Autosomal recessive inheritance is found in about 15% of patients with their clinical course and ultrastructural changes being similar to those in X-linked form [14]. It can be caused either by homozygous or compound heterozygous mutations in *COL4A3* or *COL4A4* [15, 16]. Patients who harbor only one pathogenic variant either in *COL4A3* or *COL4A4* gene are usually diagnosed with thin basement membrane nephropathy (TBMN): benign familial hematuria not progressing to ESRD. It is still disputable if patients bearing only one pathogenic variant in either *COL4A3* or *COL4A4* gene who suffer from hematuria, lack hearing or ocular abnormalities, and potentially can develop ESRD in late age should be categorized to autoso-

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Digenic inheritance pattern of AS has also been described, which can be caused by two mutations in *COL4A3* and *COL4A4* genes each (either on the same chromosome or on homologous chromosomes) as well as by a combination of two mutations in *COL4A5* and *COL4A3* or

AS prognosis is known to depend on the mutation type. Associations have been shown both in males with X-linked inheritance and in autosomal recessive form (both sexes). Associations of genotype and phenotype have been extensively studied for the following conditions: age at

The expected ESRD age is similar for the two types of AS and comprises 23–25 years, excluding females with X-linked AS [4]. The risk of ESRD development in patients with X-linked AS by the age of 30 years is 70% in males, whereas only 5% in females; by the age of 40 years: 90

Phenotypic and clinical features in AS are known to depend on mutation type. Among patients with *COL4A5* variants, in males, the risk of development of ESRD by 30 years of age is the highest for subjects bearing "severe" mutations (nonsense, frameshift mutations, and multiexon deletions): 90%; lower for subjects bearing splice site variants: 70%; the lowest for patients bearing missense variants: 50% [19]. In females, there is no statistically significant difference neither for age at onset of ESRD nor for presence of hearing or ocular abnormalities [19].

Hearing and ocular abnormalities are also more prevalent in men than in women among X-linked AS subjects [20, 21]. Sensorineural hearing loss by the age of 30 is found in 60% of male patients with missense mutations compared to 90% of patients with mutations of other more severe types (including splice site mutations) [19]. Ocular abnormalities were shown to be 2–4 times less prevalent in males harboring pathogenic missense variants [20, 22, 23].

In patients with autosomal recessive AS, mutation type has similar effect on the clinical prog-

Collagen type IV is composed of six different α chains, which assemble into three different protomers (α1α1α2, α3α4α5, and α5α5α6) with a tissue-specific distribution [24, 25]. The absence of any one of these type IV collagen chains can result in the absence of the whole protomer in the GBM, presumably due to an obligatory association of the three chains in

nosis [4], which is significant both for women (unlike for X-linked type) and men.

mal dominant AS or TBMN [17].

onset of ESRD, hearing loss, and ocular abnormalities.

*COL4A4* gene [18].

and 10%, respectively [19].

**2.2. Pathogenesis of Alport syndrome**

forming the type IV collagen superstructure [26].

## **2. Alport syndrome: genetic, pathogenesis, clinical presentation, prognosis, and treatment**

#### **2.1. Genetic and genotype-phenotype correlations in Alport syndrome**

Several clinical cases of patients with hereditary familial congenital hemorrhagic nephritis were published by Arthur C. Alport in 1927. He was the first to recognize the link between nephritis and deafness, and he also noted ocular changes in one of these patients [1].

Alport syndrome (AS) is a multisystem disorder including progressive renal disease, sensorineural deafness, and eye abnormalities.

Alport syndrome is caused by mutations in the *COL4A3*, *COL4A4*, and *COL4A5* genes, which encode the α3, α4, and α5 chains of collagen type IV, a component of the glomerular basement membrane (GBM) in the kidney.

*COL4A5* gene is located on chromosome X. More than 2300 *COL4A5* variants have been described to date [2]. Among the pathogenic variants, about 41% are missense mutations (with overwhelming majority of them being substitutions of glycine in the Gly-Xaa-Yaa triplet of the collagenous domain) which are the most frequent, followed by frameshift, splice site mutations, larger copy number variations, and nonsense mutations [3].

The *COL4A3* and *COL4A4* genes are located on chromosome 2 and share the same promoter (they are transcribed in opposite directions). Among *COL4A3* and *COL4A4* mutations, the distribution of mutation types is similar, in particular, glycine substitutions and missense mutations of other types still dominate. Incidence of pathogenic variants in the two genes is similar [4].

The abovementioned genes are known to have no mutation hotspots; however, some population-specific founder mutations have been described, e.g., *COL4A5* p.Leu649Arg mutation in the US [5], COL4A5 p.Gly624Asp mutation in the Slovenia, Hungary, and Greece [6–8], and deletion of exons 2–36 of COL4A5 in French Polynesia [9].

The most frequent X-linked form of AS is caused by *COL4A5* gene defects and detected in 85% of patients [10] (80% according to Kashtan [11]). It is dominant and more severe in males, whereas in females, penetrance depends on X chromosome inactivation pattern [12]. Patients demonstrate end-stage renal disease (ESRD) at the age of 8–60 years and usually have microhematuria with macrohematuria episodes from childhood [13]. About 80% of affected families demonstrate sensorineural hearing loss (at least in some patients) and about 50% are characterized by ocular abnormalities [13].

Autosomal recessive inheritance is found in about 15% of patients with their clinical course and ultrastructural changes being similar to those in X-linked form [14]. It can be caused either by homozygous or compound heterozygous mutations in *COL4A3* or *COL4A4* [15, 16].

Patients who harbor only one pathogenic variant either in *COL4A3* or *COL4A4* gene are usually diagnosed with thin basement membrane nephropathy (TBMN): benign familial hematuria not progressing to ESRD. It is still disputable if patients bearing only one pathogenic variant in either *COL4A3* or *COL4A4* gene who suffer from hematuria, lack hearing or ocular abnormalities, and potentially can develop ESRD in late age should be categorized to autosomal dominant AS or TBMN [17].

Digenic inheritance pattern of AS has also been described, which can be caused by two mutations in *COL4A3* and *COL4A4* genes each (either on the same chromosome or on homologous chromosomes) as well as by a combination of two mutations in *COL4A5* and *COL4A3* or *COL4A4* gene [18].

AS prognosis is known to depend on the mutation type. Associations have been shown both in males with X-linked inheritance and in autosomal recessive form (both sexes). Associations of genotype and phenotype have been extensively studied for the following conditions: age at onset of ESRD, hearing loss, and ocular abnormalities.

The expected ESRD age is similar for the two types of AS and comprises 23–25 years, excluding females with X-linked AS [4]. The risk of ESRD development in patients with X-linked AS by the age of 30 years is 70% in males, whereas only 5% in females; by the age of 40 years: 90 and 10%, respectively [19].

Phenotypic and clinical features in AS are known to depend on mutation type. Among patients with *COL4A5* variants, in males, the risk of development of ESRD by 30 years of age is the highest for subjects bearing "severe" mutations (nonsense, frameshift mutations, and multiexon deletions): 90%; lower for subjects bearing splice site variants: 70%; the lowest for patients bearing missense variants: 50% [19]. In females, there is no statistically significant difference neither for age at onset of ESRD nor for presence of hearing or ocular abnormalities [19].

Hearing and ocular abnormalities are also more prevalent in men than in women among X-linked AS subjects [20, 21]. Sensorineural hearing loss by the age of 30 is found in 60% of male patients with missense mutations compared to 90% of patients with mutations of other more severe types (including splice site mutations) [19]. Ocular abnormalities were shown to be 2–4 times less prevalent in males harboring pathogenic missense variants [20, 22, 23].

In patients with autosomal recessive AS, mutation type has similar effect on the clinical prognosis [4], which is significant both for women (unlike for X-linked type) and men.

#### **2.2. Pathogenesis of Alport syndrome**

detected in 85% of patients. Autosomal recessive inheritance is found in about 15% of patients; it can be caused either by homozygous or compound heterozygous mutations in *COL4A3* or *COL4A4.* Patients who harbor only one pathogenic variant either in *COL4A3* or *COL4A4* gene are usually diagnosed with thin basement membrane nephropathy or autosomal dominant Alport syndrome. Digenic inheritance pattern of Alport syndrome has also been described. Current knowledge and future directions on Alport syndrome will be explored in this review.

Several clinical cases of patients with hereditary familial congenital hemorrhagic nephritis were published by Arthur C. Alport in 1927. He was the first to recognize the link between

Alport syndrome (AS) is a multisystem disorder including progressive renal disease, sensori-

Alport syndrome is caused by mutations in the *COL4A3*, *COL4A4*, and *COL4A5* genes, which encode the α3, α4, and α5 chains of collagen type IV, a component of the glomerular basement

*COL4A5* gene is located on chromosome X. More than 2300 *COL4A5* variants have been described to date [2]. Among the pathogenic variants, about 41% are missense mutations (with overwhelming majority of them being substitutions of glycine in the Gly-Xaa-Yaa triplet of the collagenous domain) which are the most frequent, followed by frameshift, splice site

The *COL4A3* and *COL4A4* genes are located on chromosome 2 and share the same promoter (they are transcribed in opposite directions). Among *COL4A3* and *COL4A4* mutations, the distribution of mutation types is similar, in particular, glycine substitutions and missense mutations of other types still dominate. Incidence of pathogenic variants in the two genes is

The abovementioned genes are known to have no mutation hotspots; however, some population-specific founder mutations have been described, e.g., *COL4A5* p.Leu649Arg mutation in the US [5], COL4A5 p.Gly624Asp mutation in the Slovenia, Hungary, and Greece [6–8], and

The most frequent X-linked form of AS is caused by *COL4A5* gene defects and detected in 85% of patients [10] (80% according to Kashtan [11]). It is dominant and more severe in males, whereas in females, penetrance depends on X chromosome inactivation pattern [12]. Patients demonstrate end-stage renal disease (ESRD) at the age of 8–60 years and usually have microhematuria with macrohematuria episodes from childhood [13]. About 80% of affected families demonstrate sensorineural hearing loss (at least in some patients) and about 50% are

nephritis and deafness, and he also noted ocular changes in one of these patients [1].

**2. Alport syndrome: genetic, pathogenesis, clinical presentation,**

**2.1. Genetic and genotype-phenotype correlations in Alport syndrome**

mutations, larger copy number variations, and nonsense mutations [3].

deletion of exons 2–36 of COL4A5 in French Polynesia [9].

characterized by ocular abnormalities [13].

**prognosis, and treatment**

112 Advances in Nephropathy

neural deafness, and eye abnormalities.

membrane (GBM) in the kidney.

similar [4].

Collagen type IV is composed of six different α chains, which assemble into three different protomers (α1α1α2, α3α4α5, and α5α5α6) with a tissue-specific distribution [24, 25]. The absence of any one of these type IV collagen chains can result in the absence of the whole protomer in the GBM, presumably due to an obligatory association of the three chains in forming the type IV collagen superstructure [26].

The heterotrimers are essential for basement membrane structure and function [26]. The α3α4α5 protomer in the GBM is only produced by podocytes [27], that is why podocytes are a key cell type affected by Alport syndrome pathology. The α1α1α2 heterotrimer is produced by three cell types in the glomerulus: podocytes, endothelial cells, and mesangial cells [27]. The α1α1α2 heterotrimer is ubiquitously distributed in all basement membranes during embryogenesis, and then in the process of development, there is a partial replacement of α1α1α2 protomer by α3α4α5 heterotrimer in the GBM, lungs, eyes, cochlea, and testes, and by the α5α5α6 heterotrimer in skin, smooth muscle cells, kidney's Bowman capsules, and esophagus [28]. This substitution of collagen type IV heterotrimers leads to mechanical stability of basement membrane, as the α1α1α2 chains are highly susceptible to endoproteolysis. In AS, assembly of the α3α4α5 heterotrimer does not occur, resulting in decreased mechanical stability and splitting of the GBM, as well as other tissue-specific pathological changes.

