After histologic processing and paraffin embedding, the tissues are sectioned by microtome. These sections are prepared as thin as 3 µm or less for light microscopy. Thicker sections in needed in congo red and Immunohistochemistry staining.

The most helpful stains for light microscopy are Hematoxiline and Eosine (H&E), methenamine silver, Periodic Acid Schiff (PAS), trichrome, congo red and reticulin.

H&E highlights the cells well, while methenamine silver shows basement membrane and matrix of connective tissues. In PAS staining, there is good highlighting of cells and basement membrane. Trichrome staining is suitable for basement membrane, fibrosis, and deposit assessment. Congo red is recommended for amyloid discovering while elastin stains such as reticulin are helpful in vascular lesions.

Table 1 provides a comparison of the characteristics of tissues provided by three different stains (8).

Table 1.

#### **12. Specimen adequacy**

There is a question of how much tissue is necessary for diagnosis of renal disease. In diffuse glomerular diseases, such as amyloidosis and membranous glomerulopathy, one glomerulus is enough for diagnosis. In focal diseases, considering the random distribution of abnormalities, the probability of finding any glomerulopathy is represented by bionial equation (65). For example, if we have 10 glomeruli and the disease exist in 10% and 35% of them, we will have 65% and 95% positive report in the biopsy, respectively.

Fogo mentioned 25 and 10 glomeruli are needed for most accurate diagnosis in light microscopy for native and transplanted kidneys respectively (52). In one study it was found that a specimen with at least 25 glomeruli is needed for the biopsies of chronic lesions of the kidney (66).

There is semi-quantification of pathologic findings including glomeruli number, percentage of affected glomeruli, mesangial matrix volume, inflammatory cell infiltrate, percentage of fibrosis and atrophy in different patients. Also quantification techniques in renal biopsy have been reported (67,68,69,70). These quantifications ha help in monitoring the patients

Renal Biopsy Interpretation 53

There is a standard terminology for glomerular involving lesions report which is stated by

**Endocapillary hypercellularity**: increased cellularity internal to the GBM composed of

**Extracapillary hypercellularity**: increased cellularity in Bowman`s space (more than one

**Crescent:** extracapillary hypercellularity other than the epithelial hyperplasia of collapsing

**Fibrinoid necrosis**: lytic destruction of cells and matrix with deposition of acidophilic

**Sclerosis**: increased collagenous extracellular matrix that is expanding the mesangium,

**Membranoprolifrative**: combined capillary wall thickening and mesangial or endocapillary

**Lobular (hypersegmented)**: expansion of segments that are demarcated by intervening

**Mesangiolysis**: detachment of the paramesangial GBM from the mesangial matrix or lysis of

Each of these terms could be categorized further according to some other parameters

*Focal glomerulonephritis*: is includes inflammatory lesions in less than 50% of glomeruli. the

*Diffuse glomerulonephritis*: affects most or all of the glomeruli and differential diagnoses

**Mesangial hypercellularity**: 4 or more nuclei in mesangial region

layer of parietal or visceral epithelial cells or monocyte/machrophage)

obliterating capillary lumens or forming adhesions to Bowman`s capsule

differential diagnoses are noted based on the age and are as follow:

mild postinfectious glomerulonephritis

mesangial prolifrative glomerulonephritis

mesangial prolifrative glomerulonephritis

membranoprolifrative glomerulonephritis

thin basement membrane disease

 thin basement membrane disease systemic lupus erythematous

postinfectious glomerulonephritis

Henoch Schoenlein purpura

Jennette and et al (10,75).

variants of FSGS

hypercellularity

mesangial matrix

((74,77,78,79,80,81 ,82,83):

IgA nephropathy

IgA nephropathy

hereditary nephritis

IgA nephropathy

according to age are:

hereditary nephritis

urinary space

<15 years:

15-40 years:

>40 years:

<15 years:

fibrin-rich material

**Focal**: less than 50% of glomeruli **Diffuse**: more than 50% of glomeruli **Segmental**: part of a glomerulus **Global**: all of a glomerulus

leukocytes, endothelial cells or mesangial cells.

**Hyaline**: glassy acidophilic extracellular material

and their response to therapy as well as comparison of different biopsies and their correlation with clinical points.

Quantification methods require a standard protocol for processing and sectioning (70).

### **13. Differential diagnosis of renal lesions**

Renal biopsies are done to determine tissue diagnosis, exclude other diagnostic possibilities, assessing the severity and activity of the lesion (grade), and the amount of irreversible scarring (stage) (63). There are some steps in renal biopsy evaluation with each step correlating to one the part of the kidney including glomeruli, tubules, Interstitium and vascular parts and correlate them with each other. Any changes in each part of the kidney may be associated with secondary changes in others. The maximum data about renal biopsies is provided by light microscopy, immunofluorescence study and electron microscopy (71,72,73).

In the future, genomics and proteomics studies will be used for the diagnosis of renal diseases.

In tubular parts, the focus should be on cellular injuries, regeneration, atrophy, cast, edema, fibrosis and crystals.

Interstitium pathology includes cellular infiltrate, edema, and fibrosis, while vascular part changes are inflammation, sclerosis, hyalinosis and thrombosis.

In overall inspection of biopsy, pathologists should know about the clinical status and chronicity of the disease. In the chronic nephron loss, there is compensatory of remained normal nephrons which leads to a mixture of hypertrophied functioning glomeruli and atrophic non-functioning nephrons (10).

The pathologist also should know that many diseases which affect the kidney, have different manifestations other than kidney.

Light microscopy is often the most important tool for detecting the primary site of injury, but to differentiate between the diseases with common manifestation , electron microscopy and immunofluorescence study are helpful (10).

In this section we used Heptinstal Renal Pathology tables and their respective authors.

#### **14. Glomerular lesions**

The clinical presentations of glomerular diseases are very different including proteinuria, hematuria, casts, nephritic syndrome, acute nephritis, renal failure (acute and chronic) and rapidly progressive nephritis. Clinical data about age and urine sediment (nephritic or nephritic), narrows differential diagnoses (74).

There are some reports on the relative frequency of renal disorders in biopsies (75). The complexity and variety of glomerular diseases is a challenge for nephropathologists (10).

Renal injuries could be categorized as acute or fibrosing. Active lesions include proliferation, necrosis, crescent, edema, and active inflammation. Fibrosing lesions include glomerulosclerosis, fibrosis, crescent, tubular atrophy, interstitial fibrosis and vascular fibrosis (76).

It should be remembered in the prognosis and monitoring of renal diseases that glomerular lesion can change over time. .

and their response to therapy as well as comparison of different biopsies and their

Renal biopsies are done to determine tissue diagnosis, exclude other diagnostic possibilities, assessing the severity and activity of the lesion (grade), and the amount of irreversible scarring (stage) (63). There are some steps in renal biopsy evaluation with each step correlating to one the part of the kidney including glomeruli, tubules, Interstitium and vascular parts and correlate them with each other. Any changes in each part of the kidney may be associated with secondary changes in others. The maximum data about renal biopsies is provided by light microscopy, immunofluorescence study and electron

In the future, genomics and proteomics studies will be used for the diagnosis of renal diseases. In glomerular evaluation, pathologists should diagnose the inflammation, glomerular basement membrane changes, scarring, spikes, fibrinoid exudates,

In tubular parts, the focus should be on cellular injuries, regeneration, atrophy, cast, edema,

Interstitium pathology includes cellular infiltrate, edema, and fibrosis, while vascular part

In overall inspection of biopsy, pathologists should know about the clinical status and chronicity of the disease. In the chronic nephron loss, there is compensatory of remained normal nephrons which leads to a mixture of hypertrophied functioning glomeruli and

The pathologist also should know that many diseases which affect the kidney, have

Light microscopy is often the most important tool for detecting the primary site of injury, but to differentiate between the diseases with common manifestation , electron microscopy

The clinical presentations of glomerular diseases are very different including proteinuria, hematuria, casts, nephritic syndrome, acute nephritis, renal failure (acute and chronic) and rapidly progressive nephritis. Clinical data about age and urine sediment (nephritic or

There are some reports on the relative frequency of renal disorders in biopsies (75). The complexity and variety of glomerular diseases is a challenge for nephropathologists (10). Renal injuries could be categorized as acute or fibrosing. Active lesions include proliferation, necrosis, crescent, edema, and active inflammation. Fibrosing lesions include glomerulosclerosis, fibrosis, crescent, tubular atrophy, interstitial fibrosis and vascular

It should be remembered in the prognosis and monitoring of renal diseases that glomerular

In this section we used Heptinstal Renal Pathology tables and their respective authors.

changes are inflammation, sclerosis, hyalinosis and thrombosis.

Quantification methods require a standard protocol for processing and sectioning (70).

correlation with clinical points.

microscopy (71,72,73).

fibrosis and crystals.

hypercellularity and deposits.

**14. Glomerular lesions**

lesion can change over time. .

fibrosis (76).

atrophic non-functioning nephrons (10).

different manifestations other than kidney.

and immunofluorescence study are helpful (10).

nephritic), narrows differential diagnoses (74).

**13. Differential diagnosis of renal lesions**

There is a standard terminology for glomerular involving lesions report which is stated by Jennette and et al (10,75).

**Focal**: less than 50% of glomeruli

**Diffuse**: more than 50% of glomeruli

**Segmental**: part of a glomerulus

**Global**: all of a glomerulus

**Mesangial hypercellularity**: 4 or more nuclei in mesangial region

**Endocapillary hypercellularity**: increased cellularity internal to the GBM composed of leukocytes, endothelial cells or mesangial cells.

**Extracapillary hypercellularity**: increased cellularity in Bowman`s space (more than one layer of parietal or visceral epithelial cells or monocyte/machrophage)

**Crescent:** extracapillary hypercellularity other than the epithelial hyperplasia of collapsing variants of FSGS

**Fibrinoid necrosis**: lytic destruction of cells and matrix with deposition of acidophilic fibrin-rich material

**Sclerosis**: increased collagenous extracellular matrix that is expanding the mesangium, obliterating capillary lumens or forming adhesions to Bowman`s capsule

**Hyaline**: glassy acidophilic extracellular material

**Membranoprolifrative**: combined capillary wall thickening and mesangial or endocapillary hypercellularity

**Lobular (hypersegmented)**: expansion of segments that are demarcated by intervening urinary space

**Mesangiolysis**: detachment of the paramesangial GBM from the mesangial matrix or lysis of mesangial matrix

Each of these terms could be categorized further according to some other parameters ((74,77,78,79,80,81 ,82,83):

*Focal glomerulonephritis*: is includes inflammatory lesions in less than 50% of glomeruli. the differential diagnoses are noted based on the age and are as follow:

<15 years:

15-40 years:

>40 years:

IgA nephropathy

*Diffuse glomerulonephritis*: affects most or all of the glomeruli and differential diagnoses according to age are:

<15 years:

Renal Biopsy Interpretation 55

In normal morphology of glomeruli, the diagnoses are minimal change disease and thin basement membrane nephropathy and electron microscopy and immunofluorescence would be helpful. Early stages of many glomerulopathies show normal features in light microscopy. If there are capillaries with thick walls, diseases such as membranous glomerulopathy, diabetic glomerulosclerosis, amyloidosis, immunoglobulin deposit disease,

Where limited sclerosis of glomeruli is observed, focal segmental glomerulosclerosis, Alport's syndrome, and sclerotic phase of different glomerulopathies are differential

Hypercellularity may result from increase in mesangial, visceral epithelial, endothelial cells

Mesangial hypercellularity is seen in mesangioprolifrative glomerulonephritis, postinfectious glomerulonephritis, and membranoprolifrative glomerulonephritis. Lobular pattern of glomeruli should remind membranoprolifrative glomerulonephritis with nodular

Diffuse sclerosis is seen in end stages of glomerular, vascular or tuboluinterstitial diseases. It should be noted that many diseases may have no changes in morphology at the beginning of their evolution while other diseases such as lupus or IgA nephropathies may result in any

Alteration of glomerular basement membrane is summarized here (82,83,84):

expansion, fibrillary glomerulonephritis, and immunotactoid glomerulopathy.

On the other hand glomerulus lesions are categorized like the following (76,80)

should be remembered.

or infiltrate of leukocytes.

diagnoses.

of the changes.

