Clinical History:
A three-year-old white boy presented to the emergency room department after he developed
"tea-colored" urine. He was given antibiotics (not specified) in the emergency department and referred
to his primary physician. There was no periorbital or extremity swelling, petechiae, purpura, rashes,
joint swelling or pain. He had upper respiratory symptoms with low-grade fever and a "croupy cough".
The past medical history was significant for a normal vaginal delivery induced four weeks before term
secondary to recurrent "UTI's" in the mother. The family history was significant for approximately 40
episodes of "UTI's" not further characterized in the mother, as well as a history of one episode of
nephrolithiasis in the mother. The father also had a history of UTI's and mild vesicoureteral reflux as
a child. The father had a history of persistent microscopic hematuria with onset in childhood, and many
episodes of gross hematuria between the ages of five and seven years. He had been followed by a
pediatric nephrologist. The father currently at age 36 still had positive dipstick for blood with
occasional RBCs, no RBC casts. He had normal renal function (Screat 0.9) and no proteinuria, blood
pressure 100/64 nm Hg on no meds. The father's mother at some point had "kidney trouble" but no details
were known. Both the father's parents were alive and well.
On examination, the child was afebrile, with blood pressure 86/59 mm Hg. He weighed 13 kg (25%), and
was 93 cm tall (25%). He appeared alert and playful and was in no apparent distress. The physical exam
was unremarkable, and further review of system was noncontributory. Laboratory examination showed WBC
7000/mm3, PCV 36%, platelets 316,000 mm3. The differential showed 31% neutrophils, 54% lymphocytes and
6% atypical lymphocytes. Electrolytes were normal, BUN was 9 mg/dl, serum creatinine 0.3 mg/dl, Ca 2+
8.8 mg/dl, phosphorus 4.5 mg/dl, glucose 69 mg/dl, albumin 3.3 g/dl, total biliubin 0.5 mg/dl, alkaline
phosphatase 154 U/L, SGOT 55 U/l. An ASO titer was <58 (<125 is neg), and streptozyme was
<1:100. C3 was slightly decreased at 84 units mg/dl (88-201 normal), C4 was 38 mg/dl (15/45), IgA
level was 52 mg/dl (25-154 mg/dl normal), and ANA and ANCA tests were negative. Urinalysis showed a
specific gravity of 1.020, pH 7, 2+ protein and large blood positivity, with negative leukocyte esterase
and nitrogen tests. The microscopic exam showed 5 to 10 WBCs and numerous dysmorphic RBCs and occasional
RBC casts. Renal ultrasound showed normal kidneys with normal shape and size (7 cm), without cysts,
stones or masses. The urine protein/creatinine ratio was 0.4.
The patient was followed up in the Pediatric Renal Clinic with close monitoring of renal function and
hematuria. The hematuria persisted over the next six months, and diagnostic procedures were performed.
Case 5 - Figure 1 - Light microscopic appearance of a representative glomerulus from the 3-year-old patient, revealing mild increase in mesangial matrix. (Jones' silver stain, X400).
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Case 5 - Figure 2 - Diffuse thinning of glomerular basement membrane with intact foot processes and no deposits. (Transmission electron microscopy, X4,000)
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Case 5 - Figure 3 - Some glomerular capillary walls have lamellated basement membranes. (Transmission electron microscopy, x5000).
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Case 5 - Figure 4 - Research immunostaining with antibody to a5 type IV collagen in control normal tissue and in the 3-year-old patient.(X200)
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Case 5 - Figure 5 - Research immunostaining with antibody to a5 type IV collagen in control normal tissue and in the father.(X200)
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Material for review:
Three Kodachromes
Renal Biopsy Findings:
The LM was unremarkable, and standard IF was negative except for 1+ arteriolar staining with C3. By
electron microscopy the GBM were diffusely thin, ranging from 75 to 124 nm in most areas (normal
thickness in a 3-year-old child in our laboratory is about 150 to 200 nm). There were also very small
segmental areas with lamellation of the lamina densa. No deposits were present.