Normally, *COL4A5* is expressed in the glomerular basement membranes, and the α5(IV) chain defect due to *COL4A5* mutation (absence or abnormal structure of α5(IV)) impairs protomer assembly and the normal collagen IV network formation. Immunohistological analysis reveals abnormal distribution of the α5(IV) chain in about two-thirds of patients with X-linked AS: in male patients, the α5(IV) antigen is not detected in the glomerular, capsular, and distal tubular basement membranes, while in female patients, the α5(IV) antigen has a discontinuous distribution. In addition, there is a lack of the α3(IV) and α4(IV) chains which participate in the α3α4α5(IV)-α3α4α5(IV) network formation. At the same time, the α1(IV) and α2(IV) chains normally confining to the mesangium and the subendothelial aspect of the GBM are widely expressed throughout the glomerular basement membrane in these patients. This immunohistological picture is typical for the fetal kidneys. But approximately, one-third of patients with AS do not have marked changes in the renal expression of α(IV) chains. The immunohistochemical picture is usually consistent within the family and correlates with the

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The distribution of α(IV) chains is also abnormal in skin basement membrane: the absence of the α5(IV) and the associated α6(IV) chains from the epidermal basement membrane is typical for male patients with X-linked AS and can be detected in about two-thirds of male patients. Observation of normal patterns of α5(IV) and α6(IV) localization does not facilitate definitive

The peculiar immunohistochemical distribution pattern of α3(IV) to α6(IV) chains is observed in the skin and kidneys of most patients with autosomal recessive AS [31, 32, 35] and is characterized by the absence of α3, α4, and α5(IV) chains from the GBM contrasting with the persistence of α5(IV) chains in Bowman's capsules, collecting ducts, and epidermal basement membranes. These findings show that in autosomal recessive Alport patients, the expression of α5(IV) chains is defective only in those basement membranes in which the three chains are

Expression of mutant collagen type IV results in splitting of the GBM, podocyte effacement,

Men who are hemizygous for mutations in *COL4A5* causing X-linked AS have similar clinical presentation and prognosis to individuals homozygous for *COL4A3* and *COL4A4* mutations causing autosomal recessive AS. The median age of ESRD onset in untreated male patients with X-linked AS has been reported to be 22 years (range 7–39 years) [37, 38]. But, owing to imprinting (random inactivation on the two X chromosomes in female individuals) [39], female carriers of *COL4A5* mutations causing X-linked AS may have a higher risk of ESRD than do individuals who are heterozygous for autosomal recessive mutations in *COL4A3* and

The diagnosis of AS should be suspected in patients with family glomerular hematuria and with family history of renal failure. Children with AS syndrome can usually be diagnosed with mild hematuria with or no episodic macrohematuria and low-grade proteinuria. As the

severity of the clinicopathological features of the disease [25, 36].

associated within the α3α4α5(IV)-α3α4α5(IV) network.

**2.3. Clinical presentation of Alport syndrome**

*COL4A4*.

glomerulosclerosis, kidney fibrosis, and ESRD progression.

diagnosis in females because of the segmental distribution of the chains.

The detailed pathogenetic mechanisms leading to progression of AS are not clear. As the GBM is a key component of the filtration barrier, patients with AS develop proteinuria [29] as a result of altered podocyte orientation, podocyte effacement, and disruption of slit diaphragms. However, interactions between integrins and the laminin α5β2γ1 heterotrimer could disrupt the actin cytoskeleton and activate signaling mechanisms that result in an increase in matrix metalloproteinase expression and massive accumulation of extracellular matrix [25] as well as kidney fibrosis due to fibroblast formation by epithelial-mesenchymal transition, initiated as a physiological repair of injury response [30]. Invasion of mesangial filopodia may be responsible for deposition of the extracellular molecules in Alport GBM [31–33]. These changes could lead to progression of glomerulosclerosis, nephron loss, and hypertension. Increasing deposition of extracellular matrix and laminin α5, upregulation of matrix metalloproteinase has been shown in the stria vascularis of ear in vivo.

Ultrastructural studies have demonstrated that the fundamental lesion of AS involves the GBM [29, 34]. The typical changes of GBM in AS are its thickening, splitting, and fragmentation of the lamina densa with formation of a characteristic "basket weave" pattern. The lesions may be focal (in the early stages of the disease) or widespread (in the late stages). The thinning or irregular appearance of the GBM (an alternation of thickening and thinning) is the prevalent changes in children with AS. Diffuse thinning of the GBM as the only morphological changes may be observed in 10–20% of patients with Alport syndrome, explaining why patients with thin basal membranes may have an unfavorable prognosis. Sometimes, it is possible to see the focal ruptures of the GBM and its reparation due to the synthesis of the new material of the glomerular basement membrane [35]. The light microscopy changes are not specific and vary from the normal renal tissue appearance or minimal glomerular changes in the early stages of the disease to focal mesangial proliferation, focal and segmental thickening of the capillary walls, and focal/global glomerulosclerosis in the late stages of the AS. These changes are associated with nonspecific tubulointerstitial lesions including foci of lipid-laden foam cells. Conventional immunofluorescence detects nothing or only faint or focal deposits of IgG, IgM, or complement C3.

The distribution of the different chains of collagen type IV in the basement membranes is very important for diagnosis of AS and especially for X-linked transmission's recognition. Normally, *COL4A5* is expressed in the glomerular basement membranes, and the α5(IV) chain defect due to *COL4A5* mutation (absence or abnormal structure of α5(IV)) impairs protomer assembly and the normal collagen IV network formation. Immunohistological analysis reveals abnormal distribution of the α5(IV) chain in about two-thirds of patients with X-linked AS: in male patients, the α5(IV) antigen is not detected in the glomerular, capsular, and distal tubular basement membranes, while in female patients, the α5(IV) antigen has a discontinuous distribution. In addition, there is a lack of the α3(IV) and α4(IV) chains which participate in the α3α4α5(IV)-α3α4α5(IV) network formation. At the same time, the α1(IV) and α2(IV) chains normally confining to the mesangium and the subendothelial aspect of the GBM are widely expressed throughout the glomerular basement membrane in these patients. This immunohistological picture is typical for the fetal kidneys. But approximately, one-third of patients with AS do not have marked changes in the renal expression of α(IV) chains. The immunohistochemical picture is usually consistent within the family and correlates with the severity of the clinicopathological features of the disease [25, 36].

The distribution of α(IV) chains is also abnormal in skin basement membrane: the absence of the α5(IV) and the associated α6(IV) chains from the epidermal basement membrane is typical for male patients with X-linked AS and can be detected in about two-thirds of male patients. Observation of normal patterns of α5(IV) and α6(IV) localization does not facilitate definitive diagnosis in females because of the segmental distribution of the chains.

The peculiar immunohistochemical distribution pattern of α3(IV) to α6(IV) chains is observed in the skin and kidneys of most patients with autosomal recessive AS [31, 32, 35] and is characterized by the absence of α3, α4, and α5(IV) chains from the GBM contrasting with the persistence of α5(IV) chains in Bowman's capsules, collecting ducts, and epidermal basement membranes. These findings show that in autosomal recessive Alport patients, the expression of α5(IV) chains is defective only in those basement membranes in which the three chains are associated within the α3α4α5(IV)-α3α4α5(IV) network.

Expression of mutant collagen type IV results in splitting of the GBM, podocyte effacement, glomerulosclerosis, kidney fibrosis, and ESRD progression.

Men who are hemizygous for mutations in *COL4A5* causing X-linked AS have similar clinical presentation and prognosis to individuals homozygous for *COL4A3* and *COL4A4* mutations causing autosomal recessive AS. The median age of ESRD onset in untreated male patients with X-linked AS has been reported to be 22 years (range 7–39 years) [37, 38]. But, owing to imprinting (random inactivation on the two X chromosomes in female individuals) [39], female carriers of *COL4A5* mutations causing X-linked AS may have a higher risk of ESRD than do individuals who are heterozygous for autosomal recessive mutations in *COL4A3* and *COL4A4*.

#### **2.3. Clinical presentation of Alport syndrome**

The heterotrimers are essential for basement membrane structure and function [26]. The α3α4α5 protomer in the GBM is only produced by podocytes [27], that is why podocytes are a key cell type affected by Alport syndrome pathology. The α1α1α2 heterotrimer is produced by three cell types in the glomerulus: podocytes, endothelial cells, and mesangial cells [27]. The α1α1α2 heterotrimer is ubiquitously distributed in all basement membranes during embryogenesis, and then in the process of development, there is a partial replacement of α1α1α2 protomer by α3α4α5 heterotrimer in the GBM, lungs, eyes, cochlea, and testes, and by the α5α5α6 heterotrimer in skin, smooth muscle cells, kidney's Bowman capsules, and esophagus [28]. This substitution of collagen type IV heterotrimers leads to mechanical stability of basement membrane, as the α1α1α2 chains are highly susceptible to endoproteolysis. In AS, assembly of the α3α4α5 heterotrimer does not occur, resulting in decreased mechanical stability and splitting of the GBM, as well as other tissue-specific pathological changes.

The detailed pathogenetic mechanisms leading to progression of AS are not clear. As the GBM is a key component of the filtration barrier, patients with AS develop proteinuria [29] as a result of altered podocyte orientation, podocyte effacement, and disruption of slit diaphragms. However, interactions between integrins and the laminin α5β2γ1 heterotrimer could disrupt the actin cytoskeleton and activate signaling mechanisms that result in an increase in matrix metalloproteinase expression and massive accumulation of extracellular matrix [25] as well as kidney fibrosis due to fibroblast formation by epithelial-mesenchymal transition, initiated as a physiological repair of injury response [30]. Invasion of mesangial filopodia may be responsible for deposition of the extracellular molecules in Alport GBM [31–33]. These changes could lead to progression of glomerulosclerosis, nephron loss, and hypertension. Increasing deposition of extracellular matrix and laminin α5, upregulation of

Ultrastructural studies have demonstrated that the fundamental lesion of AS involves the GBM [29, 34]. The typical changes of GBM in AS are its thickening, splitting, and fragmentation of the lamina densa with formation of a characteristic "basket weave" pattern. The lesions may be focal (in the early stages of the disease) or widespread (in the late stages). The thinning or irregular appearance of the GBM (an alternation of thickening and thinning) is the prevalent changes in children with AS. Diffuse thinning of the GBM as the only morphological changes may be observed in 10–20% of patients with Alport syndrome, explaining why patients with thin basal membranes may have an unfavorable prognosis. Sometimes, it is possible to see the focal ruptures of the GBM and its reparation due to the synthesis of the new material of the glomerular basement membrane [35]. The light microscopy changes are not specific and vary from the normal renal tissue appearance or minimal glomerular changes in the early stages of the disease to focal mesangial proliferation, focal and segmental thickening of the capillary walls, and focal/global glomerulosclerosis in the late stages of the AS. These changes are associated with nonspecific tubulointerstitial lesions including foci of lipid-laden foam cells. Conventional immunofluorescence detects nothing or only faint or focal deposits of IgG, IgM,

The distribution of the different chains of collagen type IV in the basement membranes is very important for diagnosis of AS and especially for X-linked transmission's recognition.

matrix metalloproteinase has been shown in the stria vascularis of ear in vivo.

or complement C3.

114 Advances in Nephropathy

The diagnosis of AS should be suspected in patients with family glomerular hematuria and with family history of renal failure. Children with AS syndrome can usually be diagnosed with mild hematuria with or no episodic macrohematuria and low-grade proteinuria. As the disease progresses, patients gradually develop severe proteinuria and progressive renal failure. Irrespective of the mode of inheritance of the disorder, proteinuria indicates an increased risk of progressive renal disease, even in heterozygous carriers. Changes in the GBM that are pathognomonic for AS include splitting and enlargement of the GBM and podocyte effacement.

capsule due to the absence of the α3α4α5 network can lead to its spontaneous ruptures and secondary cataract developments [44, 45]. After cataract formation, lenticonus ceases to progress [46]. Posterior lenticonus is less common in patients with AS. Lenticonus is usually found in early middle-age patients with renal failure. The patients complain a progressive difficulty in focusing due to of their abnormal lens shape. The presence of an oil droplet sign on direct ophthalmoscopy or slit-lamp examination confirms the diagnosis. Visual symptoms progress with a time, and most patients eventually require surgery. The treatment for both symptomatic lenticonus and cataract includes the lens removal and intraocular lens implantation.