**Sclerosis:**  - usual: - collapsing: - tip lesion of FSGS: - secondary:

**Crescent:**

**Proliferation:**

 cellular: fibrocellular: fibrous:

light chain deposition disease

idiopathic nodular sclerosis

membranoprolifrative glomerulonephritis

amyloidosis

15-40 years:

>40 years:

*Nephrotic syndrome*: is associated with proteinuria and lipiduria and its differential diagnoses according to age include:

<15 years:

15-40 years:

>40 years:

In each pattern, the injuries should be written and overall the proper diagnosis should be made. The optimum approach to pathologic diagnosis of a glomerular disease is based on the presence of features indicative of specific disease and the absence of the features indicative of other disorders (10).

One of the first steps in evaluation of glomeruli is the distinction between the primary and the secondary lesions. The systemic diseases including systemic lupus erythematous, systemic vasculitis (e.g.Wegner granulomatousis), microscopic polyangitis, Henoch Schoenlein purpura, cryoglobulinemic vasculitis, diabetes mellitus, amyloidosis, monoclonal immunoglobulin deposition disease, hypertension, hepatitis B infection and etc. affect the kidney as well as the other organs.

In light microscopy evaluation, each part of glomerulus should be noted for normal and abnormal cellularity and extracellular materials.

*Nephrotic syndrome*: is associated with proteinuria and lipiduria and its differential diagnoses

In each pattern, the injuries should be written and overall the proper diagnosis should be made. The optimum approach to pathologic diagnosis of a glomerular disease is based on the presence of features indicative of specific disease and the absence of the features

One of the first steps in evaluation of glomeruli is the distinction between the primary and the secondary lesions. The systemic diseases including systemic lupus erythematous, systemic vasculitis (e.g.Wegner granulomatousis), microscopic polyangitis, Henoch Schoenlein purpura, cryoglobulinemic vasculitis, diabetes mellitus, amyloidosis, monoclonal immunoglobulin deposition disease, hypertension, hepatitis B infection and etc.

In light microscopy evaluation, each part of glomerulus should be noted for normal and

15-40 years:

>40 years:

<15 years:

15-40 years:

>40 years:

vasculitis

according to age include:

preeclampsia

 postinfectious glomerulonephritis systemic lupus erythematous

fibrillary glomerulonephritis

fibrillary glomerulonephritis

minimal change disease

 minimal change disease membranous nephropathy diabetic nephropathy

rapid progressive glomerulonephritis

rapid progressive glomerulonephritis

postinfectious glomerulonephritis

 focal segmental glomerulosclerosis mesangioprolifrative glomerulonephritis

focal segmental glomerulosclerosis

post infectious glomerulonephritis

focal segmental glomerulosclerosis

 membranous nephropathy diabetic nephropathy minimal change disease IgA nephropathy amyloidosis

 light chain deposition disease benign nephrosclerosis

indicative of other disorders (10).

postinfectious glomerulonephritis

affect the kidney as well as the other organs.

abnormal cellularity and extracellular materials.

membranoprolifrative glomerulonephritis

In normal morphology of glomeruli, the diagnoses are minimal change disease and thin basement membrane nephropathy and electron microscopy and immunofluorescence would be helpful. Early stages of many glomerulopathies show normal features in light microscopy. If there are capillaries with thick walls, diseases such as membranous glomerulopathy, diabetic glomerulosclerosis, amyloidosis, immunoglobulin deposit disease, should be remembered.

Alteration of glomerular basement membrane is summarized here (82,83,84):

Where limited sclerosis of glomeruli is observed, focal segmental glomerulosclerosis, Alport's syndrome, and sclerotic phase of different glomerulopathies are differential diagnoses.

Hypercellularity may result from increase in mesangial, visceral epithelial, endothelial cells or infiltrate of leukocytes.

Mesangial hypercellularity is seen in mesangioprolifrative glomerulonephritis, postinfectious glomerulonephritis, and membranoprolifrative glomerulonephritis. Lobular pattern of glomeruli should remind membranoprolifrative glomerulonephritis with nodular expansion, fibrillary glomerulonephritis, and immunotactoid glomerulopathy.

Diffuse sclerosis is seen in end stages of glomerular, vascular or tuboluinterstitial diseases. It should be noted that many diseases may have no changes in morphology at the beginning of their evolution while other diseases such as lupus or IgA nephropathies may result in any of the changes.

On the other hand glomerulus lesions are categorized like the following (76,80)

#### **Sclerosis:**

#### **Crescent:**

	- cellular:
	- fibrocellular:
	- fibrous:
	- mmune etiology:
	- pauci immune:

#### **Proliferation:**

	- diabetic nephropathy
	- light chain deposition disease
	- membranoprolifrative glomerulonephritis
	- amyloidosis
	- idiopathic nodular sclerosis
	- lupus nephritis
	- IgA nephropathy

Renal Biopsy Interpretation 57

As it is emphasized before, clinical information is very important and differential diagnoses

In addition to light microscopy, immunofluorescence could be helpful in many normal morphology and also many conditions where there is thickening of membranous or expansion of mesangial. It can help to determine the location and pattern of deposits and

Nowadays the routine antibodies which are used in immunoflourescent study are including

Some diseases may be dismissed without immunofluorescence or immunohistochemistry use, including light chain associated disease, IgA nephropathy, c1q nephropathy, anti GBM

In electron microscopy, basement membrane changes such as split, existence and pattern of deposits, fibrillary changes and also mesangial deposits can be diagnosed. Some diseases are detected only by electron microscopy studies including but not limited to fibrillary/

hepatitis C

complement dysregulation

*According to decreased serum complement levels:*  - postinfectious glomerulonephritis

*According to presence of acute renal failure:*  - idiopathic minimal change disease

because of myeloma casts

IgG, IgA, IgM, kappa, lambda, c3, c4, c1q.

composition of deposits.

*According to serologic studies* (74):

chronic thrombotic microangiopathy

of diseases are possible based on those information.

monoclonal immunoglobulin deposition disease

disease, humeral (c4d) transplant rejection, and fibronectin glomerulopathy.

	- minimal change disease
	- focal segmental glomerulosclerosis
	- early diabetic nephropathy
	- membranoprolifrative glomerulonephritis
	- prolifrative lupus nephritis
	- cryoglobulinemia glomerulonephritis
	- postinfectious glomerulonephritis
	- fibrillary glomerulonephritis
	- immunotactoid glomerulopathy
	- dense deposit disease

#### **Unusual lesions:**

It should be remembered that each specific histological pattern in light microscopy could be seen in different diseases (10,74,77,80,81,83,84):

*No abnormality:*

	- minimal change disease
	- thin basement membrane nephropathy

*Thick capillary walls only*

*Thick walls with mesangial expansion without hypercellularity:*

*Focal segmental glomerulosclerosis:*

It should be remembered that each specific histological pattern in light microscopy could be

chronic infection related glomerulonephritis

membranoprolifrative glomerulonephritis

 cryoglobulinemia glomerulonephritis postinfectious glomerulonephritis fibrillary glomerulonephritis immunotactoid glomerulopathy

focal segmental glomerulosclerosis

 early diabetic nephropathy - mesangial and endocapillary

prolifrative lupus nephritis

dense deposit disease

seen in different diseases (10,74,77,80,81,83,84):

 thin basement membrane nephropathy - early lesion of glomerulus (near in all diseases)

*Thick walls with mesangial expansion without hypercellularity:*

**Unusual lesions:**  - foamy podocytes:

*No abnormality:*

*Thick capillary walls only*

*Membranous injury:*

	- systemic lupus erythematous
	- infectious endocarditis
	- hepatitis C
	- complement dysregulation
	- chronic thrombotic microangiopathy
	- monoclonal immunoglobulin deposition disease

As it is emphasized before, clinical information is very important and differential diagnoses of diseases are possible based on those information.

*According to serologic studies* (74):

*According to decreased serum complement levels:*

*According to presence of acute renal failure:*

In addition to light microscopy, immunofluorescence could be helpful in many normal morphology and also many conditions where there is thickening of membranous or expansion of mesangial. It can help to determine the location and pattern of deposits and composition of deposits.

Nowadays the routine antibodies which are used in immunoflourescent study are including IgG, IgA, IgM, kappa, lambda, c3, c4, c1q.

Some diseases may be dismissed without immunofluorescence or immunohistochemistry use, including light chain associated disease, IgA nephropathy, c1q nephropathy, anti GBM disease, humeral (c4d) transplant rejection, and fibronectin glomerulopathy.

In electron microscopy, basement membrane changes such as split, existence and pattern of deposits, fibrillary changes and also mesangial deposits can be diagnosed. Some diseases are detected only by electron microscopy studies including but not limited to fibrillary/

Renal Biopsy Interpretation 59

*Tubular basement membrane changes* (in tubular atrophy, hereditary nephritis, diabetic

As it was mentioned in previous chapter, interstitium occupies small portion of kidney tissue and is slightly larger in medullary region. This part of the kidney tissue becomes affected primarily or secondarily in renal diseases. In many lesions of glomeruli, tubular or

Interstitium is the main site that demonstrates pathologic findings in drug allergic reactions

Two main lesions are acute and chronic interstitium nephritis with the former showing reversible infiltration of inflammatory cells accompanied with edema , and the latter

The lesions of interstitium that can be observed in light microscopy are listed here (5 with

nephropathy, monoclonal immunoglobulin deposit, dense deposit disease)

vascular, interstitium also gets involved and shows pathologic features.

showing irreversible fibrosis and atrophy of other compartments. Acute phase can heal or result in chronic phase and scarring.

Expansion and edema (due to increased permeability of vessels)

**16. Interstitium lesions**

and pyelonephritis (10).

Foam cells

Hemorrhagia - acute rejection - vasculitis

modification). No pathologic changes

drug induced)

Expansion with leukocyte infiltration

immunotactoid glomerulopathy, amyloidosis, cryoglobulinemia, monoclonal immunoglobulin deposition disease, collagenofibrotic glomerulopathy, fibrinogen glomerulopathy, Alport's syndrome, dense deposit disease, thin glomerular basement membrane nephropathy, lipoprotein glomerulopathy, and nail-patella syndrome. Electron microscopy can differentiate between these diseases due to specific texture of deposits (10).

#### **15. Tubular lesions**

Kidney tubules in cortex and medulla have different architecture and also different cell types. It is worth noting that cortical tubules have few interstitial tissues, compared to medullary region.

Many changes could be seen in different renal diseases. these alterations are categorized based on different point of views. Some of these are presented below.

First tubular changes introduced (10, 85):

*Acute tubular cell injury*

*Tubular casts* (principal histologic feature of light chain disease, myoglubolnuria, hemoglubolinuria, oxalate nephropathy, urate nephropathy, nephrocalcinosis and drug induced tubular lesions)

*Tubulitis* (infiltration of inflammatory cells indicative of active tubulointerstitial inflammation or nephritis or allograft rejection)

immunotactoid glomerulopathy, amyloidosis, cryoglobulinemia, monoclonal immunoglobulin deposition disease, collagenofibrotic glomerulopathy, fibrinogen glomerulopathy, Alport's syndrome, dense deposit disease, thin glomerular basement membrane nephropathy, lipoprotein glomerulopathy, and nail-patella syndrome. Electron microscopy can differentiate between these diseases due to specific texture of deposits (10).

Kidney tubules in cortex and medulla have different architecture and also different cell types. It is worth noting that cortical tubules have few interstitial tissues, compared to

Many changes could be seen in different renal diseases. these alterations are categorized

 fatty change (cytoplasm with fine small vacuoles in base of epithelial cells in severe proteinuria or nephrotic syndrome with hyperlipidemia, Reyes` syndrome, poisoning with phosphorus or carbon tetrachloride. In non nephrotic syndrome, it is in favor of

hypokalemic nephropathy (coarse and irregular varied size vacuoles due to chronic loss

hydropic change ( in conditions with assumption of sucrose, manitol, dextrane, radio

*Tubular casts* (principal histologic feature of light chain disease, myoglubolnuria, hemoglubolinuria, oxalate nephropathy, urate nephropathy, nephrocalcinosis and drug

large hyaline fractured: light chain casts (often accompanied by giant cells and

coarse granular acidophilic or red brick: myoglobulin or hemoglobulin *Tubular atrophy*

*Tubulitis* (infiltration of inflammatory cells indicative of active tubulointerstitial

(simplified epithelial cells with thickening of basement membrane)

based on different point of views. Some of these are presented below.

because of altered permeability and absorption of proteins.)

intraluminal different casts, especially cellular ones)

of potassium like laxative abuse, rectosigmoid polyps)

polyoma virus and adenovirus and lead poisoning)

opaque materials, IVIG and cyclosporine A)

**15. Tubular lesions**

*Acute tubular cell injury*

Alport's syndrome)

induced tubular lesions)

neutrophils)

RBC: glomerular bleeding

 hyaline: renal failure or low urine output WBC: tubulointerstitial inflammation

epithelial cell or granular: acute tubular injury

inflammation or nephritis or allograft rejection)

foam cells

First tubular changes introduced (10, 85):

medullary region.