In view of the above findings, the differential diagnosis of Alport syndrome,either X-linked or
autosomal recessive, or benign familial hematuria was considered. Research studies with stains for
collagen type IV were performed. In the patient's biopsy, there was 1 to 2+ capillary, tubular basement
membrane in Bowman's capsule staining with "-1 type IV collagen antibody, a control indicating that the
tissue was well-preserved for the studies. Staining with antibody to "-3 type IV collagen was completely
negative. Staining with antibody to "-5 type IV collagen was present in Bowman's capsule and distal
collecting ducts, but was negative in the glomerular basement membrane.
Interpretation and further work-up:
As further discussed below, extensive thinning of basement membranes may be found in benign familial
hematuria, in female carriers of X-linked Alport's syndrome or in carriers of autosomal dominant or in
recessive type of Alport's syndrome, and also as an early finding of Alport's syndrome. The findings in
this biopsy are suggestive of the possibility of the autosomal recessive form of Alport's syndrome.
However, "-3 and "-5 type IV collagen staining has not been fully characterized in kindreds of benign
familial hematuria with mutations of type IV collagen as discussed further below, and therefore, this
entity could not be definitively ruled out by the studies in this patient. In some kindreds with benign
familial hematuria, mutations of the same collagen gene as in autosomal forms of Alport's syndrome have
been detected, suggesting these entities may represent a continuum. Based on this possibility, a renal
biopsy was also performed in the father. The biopsy from the father showed mild interstitial fibrosis
with proportional tubular atrophy involving approximately 15 to 20% of the biopsy, with mild arteriolar
and arterial thickening. Standard IF was negative. EM showed diffuse thinning, on average 172 nm, over
approximately 80% of the circumference of the capillary loops with very focal areas of mild lamellation
with occasional extruded organelles, but without distinct basketweave pattern. Research IF showed normal
staining for type IV collagen types.
Final Diagnosis:
In view of the normal immunostaining pattern in the
father and the immunostaining pattern in the child, and the extensive thinning of glomerular basement
membranes, the findings were felt to be most consistent with autosomal recessive
type of Alport's syndrome, with heterozygosity in the father resulting in the abnormal
phenotype of the GBM but with sufficient normal type IV collagen expressed to give normal immunostaining
pattern.
Discussion:
The differential diagnosis of hematuria includes both immune complex diseases, metabolic
abnormalities including hypercalciuria, and importantly various forms of hereditary nephritis. These two
cases illustrate the complexity of differential diagnosis of the finding of thin basement membranes,
which includes benign familial hematuria, and various forms of Alport's syndrome, as discussed below.
Alport's Syndrome
Clinical Profile
Classical Alport's Syndrome is inherited in an X-linked dominant pattern and is the most common form
of Alport's syndrome (85% of cases). The overall incidence of Alport's syndrome in the United States is
between 1:5,000 to 1:10,000. Manifestations typically include hematuria, hearing loss, ocular defects
and progression to renal failure in affected men by early adulthood. Hematuria is the initial renal
presentation of disease, although some proteinuria may also be present. Nephrotic syndrome may develop
in as many as 30 to 40% of patients with severe disease. Chronic renal failure occurs more commonly in
severely affected men and less commonly in women, occurring overall in 30 to 40% of patients. Hearing loss typically does not manifest until adulthood. Ocular defects occur in
up to a third of patients. Anterior lenticonus is the most common eye defect.
Collagen type IV is made up of heterotrimers composed of combinations of 6 possible alpha chains.
These six type IV collagen genes are arranged in pairs on three different chromosomes: COL4A1 and COL4A2
are on chromosome 13; COL4A3 and COL4A4 are on chromosome 2; and COL4A5 and COL4A6 are on the X
chromosome. The mutation in this classic form of Alport's occurs in the "5 (IV) collagen chain
(COL4A5). Most of the remaining patients with Alport's have the autosomal recessive form resulting from
mutations of "3 or "4 type IV collagen genes (COL4A3 or COL4A4). Rare cases of autosomal dominant
Alport's due to mutations in COL4A3 or COL4A4 also occur. Some patients with large deletions that span
the adjacent 5' ends of the adjacent COL4A5 and COL4A6 genes have Alport's syndrome and coexisting
diffuse leiomyomatosis. In these kindreds, the COL4A6 breakpoint always was in the second intron, but
the molecular pathogenesis for the leiomyomatosis has not been established.