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The central or perimacular fleck retinopathy and peripheral coalescing fleck retinopathy are common retinal abnormalities in patients with AS. The other retinal changes include manifestations of temporal thinning [47, 48]: lamellar and giant macular hole, a lozenge, loss of the foveal reflex, disturbances of foveal pigmentation, including a bull's eye or vitelliform maculopathy [48, 49]. Central fleck retinopathy is present in 60% of men and at least 15% of women with X-linked AS and 50% of individuals with recessive disease [50]. It is more common with early-onset renal failure and lenticonus [47]. The central retinopathy varies from scattered whitish-yellow dots and flecks to a dense, almost confluent annulus around the region of temporal retinal thinning. The fleck retinopathy is associated with an abnormal inner limiting membrane. Thinning of the inner limiting membrane/nerve fiber layer may interfere with the nutrition of the overlying cells, removal of debris, and maintenance of the watertight barrier. The central retinopathy is best seen with color photographs and red-free images centered on the macula. Specialized tests of retinal function, such as electroretinogram and electrooculogram, are normal or nearly normal. The peripheral fleck retinopathy is the most common retinal abnormality, occurring in most men and 25% of women with X-linked AS and most individuals with recessive disease [47, 48]. The asymmetric patches of confluent flecks are characteristic signs of peripheral retinopathy [50]. The appearance on optical coherence tomography (OCT) and fleck location in relation to the blood vessels suggests that their formation is related to pathological changes of the retinal pigment epithelium/Bruch's membrane. The peripheral retinopathy is a very important diagnostic and prognostic symptom of AS associated with early-onset renal failure, central retinopathy, and lenticonus. But the peripheral retinopathy can also be present in women with X-linked AS who have normal renal function [47]. The peripheral retinopathy is best seen on ophthalmic examination or with retinal photographs that extend beyond the standard views into the periphery and with red-free retinal images. Temporal retinal thinning is very common in men and women with X-linked AS, and in patients with recessive disease [41, 42, 47, 48]. The lozenge, dull macular reflex, foveopathy, and lamellar and macular holes all affect the temporal retina and reflect retinal thinning of both the inner limiting membrane and Bruch's membrane. Thinning is confirmed with retinal thickness measurements on OCT. However, thinning is common in all forms of Alport syndrome and less sensitive diagnostically than a peripheral retinopathy. Hypopigmentation occurred in Alport syndrome is often not diagnosed. It is usually present along with perimacular flecks or other ocular features and does not lead to the visual impairment. Severe forms, such as a bull's eye or vitelliform retinopathy, may be occasionally found. Lamellar, partial-thickness macular holes, and giant macular holes lamellar are uncommon in patients with X-linked and autosomal recessive AS. This sign is unspecific for

There are several clinical stages in the development of glomerulopathy in AS:

Stage 0: microscopic hematuria (albumin/creatinine rate <30 mg/g/day).

Stage 1: microalbuminuria (albumin/creatinine rate = 30–300 mg/g/day).

Stage 2: gross proteinuria (albumin/creatinine rate >300 mg/g/day).

Stage 3: impaired renal function (glomerular filtration rate <60 ml/min/1.73 m2 ).

Stage 4: end-stage renal disease.

Diagnosis of AS can be made in most patients after a clinical examination on the basis of the typical symptoms including kidney, ear, and eye.

The sensorineural hearing loss in AS, which primarily affects high tones, occurs in 30–50% of relatives with renal disease. The severity of auditory and renal features does not correlate. The molecular defects that underlie the otopathology in this disease remain poorly understood. An animal model of X-linked AS showed complete absence of the α3α4α5 network in the inner ear, suggesting that collagen type IV is vital to cochlear function as well as renal function. The generation of radial tension of the spiral ligament on the basilar membrane via the extracellular matrix is necessary for reception of high-frequency sound. The lateral aspect of the spiral ligament is populated by tension fibroblasts expressing nonmuscle myosin and α-smooth muscle actin [40]. On the basis of the foregoing, it was assumed that in AS, the loss of the α3α4α5 network eventually weakens the interaction of fibroblasts with their extracellular matrix, resulting in reduced tension on the basilar membrane and the inability to respond to high-frequency sounds [40]. Findings in Alport mice suggest that the hearing loss may arise from dysfunction of the stria vascularis mediated through endothelin-1 [40, 41].

The ocular manifestations in Alport syndrome patients are caused by loss of the collagen IV α3α4α5 network in basement membranes of the eyes due to mutations which lead to basement membrane thinning, lamellation, and a decrease in its mechanical stability. Most of the ocular features in AS do not lead to a visual impairment, but they have diagnostic and prognostic value: in some cases, the ocular symptoms suggest the mode of inheritance and the likelihood of early-onset renal failure. Such ocular features as central and peripheral retinopathy and lenticonus are typical for AS, and their presence confirms the diagnosis.

Anterior lenticonus can be detected in half of men (not women) with X-linked Alport syndrome. And its presence is associated with early-onset renal failure. The symptom is often found in autosomal recessive AS regardless of the patient's sex, and therefore, the presence of lenticonus in women with AS most likely indicates an autosomal recessive mode of disease inheritance [41, 42]. Lenticonus is the consequence of the conical protrusion of the lens anteriorly through the thinnest part of the capsule [43]; sometimes, the mechanical weakness of the capsule due to the absence of the α3α4α5 network can lead to its spontaneous ruptures and secondary cataract developments [44, 45]. After cataract formation, lenticonus ceases to progress [46]. Posterior lenticonus is less common in patients with AS. Lenticonus is usually found in early middle-age patients with renal failure. The patients complain a progressive difficulty in focusing due to of their abnormal lens shape. The presence of an oil droplet sign on direct ophthalmoscopy or slit-lamp examination confirms the diagnosis. Visual symptoms progress with a time, and most patients eventually require surgery. The treatment for both symptomatic lenticonus and cataract includes the lens removal and intraocular lens implantation.

disease progresses, patients gradually develop severe proteinuria and progressive renal failure. Irrespective of the mode of inheritance of the disorder, proteinuria indicates an increased risk of progressive renal disease, even in heterozygous carriers. Changes in the GBM that are pathognomonic for AS include splitting and enlargement of the GBM and podocyte

Diagnosis of AS can be made in most patients after a clinical examination on the basis of the

The sensorineural hearing loss in AS, which primarily affects high tones, occurs in 30–50% of relatives with renal disease. The severity of auditory and renal features does not correlate. The molecular defects that underlie the otopathology in this disease remain poorly understood. An animal model of X-linked AS showed complete absence of the α3α4α5 network in the inner ear, suggesting that collagen type IV is vital to cochlear function as well as renal function. The generation of radial tension of the spiral ligament on the basilar membrane via the extracellular matrix is necessary for reception of high-frequency sound. The lateral aspect of the spiral ligament is populated by tension fibroblasts expressing nonmuscle myosin and α-smooth muscle actin [40]. On the basis of the foregoing, it was assumed that in AS, the loss of the α3α4α5 network eventually weakens the interaction of fibroblasts with their extracellular matrix, resulting in reduced tension on the basilar membrane and the inability to respond to high-frequency sounds [40]. Findings in Alport mice suggest that the hearing loss may arise

).

There are several clinical stages in the development of glomerulopathy in AS:

Stage 3: impaired renal function (glomerular filtration rate <60 ml/min/1.73 m2

from dysfunction of the stria vascularis mediated through endothelin-1 [40, 41].

lenticonus are typical for AS, and their presence confirms the diagnosis.

The ocular manifestations in Alport syndrome patients are caused by loss of the collagen IV α3α4α5 network in basement membranes of the eyes due to mutations which lead to basement membrane thinning, lamellation, and a decrease in its mechanical stability. Most of the ocular features in AS do not lead to a visual impairment, but they have diagnostic and prognostic value: in some cases, the ocular symptoms suggest the mode of inheritance and the likelihood of early-onset renal failure. Such ocular features as central and peripheral retinopathy and

Anterior lenticonus can be detected in half of men (not women) with X-linked Alport syndrome. And its presence is associated with early-onset renal failure. The symptom is often found in autosomal recessive AS regardless of the patient's sex, and therefore, the presence of lenticonus in women with AS most likely indicates an autosomal recessive mode of disease inheritance [41, 42]. Lenticonus is the consequence of the conical protrusion of the lens anteriorly through the thinnest part of the capsule [43]; sometimes, the mechanical weakness of the

Stage 0: microscopic hematuria (albumin/creatinine rate <30 mg/g/day). Stage 1: microalbuminuria (albumin/creatinine rate = 30–300 mg/g/day).

Stage 2: gross proteinuria (albumin/creatinine rate >300 mg/g/day).

effacement.

116 Advances in Nephropathy

Stage 4: end-stage renal disease.

typical symptoms including kidney, ear, and eye.

The central or perimacular fleck retinopathy and peripheral coalescing fleck retinopathy are common retinal abnormalities in patients with AS. The other retinal changes include manifestations of temporal thinning [47, 48]: lamellar and giant macular hole, a lozenge, loss of the foveal reflex, disturbances of foveal pigmentation, including a bull's eye or vitelliform maculopathy [48, 49]. Central fleck retinopathy is present in 60% of men and at least 15% of women with X-linked AS and 50% of individuals with recessive disease [50]. It is more common with early-onset renal failure and lenticonus [47]. The central retinopathy varies from scattered whitish-yellow dots and flecks to a dense, almost confluent annulus around the region of temporal retinal thinning. The fleck retinopathy is associated with an abnormal inner limiting membrane. Thinning of the inner limiting membrane/nerve fiber layer may interfere with the nutrition of the overlying cells, removal of debris, and maintenance of the watertight barrier. The central retinopathy is best seen with color photographs and red-free images centered on the macula. Specialized tests of retinal function, such as electroretinogram and electrooculogram, are normal or nearly normal. The peripheral fleck retinopathy is the most common retinal abnormality, occurring in most men and 25% of women with X-linked AS and most individuals with recessive disease [47, 48]. The asymmetric patches of confluent flecks are characteristic signs of peripheral retinopathy [50]. The appearance on optical coherence tomography (OCT) and fleck location in relation to the blood vessels suggests that their formation is related to pathological changes of the retinal pigment epithelium/Bruch's membrane. The peripheral retinopathy is a very important diagnostic and prognostic symptom of AS associated with early-onset renal failure, central retinopathy, and lenticonus. But the peripheral retinopathy can also be present in women with X-linked AS who have normal renal function [47]. The peripheral retinopathy is best seen on ophthalmic examination or with retinal photographs that extend beyond the standard views into the periphery and with red-free retinal images. Temporal retinal thinning is very common in men and women with X-linked AS, and in patients with recessive disease [41, 42, 47, 48]. The lozenge, dull macular reflex, foveopathy, and lamellar and macular holes all affect the temporal retina and reflect retinal thinning of both the inner limiting membrane and Bruch's membrane. Thinning is confirmed with retinal thickness measurements on OCT. However, thinning is common in all forms of Alport syndrome and less sensitive diagnostically than a peripheral retinopathy. Hypopigmentation occurred in Alport syndrome is often not diagnosed. It is usually present along with perimacular flecks or other ocular features and does not lead to the visual impairment. Severe forms, such as a bull's eye or vitelliform retinopathy, may be occasionally found. Lamellar, partial-thickness macular holes, and giant macular holes lamellar are uncommon in patients with X-linked and autosomal recessive AS. This sign is unspecific for AS. But in AS, the macular holes are larger (giant holes) and occur at a younger age than the spontaneous holes in patients who do not have AS. Holes may be asymmetric, unilateral, or bilateral. Beginning with multiple small defects, they hollow out from the surface of the inner limiting membrane in the consequence of accelerated passage of fluid through the defective Bruch's membrane, and followed by microcysts formation due to membrane breaking [50]. Patients with macular holes suffer from metamorphopsia (where straight lines are distorted) and impairment of central vision. In some patients, holes only become evident when there is no improvement in vision after surgery for lenticonus. Lamellar holes might be overlooked by retinal photographs; the OCT is required for their demonstration. Holes in AS do not respond well to surgical closure and often lead to a permanent loss of vision.