*Tubular basement membrane changes* (in tubular atrophy, hereditary nephritis, diabetic nephropathy, monoclonal immunoglobulin deposit, dense deposit disease)

#### **16. Interstitium lesions**

As it was mentioned in previous chapter, interstitium occupies small portion of kidney tissue and is slightly larger in medullary region. This part of the kidney tissue becomes affected primarily or secondarily in renal diseases. In many lesions of glomeruli, tubular or vascular, interstitium also gets involved and shows pathologic features.

Interstitium is the main site that demonstrates pathologic findings in drug allergic reactions and pyelonephritis (10).

Two main lesions are acute and chronic interstitium nephritis with the former showing reversible infiltration of inflammatory cells accompanied with edema , and the latter showing irreversible fibrosis and atrophy of other compartments.

Acute phase can heal or result in chronic phase and scarring.

The lesions of interstitium that can be observed in light microscopy are listed here (5 with modification). No pathologic changes

Expansion and edema (due to increased permeability of vessels)

Expansion with leukocyte infiltration

Foam cells

Hemorrhagia

Expansion with neoplastic cells

Renal Biopsy Interpretation 61

[2] Stephen S. Sternberg . Histology for pathologists , second edition. Lippincott-Raven

[3.] Suramo I, Paivansalo M, Myllyla V. Cranio-caudal movements of the liver, pancreas and

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[5] Kaisiske BL, Umen *AI.* The influence of age, sex, race, and body habitus on kidney

[7] Graves FT. The anatomy of the intrarenal arteries and its application to segmental

[8] Agnes B. Fogo MD , Michael Kashgarian ,Diagnostic Atlas of Renal Pathology: A

[9] Oliver 1. *Nephrons and kidneys: a quantitative study of development and evolutionary*

[10] Jennette, J. Charles; Olson, Jean L.; Schwartz, Melvin M.; Silva, Fred G. Hepinstall's Pathology of the Kidney, 6th Edition, Lippincott Williams & Wilkins,2007;1-72.

[12] Clapp WM, Abrahamson DR. Development and gross anatomy of the kidney. In: Tisher

[13] Manalich R, Reyes L, Herrera M, et al. Relationship between weight at birth and the

[14] Neugarten J, Kasiske B, Silbiger SR, et al. Effects of sex on renal structure. Nephron

[15] Hughson M, Farris AB 3rd, Douglas-Denton R, et al. Glomerular number and size in autopsy kidneys: The relationship to birth weight. Kidney Int 2003;63:2113.

[17] Bowman W. On the structure and use of the Malpighian bodies of the kidney, with

[18] Fine LG. William Bowman's description of the glomerulus. *Am J NephroI1985;5:437-440*. [19] Rouiller C. General anatomy and histology of the kidney. In: Rouiller C, Muller AF, eds.

[20] Mbassa G, Elger M, Kriz W. The ultrastructural organization of the basement membrane of Bowman's capsule in the rat renal corpuscle. Cell Tissue Res 1988;253:151. [21] Dunnill MS, Halley W. Some observations on the quantitative anatomy of the kidney. *J*

[22] Newbold KM, Howie *AI,* Koram A, Adu A, MichaelJ. Assessment of glomerular size in

[23] Horvat R, Hovoka A, Dekan G, Poczewski H, Kerjaschki D. Endothelial cell membranes

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CC, Brenner BM, eds. *Renal pathology.* 2nd ed. Philadelphia: JB Lippincott; 1994:3-

number and size of renal glomeruli in humans: A histomorphometric study.

observations on the circulation through that gland. *Philos Trans R Soc Land*

The Kidney: Morphology, Biochemistry and Physiology. New York: Lippincott,

renal biopsies including minimal change nephropa, thy and single kidneys. *J*

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[6] Hodson CJ. Physiological changes in size of the kidneys. Clin Radiol 1961;12:91.

*mammalian renal architectonics.* New York: Harper & Row; 1968.

[11] Hodson CJ. The lobar structure of the kidney. *Br J Urol 1972;44:* 246-261.

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*Med 1937;23:493-500.*

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*PathoI1973;110:113-121.*

*PathoI1990;160:255-258*.

*Cell Bioi 1986;102:* 484-491.

1989:67.

59.

```
Crystals
```

It should be noted that in every pathologic changes in the interstitium, other parts should be examined carefully, as usually there are changes of overall kidney tissue.

#### **17. Vascular lesions**

Kidney vessels are susceptible to many damages. Many immune complexes reach the kidney through blood circulation and are filtered in glomeruli. Receiving about 20% of the cardiac output, the kidneys are constantly exposed to the damaging elements which circulate in blood.

The main injuries of vascular elements are listed here (5 with modification).

Vasculitis

Deposition of materials

Hypertension induced injuries

Endothelitis

Thrombus

#### **18. References**

[1] Cotran R S,Komar V, robbins and cotran pathologic basis of disease.7th edit,saunders Co,2010;905-69.

It should be noted that in every pathologic changes in the interstitium, other parts should be

Kidney vessels are susceptible to many damages. Many immune complexes reach the kidney through blood circulation and are filtered in glomeruli. Receiving about 20% of the cardiac output, the kidneys are constantly exposed to the damaging elements which

examined carefully, as usually there are changes of overall kidney tissue.

The main injuries of vascular elements are listed here (5 with modification).

addition to integration of all clinical, laboratory and pathologic data. (10)

[1] Cotran R S,Komar V, robbins and cotran pathologic basis of disease.7th edit,saunders

**17. Vascular lesions**

Hypertension induced injuries - hypertrophy of media - intimal thickening - fibrinoid necrosis

circulate in blood.

Deposition of materials - amyloidosis - immune complexes - arteriosclerosis

Vasculitis

Endothelitis - drug induced - toxins Thrombus

Emboli

**18. References**

Co,2010;905-69.

Crystals

Renal Biopsy Interpretation 63

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**5**

Şafak Güçer

*Turkey*

**Diagnostic Algorithms in Renal Biopsy**

**Interpretation Along with Case Samples**

*Hacettepe University, Faculty of Medicine Pediatric Pathology Unit Ankara*

Despite significant advances in molecular analytical techniques impacting the diagnostic approach to the renal biopsy a systematic study of morphological findings is still a magic tool to provide valuable diagnostic and prognostic information in patients with renal

Kidney has three main histological compartments including glomerular, vascular and tubulointerstitial. First, an approach should be performed to localize in which department the main injury occurs and to recognize the severity and extent of the renal injury in each department. Then, the type of injury is to be assessed such as active versus chronic. Active lesions includes cellular proliferation, crescents, edema, necrosis and acute inflammation while chronic lesions represent fibrosing conditions such as fibrous crescents, interstitial fibrosis, glomerulosclerosis, tubular atrophy or vascular sclerosis. (Walker, 2004, 2009;

The first step is to determine whether there is hypercellularity or not. No or minimal hypercellularity offers usually a limited differential diagnosis. Then, in such cases further investigations with immunofluorescence (IF) and electron microscopy(EM) are needed along with a detailed clinical history. If glomerular capillaries are abnormal three main categories are defined; glomerular capillary wall thickening, sclerosis/capillary wall

If the patient has nephrotic syndrome three main diagnosis are considered ; minimal change disease, membranous GN(early stage ) and also amyloidosis in which amyloid deposits can be very subtle to detect on light microscopy. However, these disorders can be easily

Minimal change diseases(MCD) is the most common cause of idiopathic nephrotic syndrome and characterized by absence of pathologic changes by light microscopy but

differentiated by immunofluorescence and ultrastructural microscopic findings.

**1. Introduction**

Kretzler, 2002; Fogo, 2003)

**2.1.1 Glomerular lesions**

**2.1 The native kidney biopsy lesions**

**<IP_ADDRESS> No or minimal hypercellularity (Figure 1.)**

collapse and luminal occlusion(Furness,2000;Lajoie&Silva,1996).

disease in the light of history and laboratory findings.

**2. Approach to renal biopsy interpretation with algorithms**

### **Diagnostic Algorithms in Renal Biopsy Interpretation Along with Case Samples**

Şafak Güçer

*Hacettepe University, Faculty of Medicine Pediatric Pathology Unit Ankara Turkey*

#### **1. Introduction**

64 Topics in Renal Biopsy and Pathology

[66] Wang H.J, Kjellstrand1 C.M, Cockfield S.M, and Solez K.On the influence of sample size

[67] Bohle A, Wehrmann M, Bogenschutz O, et al. The long-term prognosis of the primary

[68] Kim KH, Kim Y, Gubler MC, et al. Structural-functional relationships in Alport

[69] Mauer SM. Structural-functional correlations of diabetic nephropathy. Kidney Int

[70] Pesce C. Glomerular Number and Size: Facts and Artefacts. The anatom record 1998;

[71] Lajoie G, Silva FG. Approach to the interpretation of renal biopsy. In: Silva FG, D'Agati

[72] Chapman JR. Longitudinal analysis of chronic allograft nephropathy: Clinicopathologic

[73] Coppo R, D'Amico G. Factors predicting progression of IgA nephropathies. J Nephrol

[75] Jennette JC, Falk RJ. Nephritic syndrome and glomerulonephritis. In: Silva FG, D'Agati

[77] Couser W.G. Rapidly progressive glomerulonephritis: classification, pathogenetic,

[78] Lai K.N, Li P.K, Lui S.F, Au T.C, Tam J.S, Tong K.L, Lai F.M. Membranous nephropathy related to hepatitis B virus in adults. N Engl J Med 1991;324(21):1457-63. [79] Neugarten J, Baldwin D.S. Glomerulonephritis in bacterial endocarditis. Am J Med

[80] Haas M. Histologic subclassification of IgA nephropathy: a clinicopathologic study of

[81] Cameron J.S. Focal segmental glomerulosclerosis in adults. Nephrol Dial Transplant

[82] Jahanzad I,Amoueian S,Attaranzadeh .Familial Lecithin cholestrol Acetyl transfrase

[83] Jahanzad I,Amoueian S,Attaranzadeh A,Alport's syndrome:Ultrastructural study of 26 suspected cases.Iranian journal of pathology.2008;vol 2,No 2.p178-180 . [84] Jennette J.C. An algorithmic approach to renal biopsy interpretation of glomerular

[85] D'Agati VD, Jennette JC, Silva FG. Diseases of the renal tubules. In: D'Agati VD,

deficiency:a case report.Archives of Iranian medical journal.2009.vol,No2, P179-81.

disease. First International Renal Pathology Conference ,nephropathology workshop of European Society of Pathology.Native Kidney Pathology - Glomerular

Jennette JC, Silva FG, eds. Non-neoplastic Kidney Disease. Washington: American

Interpretation, 1st ed. New York: Churchill Livingstone, 1996: 63.

[74] Rose B.D. Differential diagnosis of glomerular disease. Uptodate.com

[76] Fogo A.B. Approach to Renal Biopsy. Am J of Kidney Dis 2003;42: 826-836.

mechanisms, and therapy. Am J Kidney Dis 1998;11(6):449-464.

VD, Nadasdy T, eds. Renal Biopsy Interpretation. Part II. Manual of Renal Biopsy

VD, Nadasty T, eds. Renal Biopsy Interpretation. New York: Churchill Livingstone,

Dial Transplant 1998; 13: 165–172.

syndrome. J Am Soc Nephrol 1995;5:1659.

correlations. Kidney Int Suppl 2005: S108.

244 cases. Am J Kidney Dis 1997;29(6):829-42.

diseases last updated at 2010-09-19 20:26:56

Registry of Pathology, 2005: 517

Res Pract 1992;188:908.

1994;45:612.

251:66–71.

2005;18:503.

1996: 71.

1984;77(2):297-304.

2003;18[suppl 6]:vi45-vi51.

on the prognostic accuracy and reproducibility of renal transplant biopsy. Nephrol

glomerulonephritides: A morphological and clinical analysis of 1747 cases. Pathol

Despite significant advances in molecular analytical techniques impacting the diagnostic approach to the renal biopsy a systematic study of morphological findings is still a magic tool to provide valuable diagnostic and prognostic information in patients with renal disease in the light of history and laboratory findings.

