Differentials of Microscopic Hematuria

1.      The Nutcracker syndrome-Compression of the left renal vein in the fork between the abdominal aorta and the proximal superior mesenteric artery. This results in left renal venous hypertension leading to the development of collateral veins with intrarenal and perirenal varicosities.

2.      Loin pain Hematuria syndrome-progressive loin pain accompanied by hematuria. It is described in young women taking oral contraceptives but can also affect young and middle-aged men. Unilateral or bilateral renal colics that do not radiate occur. This may be caused by renal vasospasm which may lead to local hypercoagulability.

3.   Among patients with microscopic hematuria found in a screening program, 3-22% were discovered to have an underlying cancer and among patients older than 50 referred to a hospital because of asymptomatic hematuria, 13% had a neoplasia of the urinary tract.

Mechanism of Hematuria

1.      The size of the gaps in the GBM, if these gaps are larger than 0.25 mm, the stretching and retracting force of the internal chamber of the GBM, combined with the capillary pulse, cause the deformed RBCs to pass through the gaps and proceed to the urinary space.

2.      During the passage the erythrocytes might modify their morphology because of their intrinsic deformability, the intraglomerular capillary pressure, the sizes of gaps and the thickening of GBM. During their passage along the nephron, red blood cells are exposed to sudden variations of pH and osmotic pressure as well as to the injurious effects of tubular enzymes.

Ultrastructure of Glomerular basement membrane (GBM)  

1.      Histologically, glomerular capillary wall is composed of endothelial cells, GBM and podocytes.

2.      GBM is the thickest and most elastic capillary wall in the body, because it has to sustain the highest capillary pressure (45 mm Hg) in the body.

3.      GBM is a sheetlike structure that supports endothelial and epithelial cells and is composed of various glycoproteins secreted by these cells.

4.      Traditional basement membranes are composed of type IV collagens, laminins, proteoglycans, and entactin.

5.      Most of these heterotrimers consist of two a1(IV) chains and one a2(IV) chain, but an important minority contains an a3(IV), an a4(IV), and an a5(IV) chain.

6.      GBM is a highly specialized basement membrane with a3(IV), a4(IV), and a5(IV) collagens represented much more abundantly than a1(IV) chains and a2(IV) chains.

7.      Basement membranes in placenta and liver exhibit a limited diversity in collagen composition and are principally formed of a1(IV) and a2(IV) collagens.

Collagens 

1.      The collagens are defined as proteins that contain one or more characteristic triple-helical domains consisting of three polypeptides with repeated Gly-X-Y amino acid triplets called a chains, that wind around each other in a right-handed helix in each molecule to form a characteristic collagen triple helix.

2.      Collagens are the most abundant molecules in the extracellular space, predominantly form either fibrillar or sheet-like structures-the two major supramolecular conformations that maintain tissue integrity.

3.      Nineteen distinct collagen types have been identified in vertebrates.

4.      The biosynthesis and self-assembly of fibrillar collagens. In the rough endoplasmic reticulum, three pro-a chains are posttranslationally glycosylated, associate through disulfide bonds in the C-propeptides, and fold into a triple helix in a zipper-like manner. The secreted procollagen is cleaved at each end by the two processing enzymes, N- and C-proteinase, to generate collagen molecules, which then self-assemble to form fibrils in the extracellular space.

5.      The six human collagen IV genes are located in pairs in a head-to-head manner on three different chromosomes. a1(IV) and a2(IV) chains are products of distinct genes located pairwise in a head-to-head fashion on chromosome 13. a3(IV) and a4(IV) chains are present on chromosome 2, and the a5(IV) and a6(IV) chains are located on the X-chromosome.

6.      The classical type IV collagen trimer isolated from the extracellular matrix has the composition of (a1)₂(a2)₁.

7.      Differential expression of collagen IV genes-Tissue distribution. The a1(IV) and a2(IV) chains are normally found in all basement membranes. The a3(IV) and a4(IV) chains are expressed in basement membranes of GBM, the cochlea, and ocular basement membranes that are involved in AS. All basement membranes that express the a3(IV) and a4(IV) chains also express a5(IV), although the converse is not true. For example, EBM express a5(IV) and a6(IV), but not a3(IV) and a4(IV).  