dysphagia. Leiomyomatosis can also affect the tracheobronchial tree and the genital tract. Leiomyomatosis in AS is a consequence of a large deletion in the *COL4A5* and *COL4A6* genes, which encode collagen α5 (IV) or α5α6 (IV) chains in smooth muscle cells of the gastroesophageal tract [51, 52]. The 5 and 6 chains of type IV collagen are also found in the basement membranes surrounding vascular smooth muscle cells in the intima and media of aorta and other arteries in mice model [53]. Seki et al. [53] believed that 5 and 6 chains of type IV collagen in the basement membranes may have particular function in the arteries which are required to tolerate strong pulse and blood pressure such as the aorta. AS is associated with aortic abnormalities including aortic dilatation, ruptured ascending aortic aneurysm, aortic dissection, aortic insufficiency, and bicuspid aortic valve and coronary artery pathology

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Furthermore, patients with AS and mitral valve prolapse or ventricular septal defects and ruptured intracranial and coronary artery aneurysms have also been described [54, 57]. Therefore, the vascular imaging techniques could be included in the examination of patients

In cases where the diagnosis of AS was not confirmed by clinical workup, morphology, or genetic test, other diseases with similar symptoms should be considered. A combination of kidney pathology and hearing loss can be associated with mutations of gene encoding mito-

Patients with mitochondriopathy associated with mutations in the *COQ6* gene develop earlyonset steroid-resistant nephrotic syndrome and sensorineural deafness [58]. The mutation leads to deficiency of ubiquinone biosynthesis monooxygenase COQ6 protein, localized in the Golgi apparatus of the stria vascularis and glomerular podocytes, and to upregulation of

Alström syndrome is characterized by a defect of the primary cilium that is caused by mutations in *ALMS1* and leads to renal failure, sensorineural deafness in young adulthood, and

*MYH-9*-related disease should be considered in patients presenting with proteinuria and thrombocytopenia [60]. *MYH-9*-related disease (the gene for the heavy chain of nonmuscle myosin IIA) is a rare inherited autosomal dominant condition characterized by progressive nephritis (39%), macrothrombocytopenia (100%), Dohle-like leukocyte inclusions (100%), high-tone sensorineural deafness (49%), and cataract (54%) [60, 61]. Renal disease ranged from microscopic hematuria to end-stage renal failure necessitating dialysis and kidney transplantation. The most striking difference between hearing loss in *MYH-9*-related disease (or Fechtner syndrome) and that in AS was that the vast majority of hearing disorders in the latter occur in male patients. Hearing loss in *MYH-9*-related disease develops from the second decade of life and progresses slowly with several episodes of sudden deafness [60]. Renal biopsy findings are consistent with those of AS. Chronic renal failure can occur at a

including spontaneous coronary artery dissection in male patients [54–56].

with AS, especially in cases of family history of vascular abnormalities.

chondrial and cytoskeletal proteins, ion channels, and receptors.

**2.4. Differential diagnosis of Alport syndrome**

young age in patients with *MYH-9*-related disease.

proapoptotic factors [58].

vision loss in adulthood [59].

Posterior polymorphous corneal dystrophy and macular hole are rare but also suggest the Alport syndrome. Corneal disease is recognized infrequently in Alport syndrome. Erosions result from an abnormal Bowman's membrane in the corneal subepithelium and posterior polymorphous corneal dystrophy from an abnormal Descemet's membrane in the subendothelium [41]. The affected membranes lack the collagen IV α3α4α5 network, are weak, and adhere poorly to the epithelium, endothelium, and underlying stroma. Superficial corneal erosions occur in 10% of patients, but they are intermittent and hence. Their onset may precede the diagnosis of AS and is often in the late teenage years. They typically occur in individuals with early-onset renal failure and other extrarenal features. Posterior polymorphous corneal dystrophy is rare and more serious than the corneal erosions. Patients may be asymptomatic or have photophobia, watering, and grittiness. The diagnosis is based on high-resolution anterior segment OCT, slit-lamp biomicroscopy, specular microscopy, or in vivo confocal microscopy: there are multiple clusters of vesicles ("doughnuts") or bands ("snail tracks") at the posterior corneal surface. The vesicles result from vacuolar degeneration of dying cells or multilayered epithelial cell protuberances from Descemet's membrane. Treatment is usually symptomatic; sometimes, the dystrophy progresses, and corneal transplantation is required.

Ocular features are less sensitive but more specific than hearing loss in diagnostic of AS, because hearing loss occurs also in other inherited renal diseases and in dialysis patients. In addition, ocular symptoms may help distinguish between X-linked and autosomal recessive inheritance. Central retinopathy, lenticonus, and macular hole are rare in women with X-linked AS. The presence of any of these futures in a woman with hematuria leads to suspected autosomal recessive AS. Furthermore, revealed peripheral retinopathy in the mother of a boy with hematuria indicates the diagnosis of AS and also X-linked inheritance of the disease. Some ocular features have a prognostic value because they are associated with earlyonset renal failure. Thus, central retinopathy and lenticonus usually indicate the onset of renal failure before the age of 30 years. Therefore, it is important to conduct a thorough ophthalmologic examination (slit-lamp examination, retinal photography, and OCT) in assessing patients with suspected AS. These tests are acceptable to patients, noninvasive, inexpensive, and widely available.

Other features of Alport syndrome include gastroesophageal leiomyomatosis and vascular abnormalities. Gastroesophageal leiomyomatosis is a very rare pathology characterized by benign nodular tumors with smooth muscle cells origin. The condition clinically presents by dysphagia. Leiomyomatosis can also affect the tracheobronchial tree and the genital tract. Leiomyomatosis in AS is a consequence of a large deletion in the *COL4A5* and *COL4A6* genes, which encode collagen α5 (IV) or α5α6 (IV) chains in smooth muscle cells of the gastroesophageal tract [51, 52]. The 5 and 6 chains of type IV collagen are also found in the basement membranes surrounding vascular smooth muscle cells in the intima and media of aorta and other arteries in mice model [53]. Seki et al. [53] believed that 5 and 6 chains of type IV collagen in the basement membranes may have particular function in the arteries which are required to tolerate strong pulse and blood pressure such as the aorta. AS is associated with aortic abnormalities including aortic dilatation, ruptured ascending aortic aneurysm, aortic dissection, aortic insufficiency, and bicuspid aortic valve and coronary artery pathology including spontaneous coronary artery dissection in male patients [54–56].

Furthermore, patients with AS and mitral valve prolapse or ventricular septal defects and ruptured intracranial and coronary artery aneurysms have also been described [54, 57]. Therefore, the vascular imaging techniques could be included in the examination of patients with AS, especially in cases of family history of vascular abnormalities.

#### **2.4. Differential diagnosis of Alport syndrome**

AS. But in AS, the macular holes are larger (giant holes) and occur at a younger age than the spontaneous holes in patients who do not have AS. Holes may be asymmetric, unilateral, or bilateral. Beginning with multiple small defects, they hollow out from the surface of the inner limiting membrane in the consequence of accelerated passage of fluid through the defective Bruch's membrane, and followed by microcysts formation due to membrane breaking [50]. Patients with macular holes suffer from metamorphopsia (where straight lines are distorted) and impairment of central vision. In some patients, holes only become evident when there is no improvement in vision after surgery for lenticonus. Lamellar holes might be overlooked by retinal photographs; the OCT is required for their demonstration. Holes in AS do not respond

Posterior polymorphous corneal dystrophy and macular hole are rare but also suggest the Alport syndrome. Corneal disease is recognized infrequently in Alport syndrome. Erosions result from an abnormal Bowman's membrane in the corneal subepithelium and posterior polymorphous corneal dystrophy from an abnormal Descemet's membrane in the subendothelium [41]. The affected membranes lack the collagen IV α3α4α5 network, are weak, and adhere poorly to the epithelium, endothelium, and underlying stroma. Superficial corneal erosions occur in 10% of patients, but they are intermittent and hence. Their onset may precede the diagnosis of AS and is often in the late teenage years. They typically occur in individuals with early-onset renal failure and other extrarenal features. Posterior polymorphous corneal dystrophy is rare and more serious than the corneal erosions. Patients may be asymptomatic or have photophobia, watering, and grittiness. The diagnosis is based on high-resolution anterior segment OCT, slit-lamp biomicroscopy, specular microscopy, or in vivo confocal microscopy: there are multiple clusters of vesicles ("doughnuts") or bands ("snail tracks") at the posterior corneal surface. The vesicles result from vacuolar degeneration of dying cells or multilayered epithelial cell protuberances from Descemet's membrane. Treatment is usually symptomatic; sometimes, the dystrophy progresses, and corneal transplantation is required.

Ocular features are less sensitive but more specific than hearing loss in diagnostic of AS, because hearing loss occurs also in other inherited renal diseases and in dialysis patients. In addition, ocular symptoms may help distinguish between X-linked and autosomal recessive inheritance. Central retinopathy, lenticonus, and macular hole are rare in women with X-linked AS. The presence of any of these futures in a woman with hematuria leads to suspected autosomal recessive AS. Furthermore, revealed peripheral retinopathy in the mother of a boy with hematuria indicates the diagnosis of AS and also X-linked inheritance of the disease. Some ocular features have a prognostic value because they are associated with earlyonset renal failure. Thus, central retinopathy and lenticonus usually indicate the onset of renal failure before the age of 30 years. Therefore, it is important to conduct a thorough ophthalmologic examination (slit-lamp examination, retinal photography, and OCT) in assessing patients with suspected AS. These tests are acceptable to patients, noninvasive, inexpensive,

Other features of Alport syndrome include gastroesophageal leiomyomatosis and vascular abnormalities. Gastroesophageal leiomyomatosis is a very rare pathology characterized by benign nodular tumors with smooth muscle cells origin. The condition clinically presents by

well to surgical closure and often lead to a permanent loss of vision.

and widely available.

118 Advances in Nephropathy

In cases where the diagnosis of AS was not confirmed by clinical workup, morphology, or genetic test, other diseases with similar symptoms should be considered. A combination of kidney pathology and hearing loss can be associated with mutations of gene encoding mitochondrial and cytoskeletal proteins, ion channels, and receptors.

Patients with mitochondriopathy associated with mutations in the *COQ6* gene develop earlyonset steroid-resistant nephrotic syndrome and sensorineural deafness [58]. The mutation leads to deficiency of ubiquinone biosynthesis monooxygenase COQ6 protein, localized in the Golgi apparatus of the stria vascularis and glomerular podocytes, and to upregulation of proapoptotic factors [58].

Alström syndrome is characterized by a defect of the primary cilium that is caused by mutations in *ALMS1* and leads to renal failure, sensorineural deafness in young adulthood, and vision loss in adulthood [59].

*MYH-9*-related disease should be considered in patients presenting with proteinuria and thrombocytopenia [60]. *MYH-9*-related disease (the gene for the heavy chain of nonmuscle myosin IIA) is a rare inherited autosomal dominant condition characterized by progressive nephritis (39%), macrothrombocytopenia (100%), Dohle-like leukocyte inclusions (100%), high-tone sensorineural deafness (49%), and cataract (54%) [60, 61]. Renal disease ranged from microscopic hematuria to end-stage renal failure necessitating dialysis and kidney transplantation. The most striking difference between hearing loss in *MYH-9*-related disease (or Fechtner syndrome) and that in AS was that the vast majority of hearing disorders in the latter occur in male patients. Hearing loss in *MYH-9*-related disease develops from the second decade of life and progresses slowly with several episodes of sudden deafness [60]. Renal biopsy findings are consistent with those of AS. Chronic renal failure can occur at a young age in patients with *MYH-9*-related disease.