#### **2. Approach to renal biopsy interpretation with algorithms**

Kidney has three main histological compartments including glomerular, vascular and tubulointerstitial. First, an approach should be performed to localize in which department the main injury occurs and to recognize the severity and extent of the renal injury in each department. Then, the type of injury is to be assessed such as active versus chronic. Active lesions includes cellular proliferation, crescents, edema, necrosis and acute inflammation while chronic lesions represent fibrosing conditions such as fibrous crescents, interstitial fibrosis, glomerulosclerosis, tubular atrophy or vascular sclerosis. (Walker, 2004, 2009; Kretzler, 2002; Fogo, 2003)

#### **2.1 The native kidney biopsy lesions 2.1.1 Glomerular lesions**

#### **<IP_ADDRESS> No or minimal hypercellularity (Figure 1.)**

The first step is to determine whether there is hypercellularity or not. No or minimal hypercellularity offers usually a limited differential diagnosis. Then, in such cases further investigations with immunofluorescence (IF) and electron microscopy(EM) are needed along with a detailed clinical history. If glomerular capillaries are abnormal three main categories are defined; glomerular capillary wall thickening, sclerosis/capillary wall collapse and luminal occlusion(Furness,2000;Lajoie&Silva,1996).

If the patient has nephrotic syndrome three main diagnosis are considered ; minimal change disease, membranous GN(early stage ) and also amyloidosis in which amyloid deposits can be very subtle to detect on light microscopy. However, these disorders can be easily differentiated by immunofluorescence and ultrastructural microscopic findings.

Minimal change diseases(MCD) is the most common cause of idiopathic nephrotic syndrome and characterized by absence of pathologic changes by light microscopy but

Diagnostic Algorithms in Renal Biopsy Interpretation Along with Case Samples 67

Urinalysis: Density 1025, Proteinuria 80 mg/m2/hr, no glucose, sediment: 3-4 white blood

Fig. 2. Minimal change disease. Note the patent glomerular capillaries with normal amount

In cases with hematuria and glomerular basement membrane(GBM) thinning one should consider first Alport syndrome (AS) or thin basement membrane nephropathy(TBMN). Diagnosis of thinning should be made by comparison of age-matched controls. A cut-off value has been reported 250 nm in adults in most series. However, care must be taken to diagnose TBMN in children since GBM thickening increases with age(Kashtan, 2009). A thinned GBM may be an early lesion in Alport syndrome. Children with Alport syndrome may show only diffuse GBM attenuation, making differentiation from TBMN a

However, in full-blown cases with AS there is also characteristic thickening of the glomerular basement membrane with splitting of the lamina densa, electron lucent areas and electron dense particles detected by EM. (Figs. 4,5)A progressive, high-frequency sensorineural deafness frequently detectable by audiometry in later childhood in boys with X-linked AS and both boys and girls with autosomal recessive AS and anterior lenticonus or perimacular retinal flecks are other characteristics of AS (Savige, 2003;

BUN 10 mg/dl,Cr:0.3 m g/dl, Total protein/Albumin: 4.9/2 g/dl

Renal ultrasonography revealed unremarkable findings. **Renal biopsy diagnosis**: Minimal change disease (Figs. 2,3)

of matrix and mesangial cells.(HEx100)

challenge.

Haas, 2009).

Serum C3 ,C4 levels, ANA, Anti-DNA, viral markers were all normal.

cells.

diffuse effacement of foot processes of podocytes by EM. (Figs. 2,3) It is usually a primary disease but it may be secondary to malignant diseases and nonsteroidal anti-inflammatory drugs(Mubarak,2011,Zhang,2011). Proteinuria is generally massive, selective and mostly of abrupt onset.

Fig. 1. An algorithm for interpretation of glomerular morphological changes with no or minimal hypercellularity.(GN:Glomerulonephritis, FSGS; Focal segmental glomerulosclerosis, NPHS2;(Podocin), CD2AP; CD-associated protein, WT1; Wilms' tumor1, LAMß2;Laminin ß2, DIC;disseminated intravascular coagulation)

#### **CASE 1.**

A 5 year-old-male was admitted to the hospital with the complaints of swelling of eyelids, legs and fatigue lasting for 2 months.

No family history for kidney disease or consanguinity between parents.

On physical exam: Blood pressure:100/60 mmHg , pretibial 4(+) pitting edema was noted. Laboratory findings: Complete blood count and electrolytes were normal , ESR 33 mm/hr

BUN 10 mg/dl,Cr:0.3 m g/dl, Total protein/Albumin: 4.9/2 g/dl

Urinalysis: Density 1025, Proteinuria 80 mg/m2/hr, no glucose, sediment: 3-4 white blood cells.

Serum C3 ,C4 levels, ANA, Anti-DNA, viral markers were all normal.

Renal ultrasonography revealed unremarkable findings.

66 Topics in Renal Biopsy and Pathology

diffuse effacement of foot processes of podocytes by EM. (Figs. 2,3) It is usually a primary disease but it may be secondary to malignant diseases and nonsteroidal anti-inflammatory drugs(Mubarak,2011,Zhang,2011). Proteinuria is generally massive, selective and mostly of

Fig. 1. An algorithm for interpretation of glomerular morphological changes with no or

glomerulosclerosis, NPHS2;(Podocin), CD2AP; CD-associated protein, WT1; Wilms' tumor1,

A 5 year-old-male was admitted to the hospital with the complaints of swelling of eyelids,

On physical exam: Blood pressure:100/60 mmHg , pretibial 4(+) pitting edema was noted. Laboratory findings: Complete blood count and electrolytes were normal , ESR 33 mm/hr

minimal hypercellularity.(GN:Glomerulonephritis, FSGS; Focal segmental

No family history for kidney disease or consanguinity between parents.

LAMß2;Laminin ß2, DIC;disseminated intravascular coagulation)

abrupt onset.

**CASE 1.**

legs and fatigue lasting for 2 months.

**Renal biopsy diagnosis**: Minimal change disease (Figs. 2,3)

Fig. 2. Minimal change disease. Note the patent glomerular capillaries with normal amount of matrix and mesangial cells.(HEx100)

In cases with hematuria and glomerular basement membrane(GBM) thinning one should consider first Alport syndrome (AS) or thin basement membrane nephropathy(TBMN). Diagnosis of thinning should be made by comparison of age-matched controls. A cut-off value has been reported 250 nm in adults in most series. However, care must be taken to diagnose TBMN in children since GBM thickening increases with age(Kashtan, 2009).

A thinned GBM may be an early lesion in Alport syndrome. Children with Alport syndrome may show only diffuse GBM attenuation, making differentiation from TBMN a challenge.

However, in full-blown cases with AS there is also characteristic thickening of the glomerular basement membrane with splitting of the lamina densa, electron lucent areas and electron dense particles detected by EM. (Figs. 4,5)A progressive, high-frequency sensorineural deafness frequently detectable by audiometry in later childhood in boys with X-linked AS and both boys and girls with autosomal recessive AS and anterior lenticonus or perimacular retinal flecks are other characteristics of AS (Savige, 2003; Haas, 2009).

Diagnostic Algorithms in Renal Biopsy Interpretation Along with Case Samples 69

Fig. 4. Alport syndrome. Note mesangial matrix increment, adhesion to Bowman capsule,

Fig. 5. Alport syndrome. Electron micrograph demonstrating thickening, irregular contours of glomerular basement membrane, splitting lamina densa and several electron dense

granules(arrows). Uranyl acetate-Lead citrate x 30000.

interstitial foam cells (arrow) and fibrosis in the interstitium. Trichromex200.

Thinning of lamina densa of GBM can be seen in normal children, IgA nephropathy, minimal change disease and in some forms of lupus nephritis. However, thinning in these disorders is mostly segmental whereas there is a diffuse involvement in patients with TBMN.

Fig. 3. Minimal change disease. Electron micrograph shows diffuse effacement of foot processes(FP).(Uranyl acetate and lead citratex10000)

A thickened GBM can be detected as a major feature in several glomerular diseases including membranous GN, membranoproliferative(mesangiocapillary) GN , fibrillary GN and diabetic nephropathy, thrombotic microangiopathy, Alport syndrome or transplant glomerulopathy. Aforementioned first three disorders have typical immunofluorescence microscopic findings while IF is expected to be negative in the others.

#### **CASE 2.**

A 7 year-old-male was admitted to the hospital with the complaints of dysuria and hearing problems. He had been suffering these symptoms for two years.

No consanguinity between parents but his grandmother died of chronic renal failure of unknown etiology.

Physical examination was unremarkable except a sensorineural hearing loss. Blood pressure:100/60 mmHg

Laboratory findings: Complete blood count and electrolytes were normal.

BUN 17.3 mg/dl,Cr:0.64 mg/dl, Total protein/Albumin: 6.9/3.6 g/dl

Urinalysis: Density 1025, Proteinuria 10 mg/m2/hr, no glucose, sediment: abundant red blood cells.

Serum C3 ,C4 levels, ANA, Anti-DNA,viral markers were all normal.

Renal USG revealed normal size and echogenicity in kidneys.

**Renal biopsy diagnosis:** Alport syndrome (Figures 4,5)

Thinning of lamina densa of GBM can be seen in normal children, IgA nephropathy, minimal change disease and in some forms of lupus nephritis. However, thinning in these disorders is mostly segmental whereas there is a diffuse involvement in patients with

**FP**

Fig. 3. Minimal change disease. Electron micrograph shows diffuse effacement of foot

A thickened GBM can be detected as a major feature in several glomerular diseases including membranous GN, membranoproliferative(mesangiocapillary) GN , fibrillary GN and diabetic nephropathy, thrombotic microangiopathy, Alport syndrome or transplant glomerulopathy. Aforementioned first three disorders have typical immunofluorescence

A 7 year-old-male was admitted to the hospital with the complaints of dysuria and hearing

No consanguinity between parents but his grandmother died of chronic renal failure of

Physical examination was unremarkable except a sensorineural hearing loss. Blood

Urinalysis: Density 1025, Proteinuria 10 mg/m2/hr, no glucose, sediment: abundant red

processes(FP).(Uranyl acetate and lead citratex10000)

microscopic findings while IF is expected to be negative in the others.

Laboratory findings: Complete blood count and electrolytes were normal. BUN 17.3 mg/dl,Cr:0.64 mg/dl, Total protein/Albumin: 6.9/3.6 g/dl

Serum C3 ,C4 levels, ANA, Anti-DNA,viral markers were all normal.

Renal USG revealed normal size and echogenicity in kidneys. **Renal biopsy diagnosis:** Alport syndrome (Figures 4,5)

problems. He had been suffering these symptoms for two years.

TBMN.

**CASE 2.**

unknown etiology.

blood cells.

pressure:100/60 mmHg

Fig. 4. Alport syndrome. Note mesangial matrix increment, adhesion to Bowman capsule, interstitial foam cells (arrow) and fibrosis in the interstitium. Trichromex200.

Fig. 5. Alport syndrome. Electron micrograph demonstrating thickening, irregular contours of glomerular basement membrane, splitting lamina densa and several electron dense granules(arrows). Uranyl acetate-Lead citrate x 30000.

 Focal segmental glomerulosclerosis in 2 glomeruli and global sclerosis and Bowman

A a 9 year-old girl admitted to the hospital because of hematuria and proteinuria lasting for

She was initially diagnosed as mitochondrial disease by the findings of ptosis, ophthalmoplegia, failure to thrive, high serum lactate and pyruvate levels, ragged red fibers

No family history for kidney disease or consanguinity between parents.

Serum C3 ,C4 levels, ANA, Anti-DNA, viral markers were all normal. Renal USG revealed renal parenchymal disease with increased echogenity. **Renal biopsy diagnosis:** Focal segmental glomerulosclerosis (Figs. 6,7)

mg/dl,Cr:0.6 mg/dl, Total protein/Albumin: 3.7/1.2 g/dl

cells, 4-5 red blood cells and a few fine granular casts.

capsule adhesions in the other. (PAS x200)

in muscle biopsy and the common 4.9 kb deletion in mtDNA.

**CASE 4.**

two months.

**CASE 3.**

3 months.

pitting edema.

In some cases, one may encounter unusual or rare lesions such as foamy podocytes and/or tubules with various storage material ,foam cells within the glomerular tuft or heavily deposited collagen Type III material detected by EM. These storage materials can be diagnostic clues of specific metabolic or hereditary diseases. Fabry disease is the most common which is characterized by foamy podocytes with myelin body type inclusions by EM in podocytes, corneal epithelium, endothelial cells, etc. (Fogo,2003).

If glomerular capillary collapse or sclerosis is the main lesion it can be global or focal. The former is usually the consequence of ischemia or a lesion of the end stage kidney. In fact, glomerulosclerosis is an end result of glomerular injury irrespective of cause.