The organs involved in Alport's syndrome reflect sites where these collagen chains are normally
expressed and are essential for function. Thus "3, "4, and "5 type IV collagen chains are normally
highly expressed in the kidney, eye, and ear. In the kidney, heterotrimers of "3, "4, and "5 type IV
collagen are expressed in the GBM, while a (1)252/ (5) 26 heterotrimers are
expressed in Bowman's capsule and in some tubular basement membranes. " (1)252 heterotrimers
are present in the immature glomerulus at birth, with gradual shift to the mature expressionpattern. " `(1)
252 remains expressed in the mesangium and also in Bowman's capsule in the normal adult.
When "5 (or "3 or "4) is mutated, there an inability to form the normal heterotrimers of the GBM.
Pathology
Early in the disease in males, and in affected women, the renal biopsy may show no significant light
microscopic abnormalities. At later stages, glomerulosclerosis, interstitial fibrosis and prominent foam
cells are typical. These foam cells are not specific for this disease, and are found in numerous
proteinuric states. Glomeruli show varying stages of matrix expansion and sclerosis.
Immunofluorescence may show non-specific trapping of IgM. By electron microscopy, the diagnostic lesion
consists of irregular thinned and thickened areas of the glomerular basement membranes with splitting and
irregular multilaminated appearance of the lamina densa, so-called "basket weaving". In between these
lamina, granular, mottled material is present. At early stages of disease, i.e. in children or women,
the basement membrane may show only thinning. To further complicate matters, some kindreds with typical
Alport's syndrome clinically have only manifested basement membrane thinning as a morphologic change,
even at advanced stages.
Clinicopathologic Correlations
Each Alport kindred reported thus far has presented its own unique mutation. The rate of progression
to end stage and deafness are mutation dependent. Large deletions, nonsense mutations or mutations that
changed the reading frame were associated with 90% risk of end stage renal disease before age 30 in
affected males with X-linked Alport's, with only 50% risk for patients with missense and 70% risk for
those with splice site mutations. Risk for hearing loss before age 30 was 60% in patients with missense
mutations, versus 90% risk for all other mutations. Ultrastructural features do not strictly correlate
with type of mutation, in that some patients with major gene rearrangements had no significant lesions,
and varying ultrastructural abnormalities were present even within the same kindred.
Transplantation in patients with Alport's syndrome has shed additional light on the molecular basis
for this disease. Some patients with Alport's receiving kidney transplants, probably around 5-10%,
develop antibodies to the normal glomerular basement membrane in the transplant. Occurrence of this
post-transplant anti-GBM disease appears more frequent in patients with more extensive deletion of the a5
type IV gene.
In kidney biopsies, about 70-80% of males with X-linked Alport's lack staining of GBM, distal tubular
basement membrane and Bowman's capsule for either "3, "4 or "5 (IV) chains, and Bowman's capsule and
distal tubular basement membrane also show lack of "6 (IV). In autosomal recessive Alport's, where "3 or
"4 is mutated, the kidney GBMs usually show no expression of "3, "4 or "5, again because there is an
inability to form the normal "3, 4 5 heterotrimer of the GBM. In contrast to X-linked cases, there is
strong expression of "5 and "6 in Bowman's capsule, distal tubular basement membrane and skin, since the
" (1)252/ (5) 26 heterotrimers can still be assembled in these patients. Female
heterozygotes for X-linked Alport's syndrome frequently show mosaic staining of GBM and distal TBM for
"3, "4 and "5 (IV) chains, and skin mosaic staining for "5(IV). Patients with autosomal dominant
Alport's have not been studied immunohistochemically. Thus, the staining pattern in our 3 year old
patient was suggestive of autosomal recessive Alport's syndrome. The staining pattern in a carrier of
autosomal recessive Alport has not been previously described, but with one normal allele, one might
expect normal immunostaining to be present, as in the father in this case.