A recent report described a *COL4A1* frameshift mutation in a family with autosomal dominant hematuria, GBM thinning, kidney cysts, and progressive kidney disease [62, 63]. The syndrome is characterized by hereditary angiopathy with retinal tortuosities, aneurysms, muscular cramps, and nephropathy manifesting as hematuria or cysts (HANAC). As such, investigation of patients with unexplained hematuria should include a search for extrarenal symptoms of HANAC, especially retinal abnormalities.

using standardized dosing regimens and monitoring microalbuminuria during the treatment [69]. The ACE inhibitor ramipril is recommended as the first-line therapy; in cases of adverse effects associated with use of ramipril, the treatment with angiotensin-1-receptor antagonists will be recommended [69, 70]. The Alport Syndrome Research Collaborative guidelines suggest, therefore, that therapy with ACE inhibitors can be considered as early as stages 0 and 1

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The age at progression from microalbuminuria to proteinuria in children with Alport syndrome is an important prognostic marker—the earlier this transition occurs, the worse the

Currently, the only recruiting trial is the prospective, randomized placebo-controlled EARLY PRO-TECT Alport trial, enrolling pediatric patients with stages 0 and 1 Alport syndrome [72]. The goal of this trial is to clarify whether an early start of therapy delays renal failure more effectively than later onset of therapy and, above all, whether therapy in the early stages of Alport syndrome is safe. The trial will also investigate a potential protective effect of ramipril against the hearing loss as a secondary end point [72]. It seems to be very important because currently there are no therapies that can prevent the ocular changes or hearing loss develop-

Studies in animal models have revealed many potential new therapies for use in Alport syndrome: inhibitors of proinflammatory factors (for example, TGF-β1 and MMPs) [73, 74]; statins [75]; BX471 (a chemokine receptor-1 blocker) [76]; and upregulation of the expression of bone morphogenetic protein 7 [77]. A phase II study of antimicroRNA-21 treatment of patients with Alport syndrome 18 years of age or older with glomerular filtration rates of

tion rate decline during therapy [78]. In transgenic Alport mice, antimicroRNA-21 treatment reduced glomerular inflammation and impaired renal fibrotic pathways. A phase II/III study of the efficacy and safety of bardoxolone methyl for Alport syndrome patients with chronic

Alport syndrome is a multisystem hereditary disorder characterized by progressive renal disease, sensorineural deafness, and eye abnormalities. Diagnosis of AS can be made on the basis of the typical symptoms, renal morphology, and/or genetic tests. Early diagnosis of Alport syndrome is very important, since therapeutic blockade of the renin-angiotensin-aldosterone

will be start; its primary goal will be determining the glomerular filtra-

of the disease [69].

prognosis [71].

45–90 ml/min/1.73 m2

**3. Conclusion**

**Conflict of interest**

ment and progression in patients with AS.

kidney disease (CARDINAL) has been started [79].

system can slow the progression to ESRD.

The authors declare no conflict of interest.

#### **2.5. Renal prognosis**

The expected ESRD age is similar for the males with X-linked and patients with autosomal recessive AS and comprises 23–25 years [4]. The risk of ESRD development in patients with X-linked AS by the age of 20 years is 70% in males, whereas only 5% in females; by the age of 40 years: 90 and 10%, respectively [19]. For many years, female members of Alport families who had hematuria were considered to be carriers who were not at risk for ESRD, despite reports of ESRD in female Alport patients. But review of clinical outcomes of nearly 300 girls and women with confirmed heterozygous *COL4A5* mutations showed that 12 and 30% of female have the probability of developing ESRD before the age of 40 and 60 years, respectively [19]. The 95% of heterozygous females had hematuria, the 75%—proteinuria; in addition, it was found that the proteinuria increased the risk of ESRD.

Patients with heterozygous mutations compared to patients with mutations in both alleles of *COL4A3* or *COL4A4* (autosomal recessive Alport syndrome) or hemizygous mutations in *COL4A5* (males with X-linked Alport syndrome) usually have milder renal involvement with late ESRD development and do not have extrarenal manifestations (ocular changes and hearing loss). But, lifetime risk of ESRD in heterozygous patients is higher than in the general population [64].

AS accounted for 0.5% of all cases of ESRD and was found to be associated with superior dialysis patient survival, superior posttransplant patient survival, and potentially superior renal allograft survival to matched controls with other causes of ESRD [65–67]. An incidence rate of anti-GBM antibody disease after kidney transplantation comprises 1–5% [65–67].

#### **2.6. Treatment of Alport syndrome**

Early diagnosis of AS is important, since therapeutic blockade of the renin-angiotensinaldosterone system can slow the progression to ESRD depending on the stage of the disease when therapy is initiated. Angiotensin-converting enzyme (ACE) inhibitors are not a specific therapy for AS. The antihypertensive and antiproteinuric properties of ACE inhibitors lead to their nephroprotective effect. The animal models of AS have shown a major role of altered composition of the GBM and of podocytes (their cytoskeleton and their collagen receptors) with activation of profibrotic factors in disease progression [32, 33]. ACE inhibitors seem to be able to downregulate profibrotic factors independently from blood pressure and the amount of proteinuria in the Alport animal model [68].

The Alport Syndrome Research Collaborative recommends use of ACE inhibitors in all affected individuals with microalbuminuria or proteinuria, and underlines the importance of using standardized dosing regimens and monitoring microalbuminuria during the treatment [69]. The ACE inhibitor ramipril is recommended as the first-line therapy; in cases of adverse effects associated with use of ramipril, the treatment with angiotensin-1-receptor antagonists will be recommended [69, 70]. The Alport Syndrome Research Collaborative guidelines suggest, therefore, that therapy with ACE inhibitors can be considered as early as stages 0 and 1 of the disease [69].

The age at progression from microalbuminuria to proteinuria in children with Alport syndrome is an important prognostic marker—the earlier this transition occurs, the worse the prognosis [71].

Currently, the only recruiting trial is the prospective, randomized placebo-controlled EARLY PRO-TECT Alport trial, enrolling pediatric patients with stages 0 and 1 Alport syndrome [72]. The goal of this trial is to clarify whether an early start of therapy delays renal failure more effectively than later onset of therapy and, above all, whether therapy in the early stages of Alport syndrome is safe. The trial will also investigate a potential protective effect of ramipril against the hearing loss as a secondary end point [72]. It seems to be very important because currently there are no therapies that can prevent the ocular changes or hearing loss development and progression in patients with AS.

Studies in animal models have revealed many potential new therapies for use in Alport syndrome: inhibitors of proinflammatory factors (for example, TGF-β1 and MMPs) [73, 74]; statins [75]; BX471 (a chemokine receptor-1 blocker) [76]; and upregulation of the expression of bone morphogenetic protein 7 [77]. A phase II study of antimicroRNA-21 treatment of patients with Alport syndrome 18 years of age or older with glomerular filtration rates of 45–90 ml/min/1.73 m2 will be start; its primary goal will be determining the glomerular filtration rate decline during therapy [78]. In transgenic Alport mice, antimicroRNA-21 treatment reduced glomerular inflammation and impaired renal fibrotic pathways. A phase II/III study of the efficacy and safety of bardoxolone methyl for Alport syndrome patients with chronic kidney disease (CARDINAL) has been started [79].

#### **3. Conclusion**

A recent report described a *COL4A1* frameshift mutation in a family with autosomal dominant hematuria, GBM thinning, kidney cysts, and progressive kidney disease [62, 63]. The syndrome is characterized by hereditary angiopathy with retinal tortuosities, aneurysms, muscular cramps, and nephropathy manifesting as hematuria or cysts (HANAC). As such, investigation of patients with unexplained hematuria should include a search for extrarenal

The expected ESRD age is similar for the males with X-linked and patients with autosomal recessive AS and comprises 23–25 years [4]. The risk of ESRD development in patients with X-linked AS by the age of 20 years is 70% in males, whereas only 5% in females; by the age of 40 years: 90 and 10%, respectively [19]. For many years, female members of Alport families who had hematuria were considered to be carriers who were not at risk for ESRD, despite reports of ESRD in female Alport patients. But review of clinical outcomes of nearly 300 girls and women with confirmed heterozygous *COL4A5* mutations showed that 12 and 30% of female have the probability of developing ESRD before the age of 40 and 60 years, respectively [19]. The 95% of heterozygous females had hematuria, the 75%—proteinuria; in addition, it

Patients with heterozygous mutations compared to patients with mutations in both alleles of *COL4A3* or *COL4A4* (autosomal recessive Alport syndrome) or hemizygous mutations in *COL4A5* (males with X-linked Alport syndrome) usually have milder renal involvement with late ESRD development and do not have extrarenal manifestations (ocular changes and hearing loss). But, lifetime risk of ESRD in heterozygous patients is higher than in the general population [64]. AS accounted for 0.5% of all cases of ESRD and was found to be associated with superior dialysis patient survival, superior posttransplant patient survival, and potentially superior renal allograft survival to matched controls with other causes of ESRD [65–67]. An incidence rate of anti-GBM antibody disease after kidney transplantation comprises 1–5% [65–67].

Early diagnosis of AS is important, since therapeutic blockade of the renin-angiotensinaldosterone system can slow the progression to ESRD depending on the stage of the disease when therapy is initiated. Angiotensin-converting enzyme (ACE) inhibitors are not a specific therapy for AS. The antihypertensive and antiproteinuric properties of ACE inhibitors lead to their nephroprotective effect. The animal models of AS have shown a major role of altered composition of the GBM and of podocytes (their cytoskeleton and their collagen receptors) with activation of profibrotic factors in disease progression [32, 33]. ACE inhibitors seem to be able to downregulate profibrotic factors independently from blood pressure and the amount

The Alport Syndrome Research Collaborative recommends use of ACE inhibitors in all affected individuals with microalbuminuria or proteinuria, and underlines the importance of

symptoms of HANAC, especially retinal abnormalities.

was found that the proteinuria increased the risk of ESRD.

**2.6. Treatment of Alport syndrome**

of proteinuria in the Alport animal model [68].

**2.5. Renal prognosis**

120 Advances in Nephropathy

Alport syndrome is a multisystem hereditary disorder characterized by progressive renal disease, sensorineural deafness, and eye abnormalities. Diagnosis of AS can be made on the basis of the typical symptoms, renal morphology, and/or genetic tests. Early diagnosis of Alport syndrome is very important, since therapeutic blockade of the renin-angiotensin-aldosterone system can slow the progression to ESRD.

## **Conflict of interest**

The authors declare no conflict of interest.

## **Appendices and Nomenclature**

#### **Author details**

Marina Aksenova\* and Lev Shagam

\*Address all correspondence to<EMAIL_ADDRESS>
Y.Veltischev Research and Clinical Institute for Pediatrics at the Pirogov Russian National Research Medical University, Moscow, Russia

[7] Kovács G, Kalmár T, Endreffy E, Ondrik Z, Iványi B, et al. Efficient targeted next generation sequencing-based workflow for differential diagnosis of Alport-related disorders.

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[8] Slajpah M, Gorinsek B, Berginc G, Vizjak A, Ferluga D, et al. Sixteen novel mutations identified in COL4A3, COL4A4, and COL4A5 genes in Slovenian families with Alport syndrome and benign familial hematuria. Kidney International. 2007;**12**(71):1287-1295.

[9] Arrondel C, Deschênes G, Le Meur Y, Viau A, Cordonnier C, et al. A large tandem duplication within the COL4A5 gene is responsible for the high prevalence of Alport syndrome in French Polynesia. Kidney International. 2004;**6**(65):2030-2040. DOI: 10.

[10] Martin P, Heiskari N, Zhou J, Leinonen A, Tumelius T, et al. High mutation detection rate in the COL4A5 collagen gene in suspected Alport syndrome using PCR and direct

[11] Kashtan C. Alport syndrome: Facts and opinions. F1000Research. 2017;**6**:50. DOI: 10.12688/

[12] Rheault M. Women and Alport syndrome. Pediatric Nephrology. 2012;**1**(27):41-46. DOI:

[13] Pierides A, Voskarides V, Kkolou M, Hadjigavriel M, Deltas C. X-linked, COL4A5 hypomorphic Alport mutations such as G624D and P628L may only exhibit thin basement membrane nephropathy with microhematuria and late onset kidney failure.