Focal segmental glomerulosclerosis is a clinicopathological entity which is histologically characterized by segmental glomerulosclerosis in some glomeruli, or tuft collapse, segmental hyalinosis, mostly negative or IgM staining on immunofluorescence and effacement of foot processes on electron microscopy. (Figs. 6,7)

Fig. 6. Focal segmental glomerulosclerosis. The sclerotic lesion involves three glomeruli in a segmental pattern. There is also tubular atrophy with mononuclear inflammatory cell infiltration in the interstitium. (HEx100)

It may be etiologically classified as primary, hereditary disease associated or secondary to infections, drugs/toxins, hyperfiltration, ischemia and as a renal involvement of mitochondrial disease (Thomas, 2009; Gbadegesin, 2011; Baskn, 2011; Emma, 2011; Güçer,2005). (Figs. 8,9) Data from the experimental and human studies has demonstrated that podocyte has a central role in the pathogenesis of FSGS. Identification of products of mutated genes located in the podocyte and its slit diagram has resulted in the recognition of the hereditary forms of FSGS, and NPHS2; (Podocin), CD2AP (CD-associated protein), WT1; (Wilms' tumor1), LAMß2; Laminin ß2 ACTN4 (Alfa-actinin4), TRPC6 (Transient receptor potential channel type6), PLCE1(Phospholipase Epsilon1), INF2(Inverted forming 2) and others are classified now genetic causes of FSGS and nephrotic syndrome (Gbadegesin,2011).

#### **CASE 3.**

70 Topics in Renal Biopsy and Pathology

In some cases, one may encounter unusual or rare lesions such as foamy podocytes and/or tubules with various storage material ,foam cells within the glomerular tuft or heavily deposited collagen Type III material detected by EM. These storage materials can be diagnostic clues of specific metabolic or hereditary diseases. Fabry disease is the most common which is characterized by foamy podocytes with myelin body type inclusions by

If glomerular capillary collapse or sclerosis is the main lesion it can be global or focal. The former is usually the consequence of ischemia or a lesion of the end stage kidney. In fact,

Focal segmental glomerulosclerosis is a clinicopathological entity which is histologically characterized by segmental glomerulosclerosis in some glomeruli, or tuft collapse, segmental hyalinosis, mostly negative or IgM staining on immunofluorescence and

Fig. 6. Focal segmental glomerulosclerosis. The sclerotic lesion involves three glomeruli in a segmental pattern. There is also tubular atrophy with mononuclear inflammatory cell

It may be etiologically classified as primary, hereditary disease associated or secondary to infections, drugs/toxins, hyperfiltration, ischemia and as a renal involvement of mitochondrial disease (Thomas, 2009; Gbadegesin, 2011; Baskn, 2011; Emma, 2011; Güçer,2005). (Figs. 8,9) Data from the experimental and human studies has demonstrated that podocyte has a central role in the pathogenesis of FSGS. Identification of products of mutated genes located in the podocyte and its slit diagram has resulted in the recognition of the hereditary forms of FSGS, and NPHS2; (Podocin), CD2AP (CD-associated protein), WT1; (Wilms' tumor1), LAMß2; Laminin ß2 ACTN4 (Alfa-actinin4), TRPC6 (Transient receptor potential channel type6), PLCE1(Phospholipase Epsilon1), INF2(Inverted forming 2) and others are classified now

genetic causes of FSGS and nephrotic syndrome (Gbadegesin,2011).

EM in podocytes, corneal epithelium, endothelial cells, etc. (Fogo,2003).

effacement of foot processes on electron microscopy. (Figs. 6,7)

infiltration in the interstitium. (HEx100)

glomerulosclerosis is an end result of glomerular injury irrespective of cause.

A 8 year-old-female presented with the complaints of swelling of eyelids and face lasting for 3 months.

No family history for kidney disease or consanguinity between parents.

Physical examination revealed a high blood pressure of 160/90 mmHg and pretibial 3(+) pitting edema.

Laboratory findings: Complete blood count was normal, triglyceride 368 mg/dl, BUN 43 mg/dl,Cr:0.6 mg/dl, Total protein/Albumin: 3.7/1.2 g/dl

Urinalysis: Density 1010, Proteinuria 160 mg/m2/hr, no glucose, sediment: 8-10 white blood cells, 4-5 red blood cells and a few fine granular casts.

Serum C3 ,C4 levels, ANA, Anti-DNA, viral markers were all normal.

Renal USG revealed renal parenchymal disease with increased echogenity.

**Renal biopsy diagnosis:** Focal segmental glomerulosclerosis (Figs. 6,7)

Fig. 7. Focal segmental glomerulosclerosis in 2 glomeruli and global sclerosis and Bowman capsule adhesions in the other. (PAS x200)

#### **CASE 4.**

A a 9 year-old girl admitted to the hospital because of hematuria and proteinuria lasting for two months.

She was initially diagnosed as mitochondrial disease by the findings of ptosis, ophthalmoplegia, failure to thrive, high serum lactate and pyruvate levels, ragged red fibers in muscle biopsy and the common 4.9 kb deletion in mtDNA.

Diagnostic Algorithms in Renal Biopsy Interpretation Along with Case Samples 73

Fig. 9. Ultrastructural details of mitochondria in high magnification: Note increased matrix density and cristae formation. Vesicular structures (arrows) representing the increased

A 60 year old man was admitted with edema and hypertension lasting for one month.

Physical examination revealed unremarkable findings except a high blood pressure of

Laboratory findings: Complete blood count was normal, triglyceride 200 mg/dl, BUN 53

Urinalysis: Density 1020, Proteinuria 3078 mg/ 24hrs, no glucose, sediment:, 10-15 red blood

Serum IgA, IgG, IgM , C3 ,C4 levels, ANA, Anti-DNA, viral markers, CA 125,CA-19-9, CA-

Renal amyloidosis must be included in differential diagnosis of proteinuria or nephrotic syndrome when the light microscopy gives no or minimal histopathological findings. Amyloid deposits in general appear as pink, homogenous and amorphous material in HE sections. Sometimes, the deposits may confine to the mesangium and be barely visible by light microscopy leading a misdiagnosis of minimal change disease. The diagnosis of amyloidosis is made by demonstration of apple-green birefringence under polarized light

cristae formation are evident. Uranyl acetate lead citrate x 20000.

**CASE 5.**

cells.

No family history for kidney disease.

170/100 mmHg and pretibial 2(+) pitting edema.

mg/dl, Cr 1.2 mg/dl, Total protein/Albumin 4/1.6 g/dl

15-3 Rheumatoid factor, ASO and CRP were all normal. **Renal biopsy diagnosis:** Membranous GN (Figs.10,11)

She had been followed up for five years without any signs of other organ involvement before she developed hematuria and proteinuria.

On this admission, physical examination revealed failure to thrive, ptosis, hypo- and hyperpigmented areas in both extremities.

Urinalysis showed 2+ proteinuria with normal blood biochemistry.

Urinary protein/creatinine ratio was 0.8, Hb 12 g/dl, WBC 7700/mm3 with normal complement components and β2 microglobulin levels.

**Renal biopsy diagnosis:** FSGS associated with mitochondrial disease(Figs.8,9)

Membranous glomerulonephritis(MGN) is mostly the disease of the adults and unusual in children. It can be primary or secondary to infections, drugs, systemic disease, malignant tumors etc. Patients may present with proteinuria, hypertension , hematuria or renal insufficiency. GBMs are the main site of injury and the characteristic pathologic features are thickened GBM along with a predominant IgG deposition of granular type. In early stages, GBM may show normal appearance and mild mesangial hypercellularity (Figs.10,11) (Cybulski,2011;Beck,2010).

Fig. 8. Renal biopsy of Case 4 shows segmental sclerosis (arrow) in two glomeruli. Note focal atrophy of periglomerular tubules (arrows) and interstitial edema. HE&JSilverx100.

Fig. 9. Ultrastructural details of mitochondria in high magnification: Note increased matrix density and cristae formation. Vesicular structures (arrows) representing the increased cristae formation are evident. Uranyl acetate lead citrate x 20000.

#### **CASE 5.**

72 Topics in Renal Biopsy and Pathology

She had been followed up for five years without any signs of other organ involvement

On this admission, physical examination revealed failure to thrive, ptosis, hypo- and hyper-

Urinary protein/creatinine ratio was 0.8, Hb 12 g/dl, WBC 7700/mm3 with normal

Membranous glomerulonephritis(MGN) is mostly the disease of the adults and unusual in children. It can be primary or secondary to infections, drugs, systemic disease, malignant tumors etc. Patients may present with proteinuria, hypertension , hematuria or renal insufficiency. GBMs are the main site of injury and the characteristic pathologic features are thickened GBM along with a predominant IgG deposition of granular type. In early stages, GBM may show normal appearance and mild mesangial hypercellularity (Figs.10,11)

Fig. 8. Renal biopsy of Case 4 shows segmental sclerosis (arrow) in two glomeruli. Note focal atrophy of periglomerular tubules (arrows) and interstitial edema. HE&JSilverx100.

before she developed hematuria and proteinuria.

complement components and β2 microglobulin levels.

Urinalysis showed 2+ proteinuria with normal blood biochemistry.

**Renal biopsy diagnosis:** FSGS associated with mitochondrial disease(Figs.8,9)

pigmented areas in both extremities.

(Cybulski,2011;Beck,2010).

A 60 year old man was admitted with edema and hypertension lasting for one month. No family history for kidney disease.

Physical examination revealed unremarkable findings except a high blood pressure of 170/100 mmHg and pretibial 2(+) pitting edema.

Laboratory findings: Complete blood count was normal, triglyceride 200 mg/dl, BUN 53 mg/dl, Cr 1.2 mg/dl, Total protein/Albumin 4/1.6 g/dl

Urinalysis: Density 1020, Proteinuria 3078 mg/ 24hrs, no glucose, sediment:, 10-15 red blood cells.

Serum IgA, IgG, IgM , C3 ,C4 levels, ANA, Anti-DNA, viral markers, CA 125,CA-19-9, CA-15-3 Rheumatoid factor, ASO and CRP were all normal.

**Renal biopsy diagnosis:** Membranous GN (Figs.10,11)

Renal amyloidosis must be included in differential diagnosis of proteinuria or nephrotic syndrome when the light microscopy gives no or minimal histopathological findings. Amyloid deposits in general appear as pink, homogenous and amorphous material in HE sections. Sometimes, the deposits may confine to the mesangium and be barely visible by light microscopy leading a misdiagnosis of minimal change disease. The diagnosis of amyloidosis is made by demonstration of apple-green birefringence under polarized light

Diagnostic Algorithms in Renal Biopsy Interpretation Along with Case Samples 75

in Congo-red stained sections which should be at least 6 microns in thickness.(Şen, 2010;

A 14 year-old-male was admitted to the hospital with the complaints of swelling of legs for 2

Urinalysis: Density 1010, Proteinuria 200 mg/m2/hr, no glucose, sediment: 1-2 white blood

Serum C3 ,C4 levels, ANA, Anti-dsDNA, viral markers were all normal. MEFV

**Renal biopsy diagnosis**: Renal amyloidosis and familial Mediterranean fever(FMF)

Fig. 12. Renal Amyloidosis. Diffuse nodular amyloid infiltration in a glomerulus. There is

also vascular involvement (upper right).Congo red stain x200.

No family history for kidney disease but 1st degree consanguinity between parents. On physical exam: Blood pressure:100/60 mmHg , pretibial 4(+) pitting edema was noted.

mutation:M694V homozygote. Renal USG revealed minimal hyperechogenicity.

months. He had had recurrent fever and abdominal pain for 5 years.

Laboratory findings: Complete blood count and electrolytes were normal , BUN 10 mg/dl,Cr:0.3 m g/dl, Total protein/Albumin: 3.9/1.89 g/dl

Hopfer, 2011) (Figure 12,13).

**CASE 6.**

cells.

(Figs.12,13)

Fig. 10. Membranous GN. Early stage. A slight thickening of glomerular basement membrane. Jones Silver+HE.x400.

Fig. 11. Membranous GN. Granular type IgG staining of basement membranes in a glomerulus. Immunofluorescence microscopyx400.

in Congo-red stained sections which should be at least 6 microns in thickness.(Şen, 2010; Hopfer, 2011) (Figure 12,13).

#### **CASE 6.**

74 Topics in Renal Biopsy and Pathology

Fig. 10. Membranous GN. Early stage. A slight thickening of glomerular basement

Fig. 11. Membranous GN. Granular type IgG staining of basement membranes in a

glomerulus. Immunofluorescence microscopyx400.

membrane. Jones Silver+HE.x400.

A 14 year-old-male was admitted to the hospital with the complaints of swelling of legs for 2 months. He had had recurrent fever and abdominal pain for 5 years.