Alport’s and Thin Basement membrane disease

1.      Isolated glomerular hematuria may occur as a familial or sporadic condition, and is often associated with a renal biopsy finding of excessively thin GBM.

2.      BFH and early AS may be difficult to distinguish histologically, because diffuse GBM attenuation is characteristic of both.

8.      However, the GBM of BFH patients remains attenuated over time, rather than undergoing the progressive thickening and multilamellation that is pathognomonic of AS.

9.      Mutations were detected in three type IV collagen genes in Alport’s syndrome. The majority was present in the X-linked type IV collagen a5(IV) gene, but recently mutations in the type IV collagen a3 and a4(IV) genes have been reported in patients with the autosomal recessive form of this disease.

10.  Considering the similarities in GBM abnormalities, autosomal Alport syndrome and BFH could be the severe and mild forms of different molecular genetic defects in the same genes.

1. Alport’s Syndrome

History

1.      First description in 1902 by Guthrie of familial hematuria.

2.      Follow-up studies of the family by Hurst and Alport described the progressive nature of the nephropathy, its association with deafness, and the poorer prognosis in affected males.

3.      Identification of GBM as the site of primary renal abnormality in early 1970s.

4.      Between 1980 and 1990, type IV collagen was identified as the defect in Alport’s syndrome.

5.      Identification of COL4A5 mutations in patients with X-linked AS in 1990.

 Genetics

1.      Alport’s syndrome is a disease of collagen that affects the kidneys always, the ears often, and the eyes occasionally. The primary chemical defect in Alport’s syndrome most commonly involves the a5(IV) chain, but faulty assembly of the a3,4,5 heterotrimer produces similar pathology in glomerular, aural, and ocular basement membranes regardless of which a chain is defective.

2.      At lease 80% of kindreds are X-linked and over half of those result from a mutation of COL4A5, the gene located at Xq22 that codes for the a5 chain of type IV collagen. Autosomal recessive inheritance occurs in 5 % of cases and autosomal dominant inheritance has been shown in a few kindreds with associated thrombocytopathy (Epstein’s syndrome).

8.      Deletions, point mutations, and splicing errors occur in COL4A5 gene causing X-linked Alport’s syndrome (XAS).

9.      Normal glomerular capillaries filter plasma through basement membrane (GBM) rich in a3(IV) and a4(IV), and a5(IV) chains of type IV collagen. In X-linked Alport syndrome (XAS), their GBM instead retain a fetal distribution of a1(IV) and a2(IV) isoforms because they fail to developmentally switch their a-chain use. The anomalous persistence of these fetal isoforms of type IV collagen in the GBM in XAS confers an unexpected increase in susceptibility to proteolytic attack by collagenases and cathepsins.

10.  Homozygotes or mixed heterozygotes for mutations of COL4A3 or COL4A4 can develop autosomal recessive Alport’s syndrome. ARAS should be suspected when an individual exhibits the typical clinical and pathologic features of the disease but lacks a positive family history, especially when a young female has findings indicative  of severe disease such as deafness, renal insufficiency, or nephrotic syndrome. Patients with COL4A3 mutations appear to progress to ESRD before age 30 and also have sensorineural deafness, regardless of gender. Some cases of autosomal recessive Alport’s syndrome may be homozygotes for a COL4A4 mutation that causes benign familial hematuria.

11.  Epstein’s syndrome is an uncommon autosomal dominant variant of Alport’s syndrome associated with moderate thrombocytopenia with severe hearing loss and renal failure in both males and females.

12.  In most kindreds with Alport’s syndrome the GBM of affected males fails to stain in the normal fashion with anti-GBM sera, and the GBM of female heterozygotes stains in an interrupted fashion.

Pathology

13.  The characteristic features are seen by electron microscopy. The GBM is thickened up to two or three times its normal thickness, split into several irregular layers, and frequently interspersed with numerous electron dense granules about 40 nm in diameter. Early in the development of the lesion, thinning of the GBM may predominate or may be the only abnormality visible.

