely seen in the first decade of life, but approximately 40% of patients with Alport syndrome had increased cortical interstitial volumes after 10 yr of age (15). In our canine model, increasing interstitial fibrosis was noted over time in affected male dogs with the result that there was a significant difference in interstitial area compared with normal male dogs. Although cyclosporine A itself can also result in interstitial fibrosis, we noticed no effect of cyclosporine A on the interstitial area in either normal dogs or affected male dogs. This may be a reflection of the drug levels maintained in this study; these values have been shown to be safe for use in dogs (26). Similarly, the patients with Alport syndrome who received cyclosporine A were reported to show no evidence of toxicity on biopsy (21), although no morphometry was done. From our results, it can also be stated that cyclosporine A did not reduce the amount of interstitial fibrosis. We noted increased concentrating ability in affected dogs treated with cyclosporine A. Assuming this reflects tubulointerstitial function, we cannot attribute this improvement to better preservation of the tubulointerstitial component of the kidney. Perhaps this improvement reflects increased blood flow to tubules because there is better preservation of glomeruli with cyclosporine A treatment.
Although most patients with Alport syndrome are treated with dialysis and/or renal transplantation, other therapies have been tried before cyclosporine A. An earlier study in this family of dogs showed that a modified diet low in protein, lipid, calcium, and phosphorus delayed the onset of renal failure, increased life span by about 50%, and delayed the progression of the multilaminar appearance of the GBM (17). Drug therapy in canine and human Alport syndrome has included the use of angiotensin-converting enzyme inhibitors (ACE-I) (18,19,28). Patients on this therapy showed a decrease in the urinary protein/creatinine ratio, stabilization of the decline of creatinine clearance (18), and decreased proteinuria in about one third of subjects, although serum creatinine continued to increase (28). We previously studied the therapeutic benefit of an ACE-I in our family of dogs with Alport syndrome (19) and found this drug increased the lifespan of the affected dogs by about 35% and reduced the rise of serum creatinine concentration, the degree of proteinuria, and the urinary protein/creatinine ratios. There was a significant delay in the extent of multilaminar splitting of the GBM. Overall, ACE-I was as effective as cyclosporine A in improving the renal outcome in this family of Alport dogs, and both drugs were associated with a delay in glomerular deterioration. It is unknown whether combined therapy using more than one drug or adding dietary modification would be synergistic in the treatment of Alport syndrome.
Multimodal therapy is more likely to succeed if the individual therapies work through different mechanisms. The mechanism of action of cyclosporine A in canine or human Alport syndrome is not well understood. On the basis of animal studies and treatment of other glomerular diseases with proteinuria in humans, suggested mechanisms of action have included alterations in (1) renal hemodynamics, (2) GBM permselectivity, and (3) lymphokine secretion. Changes in renal hemodynamics could account for many of the changes we observed in the present study. In rats, cyclosporine A has been shown to reduce renal blood flow and increase renal vascular resistance mainly at the afferent glomerular level, leading to reduced filtration pressure and a lowered GFR (29–32). Reduced filtration pressure would decrease the amount of protein passing through the GBM (i.e., decreased proteinuria) and reduce mechanical stress on the GBM. The latter could account for the delay in multilaminar splitting seen in treated affected dogs. However, we did not find a decrease in proteinuria, and GFR was actually improved in affected dogs on cyclosporine A. This in turn may be related to the improved glomerular morphology.
In the normal GBM, there are two networks of collagen type IV, one containing the 1 and 2 chains and the other containing the 3, 4, and 5 chains (33). The 345 network is more highly cross-linked and is believed to confer added structural integrity to the GBM. In canine X-linked Alport syndrome (25) and most cases of human Alport syndrome (11–13), the GBM contains only the 1 and 2 chains and lacks the 3, 4, and 5 chains. The Alport GBM is likely structurally weaker than the normal GBM and deteriorates progressively under the continued exposure of normal glomerular filtration pressures. This deterioration is reflected at the ultrastructural level as a multilaminar appearance that is not present at birth (25) but gradually increases in extent over time (22). It follows that a reduction in glomerular filtration pressure might delay this deterioration, as we observed. The mechanism by which cyclosporine A lowers glomerular filtration pressure is also not understood. Some have suggested that this effect is mediated through angiotensin II (30), but others have refuted this (29). Our previous study using ACE-I would support the latter (19). We found that administration of ACE-I duplicated many of the effects of cyclosporine A in affected dogs. ACE-I would block the effects of angiotensin II; it therefore follows that the effects of cyclosporine A are not likely mediated through the action of angiotensin II.