No family history for kidney disease but 1st degree consanguinity between parents.

On physical exam: Blood pressure:100/60 mmHg , pretibial 4(+) pitting edema was noted.

Laboratory findings: Complete blood count and electrolytes were normal ,

BUN 10 mg/dl,Cr:0.3 m g/dl, Total protein/Albumin: 3.9/1.89 g/dl

Urinalysis: Density 1010, Proteinuria 200 mg/m2/hr, no glucose, sediment: 1-2 white blood cells.

Serum C3 ,C4 levels, ANA, Anti-dsDNA, viral markers were all normal. MEFV mutation:M694V homozygote. Renal USG revealed minimal hyperechogenicity.

**Renal biopsy diagnosis**: Renal amyloidosis and familial Mediterranean fever(FMF) (Figs.12,13)

Fig. 12. Renal Amyloidosis. Diffuse nodular amyloid infiltration in a glomerulus. There is also vascular involvement (upper right).Congo red stain x200.

Diagnostic Algorithms in Renal Biopsy Interpretation Along with Case Samples 77

If the glomerular cellularity is increased the algorithm in Fig. 2 is used. At this point one should detect whether the glomerular hypercellularity is purely mesangial , endocapillary (occluding the capillary lumina) or extracapillary. Glomerular lesions may be focal or diffuse based on the percentage of involved glomeruli (i.e focal: the lesion involving less than 50% of number of glomeruli). Mesangial and Endocapillary proliferation may be segmental or diffuse while extracapillary proliferation may affect the glomerulus partially or

The differential diagnosis of mesangial proliferation depends on mainly immunofluorescence findings. IgA nephropathy is one of glomerular diseases which can present mesangial proliferation and is diagnosed by dominant or co-dominant IgA deposition in glomeruli by immunofluorescence microscopy and electron microscopy. (Cattran, 2009; Bellu, 2011)(Figs

A 20 year-old-male was admitted to the hospital with the complaints of flank pain and recurrent bouts of hematuria. He had a history of upper respiratory tract infection two days

Fig. 15. Microphoto showing segmental endocapillary proliferation(arrow) a case with IgA

**<IP_ADDRESS> Pure mesangial, endocapillary or extracapillary proliferative lesions**

globally.(Fogo, 2003; Lajoie & Silva,1996)

prior to the urinary symptoms.

nephropathy. HEx200.

15-17) **CASE 7.**

Fig. 13. Apple green birefringence of Congo red stained deposits under polarized light confirming the amyloid. Congo redx400

Fig. 14. An algorithm for interpretation of glomerular morphological changes with glomerular hypercellularity.(GN: Glomerulonephritis, MPGN: membranoproliferative GN, APIGN; acute postinfectious GN,ANCA; anti-neutrophil cytoplasmic autoantibodies,PAN; polyarteritis nodosa)

#### **<IP_ADDRESS> Pure mesangial, endocapillary or extracapillary proliferative lesions**

If the glomerular cellularity is increased the algorithm in Fig. 2 is used. At this point one should detect whether the glomerular hypercellularity is purely mesangial , endocapillary (occluding the capillary lumina) or extracapillary. Glomerular lesions may be focal or diffuse based on the percentage of involved glomeruli (i.e focal: the lesion involving less than 50% of number of glomeruli). Mesangial and Endocapillary proliferation may be segmental or diffuse while extracapillary proliferation may affect the glomerulus partially or globally.(Fogo, 2003; Lajoie & Silva,1996)

The differential diagnosis of mesangial proliferation depends on mainly immunofluorescence findings. IgA nephropathy is one of glomerular diseases which can present mesangial proliferation and is diagnosed by dominant or co-dominant IgA deposition in glomeruli by immunofluorescence microscopy and electron microscopy. (Cattran, 2009; Bellu, 2011)(Figs 15-17)

#### **CASE 7.**

76 Topics in Renal Biopsy and Pathology

Fig. 13. Apple green birefringence of Congo red stained deposits under polarized light

Fig. 14. An algorithm for interpretation of glomerular morphological changes with

glomerular hypercellularity.(GN: Glomerulonephritis, MPGN: membranoproliferative GN, APIGN; acute postinfectious GN,ANCA; anti-neutrophil cytoplasmic autoantibodies,PAN;

confirming the amyloid. Congo redx400

polyarteritis nodosa)

A 20 year-old-male was admitted to the hospital with the complaints of flank pain and recurrent bouts of hematuria. He had a history of upper respiratory tract infection two days prior to the urinary symptoms.

Fig. 15. Microphoto showing segmental endocapillary proliferation(arrow) a case with IgA nephropathy. HEx200.

Diagnostic Algorithms in Renal Biopsy Interpretation Along with Case Samples 79

Fig. 17. IgA nephropathy. Electron microscopy shows immune deposits in the mesangial

immune mediated(lupus nephritis, acute postinfectious , anti GBM nephritis, IgA nephropathy etc.) or pauci-immune (Wegener's granulomatosis, microscopic PAN

A 17 year-old-male presented with the complaints of hematuria and swelling of whole body.

On physical exam was unremarkable except a blood pressure of 160/100 mmHg and

Laboratory findings: Complete blood count and electrolytes were normal , CA 7.8 mg/dl,P

Urinalysis: Density 1027, Proteinuria 500 mg/24hrs, no glucose, sediment: abundant

cell(arrows) uranyl acetate lead citrate x 8000.

**CASE 8.**

pretibial 1+ edema.

(Figs.15-17)

etc.)(Alchi,2010;Colucci,2011;Nasr,2008).(Figures 19-22)

erythrocytes and white blood cells with fine granular casts.

No family history for kidney disease or consanguinity between parents.

5.2 mg/dl, BUN 40 mg/dl,Cr:1.2 mg/dl, Total protein/Albumin: 4.14/2.7 g/dl

Serum C3 40 mg/dl, ,C4 12.1mg/dl, ANA, Anti-DNA, viral markers were all normal. Renal USG revealed minimal increase in echogenicity and no evidence of urinary stone. **Renal biopsy diagnosis**: Membranoproliferative glomerulonephritis(MPGN) type 1

No family history for kidney disease or consanguinity between parents.

On physical exam was unremarkable with a blood pressure of 100/60 mmHg .

Serum C3 ,C4 levels, ANA, Anti-DNA, viral markers were all normal.

Renal USG revealed minimal increase in echogenicity and no evidence of urinary stone. **Renal biopsy diagnosis**: IgA nephropathy (Figs. 15-17)

Fig. 16. IgA nephropathy. Mesangial distrubution of IgA deposits in two glomeruli. Immunofluorescence microscopy x200.

Mesangial and endocapillary proliferation can be seen together in membranoproliferative glomerulonephritis(MPGN) type 1, dense deposit disease(MPGN type 2), lupus nephritis, cryoglobulinemic GN, postinfectious GN, fibrillary GN or immunotactoid glomerulopathy. Then, immunofluorescence and electron microscopy are of vital importance for differential diagnosis. On the other hand, crescent formation not uncommonly associates to endocapillary proliferative ,necrotic or vasculitic lesions. The lesion is the consequence of capillary wall injury and proliferation of parietal epithelial cells. It can be categorized as

Fig. 17. IgA nephropathy. Electron microscopy shows immune deposits in the mesangial cell(arrows) uranyl acetate lead citrate x 8000.

immune mediated(lupus nephritis, acute postinfectious , anti GBM nephritis, IgA nephropathy etc.) or pauci-immune (Wegener's granulomatosis, microscopic PAN etc.)(Alchi,2010;Colucci,2011;Nasr,2008).(Figures 19-22)

#### **CASE 8.**

78 Topics in Renal Biopsy and Pathology

Laboratory findings: Complete blood count and electrolytes were normal , ESR 30 mm/hr

Renal USG revealed minimal increase in echogenicity and no evidence of urinary stone.

Fig. 16. IgA nephropathy. Mesangial distrubution of IgA deposits in two glomeruli.

Mesangial and endocapillary proliferation can be seen together in membranoproliferative glomerulonephritis(MPGN) type 1, dense deposit disease(MPGN type 2), lupus nephritis, cryoglobulinemic GN, postinfectious GN, fibrillary GN or immunotactoid glomerulopathy. Then, immunofluorescence and electron microscopy are of vital importance for differential diagnosis. On the other hand, crescent formation not uncommonly associates to endocapillary proliferative ,necrotic or vasculitic lesions. The lesion is the consequence of capillary wall injury and proliferation of parietal epithelial cells. It can be categorized as

Immunofluorescence microscopy x200.

Urinalysis: Density 1020, Proteinuria 30 mg/m2/hr, no glucose, sediment: abundant

No family history for kidney disease or consanguinity between parents. On physical exam was unremarkable with a blood pressure of 100/60 mmHg .

**Renal biopsy diagnosis**: IgA nephropathy (Figs. 15-17)

A 17 year-old-male presented with the complaints of hematuria and swelling of whole body. No family history for kidney disease or consanguinity between parents.

On physical exam was unremarkable except a blood pressure of 160/100 mmHg and pretibial 1+ edema.

Laboratory findings: Complete blood count and electrolytes were normal , CA 7.8 mg/dl,P 5.2 mg/dl, BUN 40 mg/dl,Cr:1.2 mg/dl, Total protein/Albumin: 4.14/2.7 g/dl

Urinalysis: Density 1027, Proteinuria 500 mg/24hrs, no glucose, sediment: abundant erythrocytes and white blood cells with fine granular casts.

Serum C3 40 mg/dl, ,C4 12.1mg/dl, ANA, Anti-DNA, viral markers were all normal.

Renal USG revealed minimal increase in echogenicity and no evidence of urinary stone.

**Renal biopsy diagnosis**: Membranoproliferative glomerulonephritis(MPGN) type 1 (Figs.15-17)

Diagnostic Algorithms in Renal Biopsy Interpretation Along with Case Samples 81

A 15 year-old-male presented with the complaints of hematuria, oliguria and swelling of his face and eyelids. He suffered a throat infection 10 days prior to admission. No family history

On physical exam was unremarkable except a blood pressure of 160/100 mmHg and

Laboratory findings: Complete blood count and electrolytes were normal , BUN 98

Urinalysis: Density 1018, Proteinuria 1200 mg/24hrs, no glucose, sediment: abundant

Renal USG revealed minimal increase in parenchymal echogenicity . Urine culture was

Serum C3 15 mg/dl, ,C4 6.1mg/dl, ANA, Anti-DNA, viral markers were all normal.

**Renal biopsy diagnosis**: Acute postinfectious glomerulonephritis(APIGN) (Figs.21-22)

Fig. 21. Acute postinfectious glomerulonephritis. Light micrograph shows endocapillary proliferation with numerous neutrophils. There are also several neutrophils in tubular

for kidney disease or consanguinity between parents.

mg/dl,Cr:9.34 mg/dl, Total protein/Albumin: 6.14/3.2 g/dl

erythrocytes and white blood cells and fine leukocytic and granular casts.

**CASE 9.**

negative.

lumina.HEx200.

pretibial 1+ edema.

Fig. 18. Membranoproliferative glomerulonephritis. Note endocapillary proliferation, lobulation and thickening of glomerular capillary walls. A mononuclear inflammatory cell infiltration with tubular atrophy in the interstitium is also seen. HEx200.

Fig. 20. Membranoproliferative type 1 GN. Granular frame-like staining of C3 the basement membrane and mesangium in 2 glomeruli. Immunofluorescence microscopyx200.

#### **CASE 9.**

80 Topics in Renal Biopsy and Pathology

Fig. 18. Membranoproliferative glomerulonephritis. Note endocapillary proliferation, lobulation and thickening of glomerular capillary walls. A mononuclear inflammatory cell

Fig. 20. Membranoproliferative type 1 GN. Granular frame-like staining of C3 the basement

membrane and mesangium in 2 glomeruli. Immunofluorescence microscopyx200.

infiltration with tubular atrophy in the interstitium is also seen. HEx200.

A 15 year-old-male presented with the complaints of hematuria, oliguria and swelling of his face and eyelids. He suffered a throat infection 10 days prior to admission. No family history for kidney disease or consanguinity between parents.

On physical exam was unremarkable except a blood pressure of 160/100 mmHg and pretibial 1+ edema.

Laboratory findings: Complete blood count and electrolytes were normal , BUN 98 mg/dl,Cr:9.34 mg/dl, Total protein/Albumin: 6.14/3.2 g/dl

Urinalysis: Density 1018, Proteinuria 1200 mg/24hrs, no glucose, sediment: abundant erythrocytes and white blood cells and fine leukocytic and granular casts.