Some cases with Alport's syndrome clinically and by renal biopsy showed apparent normal "5 type IV
pattern. About 20% of male classic Alport patients and affected homozygous autosomal recessive Alport
patients show faint or even normal staining of the skin or GBM for "3 and "5. This is postulated to
reflect a mutation that results in protein, that albeit abnormal, still expresses the epitope recognized
by the available antibodies. Thus, the absence of "5 type IV in the skin biopsy is helpful in indicating
a basement membrane abnormality, but an apparent normal staining pattern in either skin or kidney does
not definitively rule out Alport's syndrome. The possible continuum of Alport's syndrome with some cases
of apparent benign familial hematuria with thin basement membranes further complicates interpretation of
staining patterns (see below).
The mechanism(s) of progressive renal scarring in Alport's syndrome are unknown. In a recent report
of 7 patients with Alport's syndrome, decreased proteinuria occurred in response to ACEI, and, after an
initial decrease of GFR, renal function increased towards starting levels by 24 mos.
Thin Basement Membranes
This basement membrane abnormality has also been described as "benign familial hematuria". This term
has been used to distinguish these families from Alport's syndrome since affected individuals have been
thought to have a benign prognosis. However, morphology alone does not allow one to make specific
prognostic inferences (see below). Kindreds may show autosomal dominant or recessive inheritance. The
clinical manifestation is that of chronic hematuria, either macroscopic and microscopic, intermittent or
continuous. This lesion is common, and is present in 20 to 25% of patients biopsied for persistent
isolated hematuria in some series. The lesion may also coexist with other glomerular disease, commonly
diabetic nephropathy or IgA nephropathy. Occasionally patients with thin basement membranes have
nephrotic range proteinuria, with 5 of 8 reported cases showing superimposed FSGS lesions.
The diagnosis of thin basements is based on morphometric measurements from electron microscopic
examination. The glomerular basement membrane thickness normally increases with age. Normal thickness
in adults in one series was 373±42 nm in men versus 326±45 nm in women. Glomerular basement thickness
<250 nm has been used as a cutoff for diagnosis in many series. In children, the diagnosis of thin
basement membranes must be made with caution, establishing normal age-matched controls within each
laboratory. In our laboratory, we found a range of GBM thickness in normal children from approximately
110 nm at age one year to 222±14 nm in seven year olds. As mentioned above, thin basement membranes
(without lamellation) may also be an early or only manifestation in some kindreds with Alport's syndrome.
Thus, the presence of thin basement membranes cannot per se be taken to categorically indicate a benign
prognosis.
An "4 type IV collagen gene mutation segregated with hematuria in a kindred with apparent benign
hematuria. The index patient had electron microscopic changes typical of Alport's syndrome by renal
biopsy at age 5 years, i.e. areas of lamellation alternating with areas of thinning. This boy's parents
both had microscopic hematuria and family histories of benign hematuria without progression in any
members. In contrast, this boy developed proteinuria at age 16 years. These findings suggest that this
patient may have inherited a disorder manifest by hematuria from both parents, resulting in a more severe
phenotype. Further, the findings in this kindred, and other studies, indicate that autosomal recessive
Alport's disease and "benign familial hematuria/thin basement membrane disease" may be the severe and
mild forms of different molecular defects in the same gene. In one study of patients with thin basement
membranes on renal biopsies, there was increased global sclerosis, with later development of hypertension
and renal insufficiency in the patients, and also in some relatives. However, these patients were not
defined molecularly, and were presumed to not have Alport's based on absence of hearing or eye
abnormalities. It is possible that a second process, such as hypertensive nephrosclerosis, was also
present in these families, or that this represents a part of a continuum of basement membrane
abnormalities. These observations further reiterate that the finding of thin basement membranes alone
does not allow one to assign the label of a "benign" process.
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