Other possible actions of cyclosporine A should be considered. Glomerular diseases treated with cyclosporine A also showed a reduction in proteinuria that was attributed to a restoration of the permselectivity of the GBM (34). Whether improved GBM permselectivity is an important effect of cyclosporine A in Alport syndrome, human or experimental, is unknown. Cyclosporine A also affects the expression of other inflammatory mediators and nonstructural proteins that can affect the glomerulus. Transforming growth factor-
(TGF-) has been implicated as playing a role in the progression of glomerular disease in murine Alport syndrome (35), and blockade of TGF- was associated with improved glomerular function and GBM morphology (36). The beneficial effects of cyclosporine A are not likely mediated through blockade of TGF- because cyclosporine A increases TGF- expression (37,38), and the toxicity of cyclosporine A may be the result of increased TGF- expression (39). This would suggest, however, that combining cyclosporine A with drugs that block of TGF- might have synergistic benefits in Alport syndrome.
It has also been shown that the GBM in Alport syndrome is more susceptible to proteolysis than the normal GBM (40). This raises the possibility that, in vivo, proteolysis is contributing to the progressive deterioration of the GBM, either from activation of MMP-2 and MMP-9 or decreased activation of TIMP. Cyclosporine A has been shown to increase the expression of TIMP (39,41) and decrease the expression of MMP-2 (42) and MMP-9 (39). Hence, some of the beneficial effects of cyclosporine A observed in canine and human Alport syndrome may be the result of suppression of proteolysis of the GBM in vivo. A molecular genetic approach may provide a more detailed and informative dissection of the mechanisms of action of cyclosporine A in Alport syndrome. Newer technologies utilizing DNA microarrays would allow for screening of large numbers of genes with respect to identifying those with differential expression upon exposure to cyclosporine A.
In conclusion, we found that cyclosporine A had beneficial effects in improving renal function and delaying the onset of renal failure in canine X-linked Alport syndrome. At least part of this effect was mediated through improved glomerular morphology. Postulated mechanisms of action include a decrease in mechanical damage of the GBM from reduced glomerular filtration pressures and reduced matrix degradation from altered expression of metalloproteinases and their inhibitors. In these ways, cyclosporine A could effect an improvement in glomerular function and morphology. It must be borne in mind, however, that proteinuria was unchanged by cyclosporine A, and this may ultimately contribute to the renal failure that ensues despite drug treatment. Proteinuria can mediate damage in both glomerular and tubulointerstitial components; therefore, a more detailed analysis of the latter would also be informative.
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Acknowledgments |
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References |
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3(IV) and 4(IV) collagen genes in autosomal recessive Alport syndrome. Nature Genet 8: 77–82, 1994[CrossRef][Medline]
1(IV), 5(IV), and 6(IV) collagen chains in normal human adult and fetal tissues and in kidneys from X-linked Alport syndrome patients. J Clin Invest 96: 1948–1957, 1995
5 chain: Normal distribution and abnormalities in X-linked Alport syndrome revealed by monoclonal antibody. Am J Pathol 144: 986–996, 1994[Abstract]
3(IV):Alport antigen heterodimer. J Biol Chem 267: 4137–414, 19925 chain of collagen type IV. Proc Acad Natl Sci USA 91: 3989–3993, 19943, 4 and 5 chains of type IV collagen and its implications for the pathogenesis of Alport syndrome. J Biol Chem 273: 8767–8775, 19981 in Alport renal disease progression. Kidney Int 56: 1662–1673, 1999[CrossRef][Medline]
11 and transforming growth factor-1 play distinct roles in Alport glomerular pathogenesis and serve as dual targets for metabolic therapy. Am J Pathol 157: 1649–1659, 2000This article has been cited by other articles:
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M. N. Rheault, S. M. Kren, B. K. Thielen, H. A. Mesa, J. T. Crosson, W. Thomas, Y. Sado, C. E. Kashtan, and Y. Segal Mouse Model of X-Linked Alport Syndrome J. Am. Soc. Nephrol., June 1, 2004; 15(6): 1466 - 1474. [Abstract] [Full Text] [PDF]
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, n = 9), and normal male dogs with or without cyclosporine A (
, n = 5). Treatment with cyclosporine A commenced at 1 mo of age. Data points represent the mean ± SE. Untreated male dogs did not gain weight after 4 mo of age. Rates of gains in treated dogs were not significantly different from normal dogs.
, n = 6). Values are expressed as the fraction of area occupied by the interstitium over the entire area of cortex measured. Data points represent the mean of duplicate determinations. The difference in interstitial area was significantly greater in affected male dogs compared with normal dogs. (P = 0.005). Over time, there was an increase in the interstitial area in affected dogs, but this was not significantly altered by cyclosporine A treatment (P = 0.99). Normal dogs did not develop interstitial area increases, and this was not significantly changed by cyclosporine A treatment (P = 0.21).