Serum C3 15 mg/dl, ,C4 6.1mg/dl, ANA, Anti-DNA, viral markers were all normal.

Renal USG revealed minimal increase in parenchymal echogenicity . Urine culture was negative.

**Renal biopsy diagnosis**: Acute postinfectious glomerulonephritis(APIGN) (Figs.21-22)

Fig. 21. Acute postinfectious glomerulonephritis. Light micrograph shows endocapillary proliferation with numerous neutrophils. There are also several neutrophils in tubular lumina.HEx200.

Diagnostic Algorithms in Renal Biopsy Interpretation Along with Case Samples 83

Vascular lesions can be divided into five broad groups. Intimal fibrosis/medial fibrosis is associated with hypertension whereas necrosis may be a sign of progressive systemic sclerosis, vasculitis, drug hypersensitivity or radiation. Thrombotic lesions can be due to thrombotic microangiopathy/ hemolytic uremic syndrome, lupus nephritis + antiphospholipid syndrome, malignant hypertension and progressive systemic sclerosis.

Fig. 24. Lupus nephritis+ thrombotic microangiopathy. Note 'wire-loops(down left) and

Tubulointerstitial lesions can be divided into five main categories including interstitial cellular infiltrates, necrosis, edema, tubular atrophy/fibrosis and tubular casts/deposits. If there is a significant infiltration in the tubulointerstitial area it is crucial to determine whether it is benign or malignant.(Figure 26). If benign, then the predominant cell type in the infiltration should be assessed or if there is no significant inflammation, necrosis, edema, tubular atrophy /fibrosis, tubular casts and deposits are investigated according to the main tubular abnormalities.(Figures 27,28) **(**Kowalewska,2011; Simms,2011; Wolf,2011;

fibrin thrombi obliterating glomerular capillary lumina. HEx100

**2.1.3 Tubulointerstitial lesions**

Midgley,2011;Chandra,2010)

**2.1.2 Vascular lesions**

(Silvarin, 2011; Lindsay, 2011; Benz,2010) (Figures 24,25)

Fig. 22. Acute postinfectious GN. Immunofluorescence microphoto shows a starry sky pattern. There is a diffuse fine or coarse granular deposits of C3 in the GBM and mesangium. Immunofluorescence microscopyx200.

Fig. 23. An algorithm for interpretation of vascular lesions.(HUS: Hemolytic uremic syndrome, TTP: Thrombotic Thrombocytopenic purpura, SLE: Systemic lupus erythematosus)

#### **2.1.2 Vascular lesions**

82 Topics in Renal Biopsy and Pathology

Fig. 22. Acute postinfectious GN. Immunofluorescence microphoto shows a starry sky pattern. There is a diffuse fine or coarse granular deposits of C3 in the GBM and

Fig. 23. An algorithm for interpretation of vascular lesions.(HUS: Hemolytic uremic syndrome, TTP: Thrombotic Thrombocytopenic purpura, SLE: Systemic lupus

mesangium. Immunofluorescence microscopyx200.

erythematosus)

Vascular lesions can be divided into five broad groups. Intimal fibrosis/medial fibrosis is associated with hypertension whereas necrosis may be a sign of progressive systemic sclerosis, vasculitis, drug hypersensitivity or radiation. Thrombotic lesions can be due to thrombotic microangiopathy/ hemolytic uremic syndrome, lupus nephritis + antiphospholipid syndrome, malignant hypertension and progressive systemic sclerosis. (Silvarin, 2011; Lindsay, 2011; Benz,2010) (Figures 24,25)

Fig. 24. Lupus nephritis+ thrombotic microangiopathy. Note 'wire-loops(down left) and fibrin thrombi obliterating glomerular capillary lumina. HEx100

#### **2.1.3 Tubulointerstitial lesions**

Tubulointerstitial lesions can be divided into five main categories including interstitial cellular infiltrates, necrosis, edema, tubular atrophy/fibrosis and tubular casts/deposits. If there is a significant infiltration in the tubulointerstitial area it is crucial to determine whether it is benign or malignant.(Figure 26). If benign, then the predominant cell type in the infiltration should be assessed or if there is no significant inflammation, necrosis, edema, tubular atrophy /fibrosis, tubular casts and deposits are investigated according to the main tubular abnormalities.(Figures 27,28) **(**Kowalewska,2011; Simms,2011; Wolf,2011; Midgley,2011;Chandra,2010)

Diagnostic Algorithms in Renal Biopsy Interpretation Along with Case Samples 85

Fig. 27. Cystinosis. Multinuclear podocytes may be an early sign of cystinosis(arrow).

Fig. 28. Cystinosis. Cystine crystals in the interstitium under polarized light.HEx400.

HEx200.

Fig. 25. Lupus nephritis+ thrombotic microangiopathy. IgG staining of glomerular basement membrane and mesangium. Immunofluorescence microscopyx400.

Fig. 26. An algorithm for interpretation of tubulointerstitial lesions. (TIN: Tubulointerstitial nephropathy, SLE:Systemic lupus erythematosus,HIV; Human immunodeficiency virus)

Fig. 25. Lupus nephritis+ thrombotic microangiopathy. IgG staining of glomerular basement

Fig. 26. An algorithm for interpretation of tubulointerstitial lesions. (TIN: Tubulointerstitial nephropathy, SLE:Systemic lupus erythematosus,HIV; Human immunodeficiency virus)

membrane and mesangium. Immunofluorescence microscopyx400.

Fig. 27. Cystinosis. Multinuclear podocytes may be an early sign of cystinosis(arrow). HEx200.

Fig. 28. Cystinosis. Cystine crystals in the interstitium under polarized light.HEx400.

Diagnostic Algorithms in Renal Biopsy Interpretation Along with Case Samples 87

Fig. 30. Types of lesions commonly seen in transplanted kidneys. (CMV: Cytomegalovirus)

It should be remembered that these algorithms are designed for the diseases presenting with their classical patterns. Therefore, very rarely seen diseases or those without fully developed features are not included in the aforementioned algorithms. Since a number of disease may belong to more than one category because of diversity of the clinical and morphologic findings they may not clearly fall into any category. In addition, the best way to reach a correct diagnosis in a renal biopsy needs making a good correlation of morphological

In the future, a considerable amount of molecular genetic data are anticipated to be added. The aim is to use these data validated in large populations not only to create new algorithms providing the best approach to classify renal diseases but also to obtain complementary knowledge for appropriate prediction concerning diagnosis, therapy and prognosis of renal

Walker PD(2009). The renal biopsy. Archieves of Pathology & Laboratory Medicine, Vol.

Walker PD, Cavallo T& Bonsib SM, (2004).The Ad Hoc Committee on renal biopsy

Fogo AB.(2003). Approach to Renal Biopsy. American Journal of Kidney Diseases, Vol.42,

guidelines of the Renal Pathology Society. Practice Guidelines for the renal biopsy. Modern Pathology, Vol.17, No.12 ( December 2004), pp.1555-1563, ISSN 0893-3952 Kretzler M, Cohen CD, Doran P, Henger A, Madden S, Gröne EF, Nelson PJ, Schlöndorff D&

Gröne HJ.(2002). Repuncturing the renal biopsy: strategies for molecular diagnosis in nephrology. Journal of the American Society of Nephrology, Vol.13 No.7, (July

133, No.2 ( February 2009), pp181-188, ISSN 0003-9985

2002), pp 1961-1972, ISSN 1555-9041

No.4, (October 2003), pp 826-836, ISSN 0272-6386

**3. Conclusion**

diseases.

**4. References**

findings with appropriate clinical data.

#### **CASE 10.**

A 3 year-old-male presented with the complaints of polyuria, polydipsia and oliguria lasting eight months. No family history for kidney disease but 2nd degree consanguinity between parents.

Physical examination revealed a malnourished infant with failure to thrive.

Laboratory findings: Complete blood count and electrolytes were normal , Ca 9.8 mg/dl, P 3.97 mg/dl, BUN 30 mg/dl,Cr:0.7 mg/dl, Total protein/Albumin: 7.4/4.3 g/dl

Urinalysis: Density 1006, Proteinuria 200 mg/24hrs, glucose(+), sediment: 7-8 erythrocytes and 5-6 white blood cells. Serum C3 90 mg/dl, ,C20.1mg/dl, ANA, Anti-DNA, viral markers were all normal. Ultrasonography revealed increase in renal parenchymal echogenicity.

**Renal biopsy diagnosis**: Cystinosis (Figs.27-28)

#### **2.2 Transplant kidney lesions**

Renal allograft biopsy is still the gold standard for diagnosis of transplant rejection. A number of both immune complex( such as MPGN, IgA nephropathy etc.) or non-immune disease(FSGS,diabetic nephropathy etc.) can recur in transplanted kidneys. (Sun,2011; John,2010)(Figure 29)

Fig. 29. Acute cellular rejection, Banff type 2. Light microscopic photo showing mildmoderate tubulitis along with tubulointerstitial inflammation. Note that the morphology of glomerulus is unremarkable.HEx100.

Fig. 30. Types of lesions commonly seen in transplanted kidneys. (CMV: Cytomegalovirus)

#### **3. Conclusion**

86 Topics in Renal Biopsy and Pathology

A 3 year-old-male presented with the complaints of polyuria, polydipsia and oliguria lasting eight months. No family history for kidney disease but 2nd degree consanguinity between

Laboratory findings: Complete blood count and electrolytes were normal , Ca 9.8 mg/dl, P

Urinalysis: Density 1006, Proteinuria 200 mg/24hrs, glucose(+), sediment: 7-8 erythrocytes and 5-6 white blood cells. Serum C3 90 mg/dl, ,C20.1mg/dl, ANA, Anti-DNA, viral markers were all normal. Ultrasonography revealed increase in renal parenchymal

Renal allograft biopsy is still the gold standard for diagnosis of transplant rejection. A number of both immune complex( such as MPGN, IgA nephropathy etc.) or non-immune disease(FSGS,diabetic nephropathy etc.) can recur in transplanted kidneys. (Sun,2011;

Fig. 29. Acute cellular rejection, Banff type 2. Light microscopic photo showing mildmoderate tubulitis along with tubulointerstitial inflammation. Note that the morphology of

Physical examination revealed a malnourished infant with failure to thrive.

3.97 mg/dl, BUN 30 mg/dl,Cr:0.7 mg/dl, Total protein/Albumin: 7.4/4.3 g/dl

**CASE 10.**

parents.

echogenicity.

**Renal biopsy diagnosis**: Cystinosis (Figs.27-28)

**2.2 Transplant kidney lesions**

glomerulus is unremarkable.HEx100.

John,2010)(Figure 29)

It should be remembered that these algorithms are designed for the diseases presenting with their classical patterns. Therefore, very rarely seen diseases or those without fully developed features are not included in the aforementioned algorithms. Since a number of disease may belong to more than one category because of diversity of the clinical and morphologic findings they may not clearly fall into any category. In addition, the best way to reach a correct diagnosis in a renal biopsy needs making a good correlation of morphological findings with appropriate clinical data.

In the future, a considerable amount of molecular genetic data are anticipated to be added. The aim is to use these data validated in large populations not only to create new algorithms providing the best approach to classify renal diseases but also to obtain complementary knowledge for appropriate prediction concerning diagnosis, therapy and prognosis of renal diseases.

#### **4. References**

Diagnostic Algorithms in Renal Biopsy Interpretation Along with Case Samples 89

Cattran DC, Coppo R, Cook HT, Feehally J, Roberts IS, Troyanov S, Alpers CE, Amore A,

Bellu SS, Troyanov, S, Hook T, Roberts ISD, on behalf of a Working Group of the

Alchi B, Jayne D.(2010). Membranoproliferative glomerulonephritis. Pediatric Nephrology,

Colucci G, Manno C, Grandaliano G, Schena FP. (2011). Cryoglobulinemic

Wen YK, Chen ML.(2010). The significance of atypical morphology in the changes of

Silvariño R, Sant F, Espinosa G, Pons-Estel G, Solé M, Cervera R, Arrizabalaga P.(2011).