[14] Haas M. Alport syndrome and thin glomerular basement membrane nephropathy: A practical approach to diagnosis. Archives of Pathology & Laboratory Medicine. 2009;

[15] Lemmink H, Hughes A, Hill C, Smeets H, Doherty C, et al. Mutations in the type IV collagen alpha 3 (COL4A3) gene in autosomal recessive Alport syndrome. Human Molecu-

[16] Mochizuki T, Lemmink H, Mariyama M, Antignac C, Gubler M, et al. Identification of mutations in the alpha 3(IV) and alpha 4(IV) collagen genes in autosomal recessive Alport

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10.1136/jmedgenet-2014-102822

#### **References**

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**Appendices and Nomenclature**

ACE angiotensin-converting enzyme

GBM glomerular basement membrane

OCT optical coherence tomography

Marina Aksenova\* and Lev Shagam

TBMN thin basement membrane nephropathy

Research Medical University, Moscow, Russia

nal. 1927;**1**:504-506. PMID: 20773074

DOI: 10.1111/j.1399-0004.2011.01647

\*Address all correspondence to<EMAIL_ADDRESS>
Y.Veltischev Research and Clinical Institute for Pediatrics at the Pirogov Russian National

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**Chapter 6**

Provisional chapter

**Antibodies Against M-Type Phospholipase A2 Receptor**

DOI: 10.5772/intechopen.78955

Membranous nephropathy (MN) is a very common disease of male adults with nephrotic syndrome. The disease can be primary, when the cause is not known, or secondary associated with infections, drugs, neoplasias and autoimmune systemic diseases, such as systemic lupus erythematosus (SLE). The primary form accounts for 70–80% of the cases. SLE is a common cause of secondary MN affecting young women. The differential diagnosis from primary and lupus MN by clinical and morphological findings can be difficult. The search for autoantibodies against podocyte antigen M-type phospholipase A2 receptor (PLA2R) has demonstrated high positivity in the serum and renal biopsies in the primary MN and negativity in lupus MN (WHO class V). There is a large literature on the role of anti-PLA2R antibody in the diagnosis and follow-up of patients with membranous nephropathy. The aim of this review is to summarize the literature data on the etiopathogenesis of MN and the value of anti-PLA2R antibody screening for the diagnosis and management of patients.

Keywords: primary membranous nephropathy, lupus membranous nephropathy, podocyte antigen M-type anti–phospholipase A2 receptor, podocyte autoantibodies,

Membranous nephropathy is a frequent cause of nephrotic syndrome in adults. MN can manifest as an autoimmune primary disease or can be secondary to a wide variety of conditions such as infections, neoplasias, drugs and autoimmune diseases. The differential diagnosis

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Antibodies Against M-Type Phospholipase A2 Receptor

**Nephropathy and Lupus Nephritis Class V: A Review**

Nephropathy and Lupus Nephritis Class V: A Review

**(PLA2R) in Patients with Primary Membranous**

(PLA2R) in Patients with Primary Membranous

Rosa M. Viero, Bruno M. Miamoto,

Rosa M. Viero, Bruno M. Miamoto,

http://dx.doi.org/10.5772/intechopen.78955

differential diagnosis, disease activity

Abstract

1. Introduction

Vanessa dos S. Silva and Daniela C. dos Santos

Vanessa dos S. Silva and Daniela C. dos Santos

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

#### **Antibodies Against M-Type Phospholipase A2 Receptor (PLA2R) in Patients with Primary Membranous Nephropathy and Lupus Nephritis Class V: A Review** Antibodies Against M-Type Phospholipase A2 Receptor (PLA2R) in Patients with Primary Membranous Nephropathy and Lupus Nephritis Class V: A Review

DOI: 10.5772/intechopen.78955

Rosa M. Viero, Bruno M. Miamoto, Vanessa dos S. Silva and Daniela C. dos Santos Rosa M. Viero, Bruno M. Miamoto, Vanessa dos S. Silva and Daniela C. dos Santos

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.78955

#### Abstract

[74] Zeisberg M, Khurana M, Rao V, Cosgrove D, Rougier J-P, et al. Stage-specific action of matrix metalloproteinases influences progressive hereditary kidney disease. PLoS

[75] Koepke M, Weber M, Schulze-Lohoff E, Beirowski B, Segerer S, Gross O. Nephroprotective effect of the HMG CoA reductase inhibitor cerivastatin in a mouse model of progressive renal fibrosis in Alport syndrome. Nephrology, Dialysis, Transplantation. 2007;

[76] Ninichuk V, Gross O, Reichel C, Khandoga A, Pawar R, et al. Delayed chemokine receptor 1 blockade prolongs survival in collagen 4A3-deficient mice with Alport disease.

[77] Zeisberg M, Bottiglio C, Kumar N, Maeshima Y, Strutz F, et al. Bone morphogenic protein 7 inhibits progression of chronic renal fibrosis associated with two genetic mouse models. American Journal of Physiology. Renal Physiology. 2003;**285**:F1060-F1067. DOI:

[78] Study of RG-012 in Male Subjects With Alport Syndrome (HERA). [Internet]. Available from: https://clinicaltrials.gov/ct2/show/NCT02855268 [Accessed: 2018.05.16]

[79] A Phase 2/3 Trial of the Efficacy and Safety of Bardoxolone Methyl in Patients With Alport Syndrome - CARDINAL (CARDINAL). [Internet]. Available from: https://clini-

Medicine. 2006;**3**:e100. DOI: 10.1371/journal.pmed.0030100

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caltrials.gov/ct2/show/NCT03019185 [Accessed: 2018.05.16]

**22**:1062-1069. DOI: 10.1093/ndt/gfl810

10.1152/ajprenal.00191.2002

128 Advances in Nephropathy

Membranous nephropathy (MN) is a very common disease of male adults with nephrotic syndrome. The disease can be primary, when the cause is not known, or secondary associated with infections, drugs, neoplasias and autoimmune systemic diseases, such as systemic lupus erythematosus (SLE). The primary form accounts for 70–80% of the cases. SLE is a common cause of secondary MN affecting young women. The differential diagnosis from primary and lupus MN by clinical and morphological findings can be difficult. The search for autoantibodies against podocyte antigen M-type phospholipase A2 receptor (PLA2R) has demonstrated high positivity in the serum and renal biopsies in the primary MN and negativity in lupus MN (WHO class V). There is a large literature on the role of anti-PLA2R antibody in the diagnosis and follow-up of patients with membranous nephropathy. The aim of this review is to summarize the literature data on the etiopathogenesis of MN and the value of anti-PLA2R antibody screening for the diagnosis and management of patients.

Keywords: primary membranous nephropathy, lupus membranous nephropathy, podocyte antigen M-type anti–phospholipase A2 receptor, podocyte autoantibodies, differential diagnosis, disease activity

#### 1. Introduction

Membranous nephropathy is a frequent cause of nephrotic syndrome in adults. MN can manifest as an autoimmune primary disease or can be secondary to a wide variety of conditions such as infections, neoplasias, drugs and autoimmune diseases. The differential diagnosis

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

between primary and secondary forms must be made after an extensive clinical and serological investigation. The clinical course of primary MN is variable, some patients have spontaneous remission and others show progressive course to chronic renal failure. There is no specific marker that identifies which patients will develop chronic renal failure. The treatment can be conservative but sometimes immunosuppressive therapy is required. On the other hand, the secondary forms ameliorate after the associate condition is removed [1].

Recently, a new autoantigen in the podocyte membrane, an M-type phospholipase A2 receptor (PLA2R), was discovered in the serum and in the renal biopsies in 70–80% of primary MN patients. The presence of anti-PLA2R in the glomerular capillary walls by immunohistochemistry favors the diagnosis of primary form of MN and excluded the secondary causes. Anti-PLA2R positive has been very rare in the secondary MN. There are many studies showing anti-PLA2R negative in the serum and biopsies of membranous lupus nephritis. The serum titers of anti-PLA2R are usually elevated in the active phase of primary MN and decline in the remission of disease. In this way, the anti-PLA2R antibody has been used in clinical practice in the differential diagnosis of primary and secondary forms as well as in the monitoring of disease progression [2].

#### 2. The disease

Membranous nephropathy is a glomerular disease characterized by diffuse and uniform thickening of the glomerular capillary walls caused by the deposition of immune complexes, which appears by immunofluorescence as granular deposits of IgG and in the ultrastructure as subepithelial deposits [1] (Figures 1–3).

It is one of the main causes of primary glomerulonephritis worldwide [3, 4]. In the São Paulo Registry of Glomerulopathies [5] and, in a retrospective epidemiological study conducted in Brazil with 9617 renal biopsies [6], MN was indicated as the second most prevalent primary glomerular lesion. In our service, the incidence is 33% [7]. It mainly affects male adults, between the fourth and fifth decades [8], and is the main glomerulopathy in the elderly [9].

MN presents with high levels of proteinuria of insidious appearance. In 70 to 80% of the cases, there is nephrotic syndrome with hypoalbuminemia, hypercholesterolemia and edema. Microscopic hematuria is common, and the presence of hypertension varies around 50% of cases. Renal function is usually preserved; however, chronic renal failure occurs in 40% of cases within 10 years of evolution. The diagnosis of the disease is made by renal biopsy in the clinical

Figure 3. Electron-dense epimembranous deposits in the glomerular basement membrane. D = deposit, S = spikes,

Antibodies Against M-Type Phospholipase A2 Receptor (PLA2R) in Patients with Primary Membranous Nephropathy…

http://dx.doi.org/10.5772/intechopen.78955

131

GBM = glomerular basement membrane, P = podocyte, and E = endothelial cell (EM-21,000).

Figure 2. Diffuse granular deposits of IgG in the glomerular capillary walls (IF-200).

MN can evolve over the years in different ways, from spontaneous remission to persistence of the nephrotic syndrome with a slow and progressive course for chronic renal failure.

suspicion of proteinuria or nephrotic syndrome [10–14].

Figure 1. Diffuse thickening of the glomerular capillary walls (HE-200).

Antibodies Against M-Type Phospholipase A2 Receptor (PLA2R) in Patients with Primary Membranous Nephropathy… http://dx.doi.org/10.5772/intechopen.78955 131

Figure 2. Diffuse granular deposits of IgG in the glomerular capillary walls (IF-200).

between primary and secondary forms must be made after an extensive clinical and serological investigation. The clinical course of primary MN is variable, some patients have spontaneous remission and others show progressive course to chronic renal failure. There is no specific marker that identifies which patients will develop chronic renal failure. The treatment can be conservative but sometimes immunosuppressive therapy is required. On the other hand, the

Recently, a new autoantigen in the podocyte membrane, an M-type phospholipase A2 receptor (PLA2R), was discovered in the serum and in the renal biopsies in 70–80% of primary MN patients. The presence of anti-PLA2R in the glomerular capillary walls by immunohistochemistry favors the diagnosis of primary form of MN and excluded the secondary causes. Anti-PLA2R positive has been very rare in the secondary MN. There are many studies showing anti-PLA2R negative in the serum and biopsies of membranous lupus nephritis. The serum titers of anti-PLA2R are usually elevated in the active phase of primary MN and decline in the remission of disease. In this way, the anti-PLA2R antibody has been used in clinical practice in the differential diagnosis of primary and secondary forms as well as in the monitoring of disease progression [2].

Membranous nephropathy is a glomerular disease characterized by diffuse and uniform thickening of the glomerular capillary walls caused by the deposition of immune complexes, which appears by immunofluorescence as granular deposits of IgG and in the ultrastructure as

It is one of the main causes of primary glomerulonephritis worldwide [3, 4]. In the São Paulo Registry of Glomerulopathies [5] and, in a retrospective epidemiological study conducted in Brazil with 9617 renal biopsies [6], MN was indicated as the second most prevalent primary glomerular lesion. In our service, the incidence is 33% [7]. It mainly affects male adults, between the fourth and fifth decades [8], and is the main glomerulopathy in the elderly [9].

secondary forms ameliorate after the associate condition is removed [1].

2. The disease

130 Advances in Nephropathy

subepithelial deposits [1] (Figures 1–3).