14.  A mutation affecting one of the chains involved in the putative a3-a4- a5(IV) network can prevent GBM expression not only of that chain, but also of the other two chains. The mechanism is unknown, yet.

15.  In males with XLAS, the GBM, distal tubular basement membrane (TBM), and Bowman’s capsules of males with XLAS usually fail to stain for the a3(IV) and a4(IV), and a5(IV) chains, but do express the a1(IV) and a2(IV) chains. Women who are heterozygous for XLAS mutations frequently exhibit mosaicism of GBM expression of the a3(IV) and a4(IV), and a5(IV) chains.

16.  In patients with ARAS, GBM usually show no expression of the a3(IV) and a4(IV), and a5(IV) chains, but a5(IV) and a6(IV) chains are expressed in Bowman’s capsule, distal TBM, and EBM. Therefore, XLAS and ARAS may be distinguishable by immunohistochemical analysis of renal biopsy specimens.

Clinical Presentation

17.  Uninterrupted microscopic hematuria is present from birth in affected males. Urinary RBCs are dysmorphic, and RBC casts can usually be found. Proteinuria is variable; occasionally it reaches nephrotic levels. Hemizygous males inevitably progress to end stage renal disease (ESRD). Heterozygous females are generally much less severely affected. Around one fifth of them will develop ESRD after the age of 50.

18.  The families with early onset of renal failure in males are termed “juvenile” and those with renal failure in middle age are called “adult” type nephritis. Extrarenal manifestations tend to be more prominent in the juvenile kindreds. In families with juvenile type disease, hearing loss is almost universal in male hemizygotes and common in severly affected female heterozygotes.

19.  In Alport syndrome, renal disease progresses largely due to the increased susceptibility to proteolysis and unrestrained deposition of certain collagens, most likely, a1(IV) and a2(IV), resulting in glomerulosclerosis.

20.  After transplantation, about 10% of Alport’s males develop anti-GBM nephritis. The “Alport’s antigen” is a 26-kDa monomer belonging to the a3(IV) chain. The failure of normal heterotrimer formation due to the genetic defect in the gene coding for the a5(IV) chain causes the absence of demonstrable a3(IV) chains in GBM.

2. Benign Familial Hematuria (Thin GBM Disease)

1.      Thin GBM is a descriptive term, and because it is likely that several disorders that differ at the molecular level can be associated with this abnormality, benign familial hematuria (BFH) is appropriate for familial, non-progressive hematuria.

2.      BFH is a genetically heterogeneous condition and it is usually autosomal dominant disease.

3.      Lemmink et al. in 1996 described a first genetic defect explaining BFH. In a large Dutch BFH kindred, the disease locus was mapped to chromosome 2 in the region of COL4A3 and COL4A4 genes. A pathogenic mutation in the COL4A4 was identified.

4.      Immunohistological evaluation of GBM type IV collagen may be useful in the differentiation of BFH and AS.

5.      Overt proteinuria and hypertension are unusual in BFH, but have been described. Some of these cases may represent variants of AS, rather than thickening and multilamellation.

6.      Light and immunofluorescence microscopy is unremarkable in typical cases of BFH or thin GBM disease. Ultrastructurally, most patients with BFH exhibit diffuse thinning of the GBM as a whole, and of the lamina densa. There is no disruption or lamellation of GBM.

7.      The cutoff value of 250 nm will accurately separate adults with normal GBM from those with thin GBM. It is useful to note that the intraglomerular variability in GBM width is small in thin GBM disease. Marked variability in GBM width within a glomerulus, in a patient with persistent microhematuria, should raise suspicion of Alport’s syndrome.

8.      If the patient’s family history indicates autosomal dominant transmission of hematuria, and there is no history of chronic renal failure, a presumptive diagnosis of BFH can often be made without kidney biopsy. When family history is negative or unknown, or there are atypical coexisting features such as proteinuria or deafness, renal biopsy may be extremely informative.

9.      Normal distribution of type IV collagen alpha chains provides supportive evidence for a diagnosis of BFH or thin GBM disease.

10.  Patients who are given a diagnosis of BFH or thin GBM disease should be reassured, but not lost to follow-up exam every year, since the risk of chronic renal insufficiency appears to be small but real.