Figure 1. Diffuse thickening of the glomerular capillary walls (HE-200).

Figure 3. Electron-dense epimembranous deposits in the glomerular basement membrane. D = deposit, S = spikes, GBM = glomerular basement membrane, P = podocyte, and E = endothelial cell (EM-21,000).

MN presents with high levels of proteinuria of insidious appearance. In 70 to 80% of the cases, there is nephrotic syndrome with hypoalbuminemia, hypercholesterolemia and edema. Microscopic hematuria is common, and the presence of hypertension varies around 50% of cases. Renal function is usually preserved; however, chronic renal failure occurs in 40% of cases within 10 years of evolution. The diagnosis of the disease is made by renal biopsy in the clinical suspicion of proteinuria or nephrotic syndrome [10–14].

MN can evolve over the years in different ways, from spontaneous remission to persistence of the nephrotic syndrome with a slow and progressive course for chronic renal failure. Spontaneous remission occurs in about 25–30%. The progression to renal insufficiency is around 12–18% of the cases [15]. Due to the high rate of spontaneous remission of the disease and the aggressive specific treatment, it is important to identify prognostic factors that are associated with progression to renal failure. Among the prognostic factors, age over 50 years, male gender, presence of arterial hypertension, proteinuria intensity, elevated serum creatinine, advanced glomerular stage and presence of interstitial fibrosis in renal biopsy are indicators of disease progression [10–12, 15].

## 3. The primary or idiopathic form and etiopathogenic aspects

The cause of MN is unknown in 80% of the cases being considered primary or idiopathic [16].

Since the mid-1950s, the understanding of the etiopathogenic mechanisms of MN in experimental models has been sought. The first formulated hypothesis was the deposition of circulating immune complexes in the kidney.

MN. Beck et al. Salant is the responsible for this Research Group kidney from deceased donors, not used in transplantation, to extract glomerular proteins that were used in western blot reaction with circulating antibodies from MN patients. A 185-kD protein was detected in 70% of patients with primary MN, whereas sera from patients with secondary MN or other glomerular diseases did not react with this protein. The protein was identified by spectrometry as a transmembrane type M receptor of secretory phospholipase A2 (sPLA2R). The PLA2R superfamily is a heterogeneous group of proteins and enzymes that is present in normal podocytes and includes the secretory (sPLA2R), cytoplasmic (cPLA2R), and lysosomal (IPLA2R) forms. They are ubiquitous in nature as an intra- and extracellular form, hydrolyze several phospholipids and act in various biological activities [22, 23]. In the biopsies from patients with idiopathic MN, the receptor is located next to IgG4, a place consistent with the localization of the immunoc-

Antibodies Against M-Type Phospholipase A2 Receptor (PLA2R) in Patients with Primary Membranous Nephropathy…

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133

PLA2R is considered the autoantigen responsible for the development of primary MN in 70– 80% of cases, but there were still 20–30% left without a specific marker. In 2014, a group of researchers from several countries demonstrated the presence of thrombospondin type 1, with a 7A domain (THSD7A), another antigenic target in patients with NM that respond for about 10% of the primary cases [24]. More recently, the same group induced MN in animal model with autoantibodies against THSD7A eluted from renal biopsy of a patient with recurrent MN

There are reports of other podocyte antigens with antigenic and targeting capacity for autoan-

In conclusion, the findings of NEP, PLA2R, and THSD7A autoantigens in primary MN demonstrated that subepithelial deposits form in situ through the binding of circulating autoantibodies to autoantigens expressed on the surface of podocytes. Heymann's nephritis was the

However, the stimulus that initiates the autoimmune response to podocyte antigens with

tibody production, but their role is still uncertain and more studies are needed [26].

omplexes of idiopathic membranous nephropathy [2] (Figure 4).

Figure 4. Immunofluorescence staining of PLA2R in glomerular deposits (IF-400).

basis for understanding the pathogenesis of primary MN.

disease development is still unknown.

in the allograft [25].

Heymann et al. [17] reproduced in the rat a glomerular lesion similar to that of human MN. Heymann's active nephritis was induced by the immunization of rats with rat kidney extracts in Freund's adjuvant. The deposits in the glomerular basement membrane were interpreted as resulting from the passive deposition of circulating immune complexes. Subsequent studies have shown that glomerular deposits in the subepithelial region were composed of antigens normally present on the brush border of the proximal tubules and autoantibodies [18]. And in the Heymann's passive nephritis, the same disease was reproduced in the rat, using the injection of the antiserum obtained by immunizing rabbits with brush border proteins of rat kidney [19]. The hypothesis of circulating immune complexes started to be questioned, and a new hypothesis of local formation of immune deposits by an endogenous antigen localized at subepithelial side of glomerular basement membrane and a circulating autoantibody was formulated (in situ immune complexes). Kerjaschki and Farquhar [20] subsequently identified the target autoantigen in the mouse, a protein called megalin, located on the brush border of the proximal tubules and on the cytoplasmic membrane of the podocyte.

In conclusion, the findings of the experimental studies demonstrated that the podocyte contributes to the formation of immunodeposits in MN, which has been considered an autoimmune disease.

Since megalin is not present in human podocytes, the antigen(s) involved in the primary human MN remains unknown.

In 2002, Debiec et al. [21] identified the first autoantigen in the human MN in a case of neonatal nephrotic syndrome. The mother with genetic deficiency of neutral endopeptidase (NEP), a normal protein in the podocytes, was immunized by the fetus with normal expression of NEP. The anti-NEP antibodies synthesized by the mother were transferred, via the placenta to the fetus, with formation of in situ immunocomplexes in the kidney. This was a rare familial case of human neonatal primary MN by an autoantigen present in the podocyte. However, MN is a very common pathology, and these findings did not explain all cases of the primary form. Subsequently, numerous studies were done in search for other autoantigens responsible for primary Antibodies Against M-Type Phospholipase A2 Receptor (PLA2R) in Patients with Primary Membranous Nephropathy… http://dx.doi.org/10.5772/intechopen.78955 133

Figure 4. Immunofluorescence staining of PLA2R in glomerular deposits (IF-400).

Spontaneous remission occurs in about 25–30%. The progression to renal insufficiency is around 12–18% of the cases [15]. Due to the high rate of spontaneous remission of the disease and the aggressive specific treatment, it is important to identify prognostic factors that are associated with progression to renal failure. Among the prognostic factors, age over 50 years, male gender, presence of arterial hypertension, proteinuria intensity, elevated serum creatinine, advanced glomerular stage and presence of interstitial fibrosis in renal biopsy are indica-

The cause of MN is unknown in 80% of the cases being considered primary or idiopathic [16]. Since the mid-1950s, the understanding of the etiopathogenic mechanisms of MN in experimental models has been sought. The first formulated hypothesis was the deposition of circu-

Heymann et al. [17] reproduced in the rat a glomerular lesion similar to that of human MN. Heymann's active nephritis was induced by the immunization of rats with rat kidney extracts in Freund's adjuvant. The deposits in the glomerular basement membrane were interpreted as resulting from the passive deposition of circulating immune complexes. Subsequent studies have shown that glomerular deposits in the subepithelial region were composed of antigens normally present on the brush border of the proximal tubules and autoantibodies [18]. And in the Heymann's passive nephritis, the same disease was reproduced in the rat, using the injection of the antiserum obtained by immunizing rabbits with brush border proteins of rat kidney [19]. The hypothesis of circulating immune complexes started to be questioned, and a new hypothesis of local formation of immune deposits by an endogenous antigen localized at subepithelial side of glomerular basement membrane and a circulating autoantibody was formulated (in situ immune complexes). Kerjaschki and Farquhar [20] subsequently identified the target autoantigen in the mouse, a protein called megalin, located on the brush border of the proximal tubules

In conclusion, the findings of the experimental studies demonstrated that the podocyte contributes to the formation of immunodeposits in MN, which has been considered an autoim-

Since megalin is not present in human podocytes, the antigen(s) involved in the primary

In 2002, Debiec et al. [21] identified the first autoantigen in the human MN in a case of neonatal nephrotic syndrome. The mother with genetic deficiency of neutral endopeptidase (NEP), a normal protein in the podocytes, was immunized by the fetus with normal expression of NEP. The anti-NEP antibodies synthesized by the mother were transferred, via the placenta to the fetus, with formation of in situ immunocomplexes in the kidney. This was a rare familial case of human neonatal primary MN by an autoantigen present in the podocyte. However, MN is a very common pathology, and these findings did not explain all cases of the primary form. Subsequently, numerous studies were done in search for other autoantigens responsible for primary

3. The primary or idiopathic form and etiopathogenic aspects

tors of disease progression [10–12, 15].

132 Advances in Nephropathy

lating immune complexes in the kidney.

and on the cytoplasmic membrane of the podocyte.

mune disease.

human MN remains unknown.

MN. Beck et al. Salant is the responsible for this Research Group kidney from deceased donors, not used in transplantation, to extract glomerular proteins that were used in western blot reaction with circulating antibodies from MN patients. A 185-kD protein was detected in 70% of patients with primary MN, whereas sera from patients with secondary MN or other glomerular diseases did not react with this protein. The protein was identified by spectrometry as a transmembrane type M receptor of secretory phospholipase A2 (sPLA2R). The PLA2R superfamily is a heterogeneous group of proteins and enzymes that is present in normal podocytes and includes the secretory (sPLA2R), cytoplasmic (cPLA2R), and lysosomal (IPLA2R) forms. They are ubiquitous in nature as an intra- and extracellular form, hydrolyze several phospholipids and act in various biological activities [22, 23]. In the biopsies from patients with idiopathic MN, the receptor is located next to IgG4, a place consistent with the localization of the immunocomplexes of idiopathic membranous nephropathy [2] (Figure 4).

PLA2R is considered the autoantigen responsible for the development of primary MN in 70– 80% of cases, but there were still 20–30% left without a specific marker. In 2014, a group of researchers from several countries demonstrated the presence of thrombospondin type 1, with a 7A domain (THSD7A), another antigenic target in patients with NM that respond for about 10% of the primary cases [24]. More recently, the same group induced MN in animal model with autoantibodies against THSD7A eluted from renal biopsy of a patient with recurrent MN in the allograft [25].

There are reports of other podocyte antigens with antigenic and targeting capacity for autoantibody production, but their role is still uncertain and more studies are needed [26].

In conclusion, the findings of NEP, PLA2R, and THSD7A autoantigens in primary MN demonstrated that subepithelial deposits form in situ through the binding of circulating autoantibodies to autoantigens expressed on the surface of podocytes. Heymann's nephritis was the basis for understanding the pathogenesis of primary MN.

However, the stimulus that initiates the autoimmune response to podocyte antigens with disease development is still unknown.

There is a strong association between idiopathic MN and HLA-DQA1, located on chromosome 6p21, and the presence of the allele would facilitate the formation of autoantibodies against PLA2R. In chromosome 2q24, the gene encoding the M-type receptor of phospholipase A2 (PLA2R1) is located [27].

as an antigenic stimulus, against which an IgG4 or IgG1 is produced, local complement activation, inflammatory and oxidative response, with podocyte disruption and lesion in the

Antibodies Against M-Type Phospholipase A2 Receptor (PLA2R) in Patients with Primary Membranous Nephropathy…

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Ronco and Debiec [33] believe that further studies should be conducted to evaluate whether PLA2R protein in human MN produces podocyte injury similar to the megalin of Heymann's nephritis. The correlation of the anti-PLA2R antibodies with the disease activity and the presence of the PLA2R protein in the immune deposits suggest that they are the cause and not consequence of the podocyte injury. On the other hand, the absence of anti-PLA2R antibodies in other pathologies with proteinuria also shows that these antibodies are not only

4. The discovery of the PLA2R antigen and the differential diagnosis of the

The secondary form of MN is more rare and is related to infections, autoimmune diseases, neoplasias and drugs. In children, hepatitis B is a frequent cause of MN and the treatment of hepatitis with alpha interferon is usually accompanied by remission of proteinuria. Systemic lupus erythematosus (SLE) often courses with glomerulonephritis, and one type of presentation is a membranous form (WHO and ISN/RPS class V). About 10% of MN patients are carriers of neoplasias. The most common sites are gastrointestinal tract, prostate, lung, and breast. The antigen finding in the biopsies of MN patients, such as HBeAg, HBsAg and HBcAg in hepatitis B virus, double helix DNA in SLE and carcinoembryonic antigen (CEA) in gastrointestinal tract neoplasias, suggests a causal relationship between the underlying disease and the manifestation of glomerulonephritis. However, the possibility of association of diseases

The investigation of secondary causes with complete clinical examination, serologies and analysis by images is necessary. After excluding secondary forms, the patients with idiopathic MN can receive supportive treatment or an immunosuppressive therapy; in the secondary form, the treatment is directed against the cause of the disease. However, in some patients, MN may appear months or years before the cause is detected. This makes treatment and clinical

The discovery of the PLA2R antigen brought a number of benefits for the diagnosis and monitoring of the primary form of the disease. The distinction between the primary and secondary forms was made easier by the detection of the anti-PLA2R autoantibody in kidney biopsy and serum of patients, and extensive procedures for differential diagnosis and unnec-

Several studies have been done with the aim of evaluating the sensitivity and specificity of the

In a Chinese study of 60 patients with primary and 46 patients with secondary MN, a specificity of 89% [35] was demonstrated. In the Iranian population, anti-PLA2R was detected in 74%

filtration barrier with consequent proteinuria.

markers of the disease or of the podocyte lesion [34].

essary exposure to immunosuppression may be excluded.

anti-PLA2R autoantibody in renal biopsy and serum of patients with MN.

of patients with primary MN and was absent in the secondary form [36].

primary and secondary MN

cannot be ruled out [1].

monitoring difficult.

A genetic predisposition can act as a trigger, the dysfunction in the innate immune response functions as the bullet and the antibody formed against PLA2R the target. As this process initiate, the disease follows its course. Recently, a publication by Ponticelli and Glassock [28] has gathered the main information about the pathogenesis of NM: the presence of the HLA-DQA1 allele predisposes to disease and with PLA2R exposure in the podocyte membrane with characteristic conformation favors its recognition as autoantigen and starts the process of autoimmunization. Cells responsible for regulating the innate immune response, such as dendritic cells, contact with the receptor and presents to CD8+ cells of the adaptive immune response. Thus, there is a production of IgG4 or IgG1 that binds to the receptor in podocytes with formation of in situ immunocomplexes. The conformation of the receptor in the cell is essential to make it more or less antigenic and enable for the formation of autoantibodies.

With the in situ deposition of immunocomplexes, there is activation of the complement system, lesion of the filtration barrier and loss of proteins to the urinary space. The binding of IgG4 to PLA2R determines local activation of the complement via mannose, since IgG4 is not able to activate complement by the classical or alternative pathway, triggering C5 cleavage, and formation of C5a and C5b. C5a can be eliminated in the urine while C5b binds to other components until formation of C5b-9. The C5b-9 fraction of the complement (attack complex) activates the inflammatory response through specific extracellular signaling pathways, with generation of reactive oxygen species induced by massive biosynthesis of NADPH oxyreductase, lipid peroxidation, and degradation of the glomerular basement membrane with consequent loss of proteins into the urinary space [29]. There is release of arachidonic acid and eicosanoids with disruption of the actin cytoskeleton of the podocytes and loss of the diaphragm of the filtration barrier, leading to detachment of the podocytes from glomerular basement membrane as well as apoptosis of the podocytes [30]. Persistent proteinuria leads to the intrarenal activation of the renin-angiotensin system with angiotensin II synthesis, which also promotes proinflammatory response, activation of transforming growth factor β (TGFβ) and platelet-derived growth factor (PDGF), recruitment of inflammatory cells, and myofibroblastic activation, which promotes interstitial fibrosis with concomitant tubular atrophy and chronic renal failure [31].

Studies have been shown that proteinuria, in addition to being directly related to complement activation, also correlates with the deposits' characteristics, the degree of podocyte lesion and CD8+ cellular response [32].

Regarding the pathogenesis of NM, there are doubts in the pathway that change the conformation of PLA2R making it antigenic, the mechanisms of proteinuria, spontaneous remission and the progression of disease. The understanding of these mechanisms may help to choose the therapeutic options more efficient and different for each phase of the disease.

In conclusion, the pathogenic mechanism leading to the deposition of immunocomplexes in the subepithelial region in idiopathic NM is the formation of immunocomplexes in situ. The expression of phospholipase A2 receptor 1 (PLA2R) on the podocyte membrane is recognized as an antigenic stimulus, against which an IgG4 or IgG1 is produced, local complement activation, inflammatory and oxidative response, with podocyte disruption and lesion in the filtration barrier with consequent proteinuria.

There is a strong association between idiopathic MN and HLA-DQA1, located on chromosome 6p21, and the presence of the allele would facilitate the formation of autoantibodies against PLA2R. In chromosome 2q24, the gene encoding the M-type receptor of phospholipase A2

A genetic predisposition can act as a trigger, the dysfunction in the innate immune response functions as the bullet and the antibody formed against PLA2R the target. As this process initiate, the disease follows its course. Recently, a publication by Ponticelli and Glassock [28] has gathered the main information about the pathogenesis of NM: the presence of the HLA-DQA1 allele predisposes to disease and with PLA2R exposure in the podocyte membrane with characteristic conformation favors its recognition as autoantigen and starts the process of autoimmunization. Cells responsible for regulating the innate immune response, such as dendritic cells, contact with the receptor and presents to CD8+ cells of the adaptive immune response. Thus, there is a production of IgG4 or IgG1 that binds to the receptor in podocytes with formation of in situ immunocomplexes. The conformation of the receptor in the cell is essential to make it more or less antigenic and enable for the formation of autoantibodies.

With the in situ deposition of immunocomplexes, there is activation of the complement system, lesion of the filtration barrier and loss of proteins to the urinary space. The binding of IgG4 to PLA2R determines local activation of the complement via mannose, since IgG4 is not able to activate complement by the classical or alternative pathway, triggering C5 cleavage, and formation of C5a and C5b. C5a can be eliminated in the urine while C5b binds to other components until formation of C5b-9. The C5b-9 fraction of the complement (attack complex) activates the inflammatory response through specific extracellular signaling pathways, with generation of reactive oxygen species induced by massive biosynthesis of NADPH oxyreductase, lipid peroxidation, and degradation of the glomerular basement membrane with consequent loss of proteins into the urinary space [29]. There is release of arachidonic acid and eicosanoids with disruption of the actin cytoskeleton of the podocytes and loss of the diaphragm of the filtration barrier, leading to detachment of the podocytes from glomerular basement membrane as well as apoptosis of the podocytes [30]. Persistent proteinuria leads to the intrarenal activation of the renin-angiotensin system with angiotensin II synthesis, which also promotes proinflammatory response, activation of transforming growth factor β (TGFβ) and platelet-derived growth factor (PDGF), recruitment of inflammatory cells, and myofibroblastic activation, which promotes

interstitial fibrosis with concomitant tubular atrophy and chronic renal failure [31].

the therapeutic options more efficient and different for each phase of the disease.

Studies have been shown that proteinuria, in addition to being directly related to complement activation, also correlates with the deposits' characteristics, the degree of podocyte lesion and

Regarding the pathogenesis of NM, there are doubts in the pathway that change the conformation of PLA2R making it antigenic, the mechanisms of proteinuria, spontaneous remission and the progression of disease. The understanding of these mechanisms may help to choose

In conclusion, the pathogenic mechanism leading to the deposition of immunocomplexes in the subepithelial region in idiopathic NM is the formation of immunocomplexes in situ. The expression of phospholipase A2 receptor 1 (PLA2R) on the podocyte membrane is recognized

(PLA2R1) is located [27].

134 Advances in Nephropathy

CD8+ cellular response [32].

Ronco and Debiec [33] believe that further studies should be conducted to evaluate whether PLA2R protein in human MN produces podocyte injury similar to the megalin of Heymann's nephritis. The correlation of the anti-PLA2R antibodies with the disease activity and the presence of the PLA2R protein in the immune deposits suggest that they are the cause and not consequence of the podocyte injury. On the other hand, the absence of anti-PLA2R antibodies in other pathologies with proteinuria also shows that these antibodies are not only markers of the disease or of the podocyte lesion [34].

## 4. The discovery of the PLA2R antigen and the differential diagnosis of the primary and secondary MN

The secondary form of MN is more rare and is related to infections, autoimmune diseases, neoplasias and drugs. In children, hepatitis B is a frequent cause of MN and the treatment of hepatitis with alpha interferon is usually accompanied by remission of proteinuria. Systemic lupus erythematosus (SLE) often courses with glomerulonephritis, and one type of presentation is a membranous form (WHO and ISN/RPS class V). About 10% of MN patients are carriers of neoplasias. The most common sites are gastrointestinal tract, prostate, lung, and breast. The antigen finding in the biopsies of MN patients, such as HBeAg, HBsAg and HBcAg in hepatitis B virus, double helix DNA in SLE and carcinoembryonic antigen (CEA) in gastrointestinal tract neoplasias, suggests a causal relationship between the underlying disease and the manifestation of glomerulonephritis. However, the possibility of association of diseases cannot be ruled out [1].

The investigation of secondary causes with complete clinical examination, serologies and analysis by images is necessary. After excluding secondary forms, the patients with idiopathic MN can receive supportive treatment or an immunosuppressive therapy; in the secondary form, the treatment is directed against the cause of the disease. However, in some patients, MN may appear months or years before the cause is detected. This makes treatment and clinical monitoring difficult.

The discovery of the PLA2R antigen brought a number of benefits for the diagnosis and monitoring of the primary form of the disease. The distinction between the primary and secondary forms was made easier by the detection of the anti-PLA2R autoantibody in kidney biopsy and serum of patients, and extensive procedures for differential diagnosis and unnecessary exposure to immunosuppression may be excluded.

Several studies have been done with the aim of evaluating the sensitivity and specificity of the anti-PLA2R autoantibody in renal biopsy and serum of patients with MN.

In a Chinese study of 60 patients with primary and 46 patients with secondary MN, a specificity of 89% [35] was demonstrated. In the Iranian population, anti-PLA2R was detected in 74% of patients with primary MN and was absent in the secondary form [36].

## 5. Relationship between anti-PLA2R antibodies and the disease activity

initiation of specific immunosuppressive therapy, there was a progressive reduction in circulating antibody levels in the first 3 months, followed by a slow but progressive reduction of proteinuria levels, which reached their lowest value after about 9 months of onset of follow-up. Thus, it can be stated that the reduction in the antibody precedes in months the reduction of

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Patients with increasing anti-PLA2R antibody titers are less prone to spontaneous remission, which, together with nephrotic proteinuria, would suggest to use immunosuppression, and patients with progressive reduction of antibody levels, which could be clinically followed, can evolve with spontaneous remission [48]. During the follow-up of the patients, the dosage of the anti-PLA2R antibody brings great contributions. The quantitative and evolutionary evaluation of the antibody can predict spontaneous remission in months, thus avoiding the initiation of specific treatment in patients with greater chances of remission. In cases where there is an increase in anti-PLA2R antibody levels, there is a greater chance of progression, which would suggest the initiation of specific treatment, avoiding waiting and decreasing the chance

The close correlation of the serum levels of the anti-PLA2 R autoantibodies with the immunological activity of the disease allows the monitoring of the treatment and evaluation of the prognosis. Patients who present remission of the disease after treatment show a reduction in antibody titers and proteinuria levels [35, 37, 44, 49] and an increase with relapse [37]. A prospective study shows that the reduction in anti-PLA2R antibody titers is greater in patients' remission of the disease. Monitoring of antibody titers after initiation of treatment is useful in estimating the time to remission of the disease [43]. Beck et al. [49] evaluated the relationship between levels of the anti-PLA2R antibody and response to Rituximab treatment. Titers

proteinuria, and that it is associated with the time to reach remission [46].

decreased in most patients and anticipated a decrease in proteinuria.

6. Antibodies to M-type phospholipase A2 receptor (PLA2R) and