Keir L, Coward RJ.(2011). Advances in our understanding of the pathogenesis of glomerular

Benz K, Amann K.(2010). Thrombotic microangiopathy: new insights. Current Opinion in

Kowalewska J, Nicosia RF, Smith KD, Kats A, Alpers CE.(2011). Patterns of glomerular

Simms RJ, Hynes AM, Eley L, Sayer JA.(2011). Nephronophthisis: a genetically diverse ciliopathy. International Journal of Nephrology, Vol. No. (2011), pp. ISSN Wolf MT, Hildebrandt F.(2011). Nephronophthisis. Pediatric Nephrology, Vol.26 No.2

Vol.42, No.6 (June 2011), pp. 896-903, ISSN 0046-8177

(February 2011), pp 181-194, ISSN 0931-041X

Nephrology, Vol. 74, No.4 (April 2010), pp. 374-379, ISSN 0301-0430 Nasr SH, Markowitz GS, Stokes MB, Said SM, Valeri AM, D'Agati VD.(2008). Acute

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**Part 3**

**Glomerular Diseases**

**Part 3**

**Glomerular Diseases**

90 Topics in Renal Biopsy and Pathology

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YA, Wu S.(2011). Macrophages and T lymphocytes are the predominant cells in intimal arteritis of resected renal allografts undergoing acute rejection. Transplant

**6**

*Cyprus*

Kyriacos Kyriacou et al.\*

**The Many Faces of Thin Basement Membrane**

Thin basement membrane nephropathy (TBMN) or benign familial hematuria, is one of the commonest kidney disorders, characterized by recurrent benign hematuria, which is often associated with a family history (Gregory, 2005). It is generally considered to be a nonprogressive, life long disorder with a rather benign course (Tryggvason & Patrakka, 2006), although data from some studies show that a proportion of patients manifest more severe symptoms, than originally described and eventually develop endstage renal disease (ESRD) (Dische, Weston, & Parsons, 1985; Frasca et al., 2005; Pierides et al., 2009; Tiebosch et al., 1989). TBMN was first described in 1926 (Baer, 1926) and at presentation is usually characterized by microscopic painless hematuria, by little or negligible proteinuria and normal renal function. TBMN is an autosomal dominant disorder and more than 50% of the cases have a family history of hematuria. In about 40% of families with a confirmed diagnosis of TBMN the condition co-segregates with heterozygous COL4A3/COL4A4 mutations (Lemmink et al., 1996). Mutations in the type IV collagen gene family which also includes the COL4A5 gene, also cause Alport syndrome (Hostikka et al., 1990). It is believed that these disorders are the result of defective, synthesis and/or assembly, of the critical glycoprotein components, that form the glomerular basement membranes (GBMs), among

Clinically TBMN must be differentially diagnosed, between IgA nephropathy and Alport syndrome which are the other two main causes of hematuria, particularly in children. The need for differential diagnosis, often necessitates the examination of a kidney biopsy at the electron microscopical level, which also enables measurement of the thickness of the GBMs. Indeed Rogers et al (Rogers, Kurtzman, Bunn, & White, 1973) were the first to associate recurrent benign hematuria with the presence of thin GBMs. Currently the most widely

Marianna Nearchou1, Ioanna Zouvani2, Christina Flouri1, Maria Loizidou1, Michael Hadjigavriel3,

*1Department of Electron Microscopy/Molecular Pathology, The Cyprus Institute of Neurology and Genetics, Nicosia,*

**1. Introduction**

 \*

*Cyprus*

which type IV collagen, is the major constituent.

Andreas Hadjisavvas1 and Kyriacos Ioannou4

*2Department of Histopathology, Nicosia General Hospital, Nicosia 3Department of Nephrology, Larnaca General Hospital, Larnaca 4Department of Nephrology, Nicosia General Hospital, Nicosia*

**Nephropathy; A Population Based Study**

*Department of Electron Microscopy; Molecular Pathology The Cyprus Institute of Neurology and Genetics, Nicosia*

### **The Many Faces of Thin Basement Membrane Nephropathy; A Population Based Study**

Kyriacos Kyriacou et al.\*

*Department of Electron Microscopy; Molecular Pathology The Cyprus Institute of Neurology and Genetics, Nicosia Cyprus*

#### **1. Introduction**

Thin basement membrane nephropathy (TBMN) or benign familial hematuria, is one of the commonest kidney disorders, characterized by recurrent benign hematuria, which is often associated with a family history (Gregory, 2005). It is generally considered to be a nonprogressive, life long disorder with a rather benign course (Tryggvason & Patrakka, 2006), although data from some studies show that a proportion of patients manifest more severe symptoms, than originally described and eventually develop endstage renal disease (ESRD) (Dische, Weston, & Parsons, 1985; Frasca et al., 2005; Pierides et al., 2009; Tiebosch et al., 1989). TBMN was first described in 1926 (Baer, 1926) and at presentation is usually characterized by microscopic painless hematuria, by little or negligible proteinuria and normal renal function. TBMN is an autosomal dominant disorder and more than 50% of the cases have a family history of hematuria. In about 40% of families with a confirmed diagnosis of TBMN the condition co-segregates with heterozygous COL4A3/COL4A4 mutations (Lemmink et al., 1996). Mutations in the type IV collagen gene family which also includes the COL4A5 gene, also cause Alport syndrome (Hostikka et al., 1990). It is believed that these disorders are the result of defective, synthesis and/or assembly, of the critical glycoprotein components, that form the glomerular basement membranes (GBMs), among which type IV collagen, is the major constituent.

Clinically TBMN must be differentially diagnosed, between IgA nephropathy and Alport syndrome which are the other two main causes of hematuria, particularly in children. The need for differential diagnosis, often necessitates the examination of a kidney biopsy at the electron microscopical level, which also enables measurement of the thickness of the GBMs. Indeed Rogers et al (Rogers, Kurtzman, Bunn, & White, 1973) were the first to associate recurrent benign hematuria with the presence of thin GBMs. Currently the most widely

<sup>\*</sup> Marianna Nearchou1, Ioanna Zouvani2, Christina Flouri1, Maria Loizidou1, Michael Hadjigavriel3, Andreas Hadjisavvas1 and Kyriacos Ioannou4

*<sup>1</sup>Department of Electron Microscopy/Molecular Pathology, The Cyprus Institute of Neurology and Genetics, Nicosia, 2Department of Histopathology, Nicosia General Hospital, Nicosia*

*<sup>3</sup>Department of Nephrology, Larnaca General Hospital, Larnaca*

*<sup>4</sup>Department of Nephrology, Nicosia General Hospital, Nicosia*

*Cyprus*

The Many Faces of Thin Basement Membrane Nephropathy; A Population Based Study 95

simultaneous occurrence of other glomerulopathies in the background of thin GBMs. In this review, the spectrum of histopathological and ultrastructural phenotypes detected in TBMN patients in our population will be described. We also illustrate the value of the direct GBM measurement method in detecting thin GBMs, in cases of TBMN presenting with more advanced glomerulosclerosis. In such cases the presence of thin GBMs may be overlooked, a factor which contributes to an underdiagnosis of this, rather common

a. To present the histopathological features of TBMN in a population based study.

b. To highlight the fact that TBMN nephropathy is a more frequent disease and has a much higher incidence, than the 1-2% that is usually quoted in the literature. c. To emphasize the need to use a standardized direct morphometric method for the

d. To highlight the observation that TBMN as a disease entity, may have different faces (phases), as histologically it is usually associated with different stages of glomerulosclerosis. Another factor that contributes to the different histological faces of TBMN is the phenomenon that TBMN is often associated with other glomerulopathies.

In the last eighteen years, 1200 renal biopsies from Cypriot patients were investigated in the department of electron microscopy and 75 of these were diagnosed as TBMN. The TBMN group included 35 males and 40 females who at the time of biopsy had microscopic hematuria. Twenty-two patients had hematuria with proteinuria and of these eight had proteinuria in the nephrotic range. The majority of patients had normal renal function (Table 1). Detailed clinical data for some of these patients were described previously (Marquez et al., 2003; Zouvani et al., 2008). In this group of 75 patients, seven patients were diagnosed with TBMN which was associated with other glomerulopathies (Table 2). All

Renal biopsies were obtained using a standard percutaneous technique under local anesthesia. The biopsies were examined and sectioned under an ordinary light microscope to select tissue containing glomeruli for light microscopy, immunofluorescence, and electron microscopy. All biopsies containing at least one glomerulus in the tissue submitted for

Tissue was fixed in 10% phosphate-buffered formalin and embedded in paraffin wax. Paraffin sections were cut at 4μm and stained with hematoxylin and eosin, periodic acid-Schiff (PAS), silver methanamine, Masson's trichrome and Congo red according to standard

genetic disorder.

The main aims of the chapter are:

**3. Materials and methods**

**3.1 Patients**

**3.2 Renal biopsies**

**3.3 Light microscopy**

protocols.

electron microscopy were included.

accurate and systematic diagnosis of TBMN.

patients included in the study were adults, above 18 years old.

**2. Aims**

accepted pathognomonic hallmark for diagnosing TBMN is the presence of diffuse thinning of the GBM, in at least 50% of glomeruli examined. Accurate evaluation of the thickness of the GBM is best performed with the use of morphometry and requires the prior establishment of a normal range of GBM thickness, at each diagnostic centre.

It is well documented that the thickness of the normal GBM varies with age, gender and method of tissue preparation; thickness is also influenced by the method of measurement. According to general agreement, the GBM thickness is determined from samples fixed in glutaraldehyde. Recently it has been shown that even the choice of intermediate solvent and type of embedding resin are variables that also affect the thickness of the GBM (Edwards, Griffiths, Morgan, Pitman, & von Ruhland, 2009). Normal GBM thickness has been estimated in several reports (Coleman, Haynes, Dimopoulos, Barratt, & Jarvis, 1986; Das, Pickett, & Tungekar, 1996; Dische et al., 1985; Haas, 2009; Jovanovic GB, 1990). Since there is a lack of a general consensus on diagnostic criteria, it is recommended that a GBM normal range should be established for each laboratory (Dische et al., 1985; Tiebosch et al., 1989). This normal range is a necessary pre-requisite that facilitates the subsequent accurate diagnosis of TBMN. In our department we developed initially a detailed morphometric technique (Marquez et al., 1999) which was subsequently simplified to a more direct method, for measuring GBM thickness in kidney biopsies (Marquez et al., 2003). In this context we are one of a few electron microscopy departments which have been applying a standardized "in house" morphometric method, to diagnose TBMN cases, prospectively for more than 15 years. This method involves surveying the glomerulus and selecting the thinnest 4-5 peripheral glomerular capillary loops. Then the thickness of the GBM is measured in at least 4 different points per loop. If the average (arithmetic mean) of the measurements is less than 300nm, then the case is diagnosed as TBMN. We believe that this approach and practice have contributed to a more accurate estimate of the incidence of TBMN in our population (Zouvani et al., 2008).

In terms of histopathology, TBMN cases have normal glomeruli but some cases are often associated with mild and often premature glomerular changes. In some reports, the coexistence of thin GBMs with lesions compatible with focal segmental glomerulosclerosis (FSGS), affecting a variable number of glomeruli (up to 25%) was noted (Nieuwhof, de Heer, de Leeuw, & van Breda Vriesman, 1997; van Paassen, van Breda Vriesman, van Rie, & Tervaert, 2004). However, in our experience the occurrence of FSGS lesions in TBMN cases is more frequent and most of the glomeruli in TBMN cases show focal glomerulosclerosis. At the same time the importance of applying ultrastructural morphometry will be highlighted, as this is an essential component of accurate diagnosis. Our department operates as a referral diagnostic centre for the whole of Cyprus so the results are based on 1200 renal biopsies, from an unselected population sample, which is considered to be representative of our population. In this cohort of patients, a total of 75 renal biopsies showed thinning of GBMs, average thickness being less than 300nm and these cases were diagnosed as TBMN. The majority of glomeruli in these patients showed FSGS of different stages (different phases as described in the title), ranging from a mild segmental phenotype to more advance lesions of glomerulosclerosis. Another important aspect of TBMN is its association with other glomerulopathies, as shown by the histological examination of renal biopsy. This simultaneous presentation of TBMN with other glomerulopathies was present in seven of the 75 cases included in our study and gave rise to the selection of the title for this chapter. Consequently this title was chosen to reflect the progressive lesions of FSGS that are associated with TBMN, as well as the simultaneous occurrence of other glomerulopathies in the background of thin GBMs. In this review, the spectrum of histopathological and ultrastructural phenotypes detected in TBMN patients in our population will be described. We also illustrate the value of the direct GBM measurement method in detecting thin GBMs, in cases of TBMN presenting with more advanced glomerulosclerosis. In such cases the presence of thin GBMs may be overlooked, a factor which contributes to an underdiagnosis of this, rather common genetic disorder.

#### **2. Aims**

94 Topics in Renal Biopsy and Pathology

accepted pathognomonic hallmark for diagnosing TBMN is the presence of diffuse thinning of the GBM, in at least 50% of glomeruli examined. Accurate evaluation of the thickness of the GBM is best performed with the use of morphometry and requires the prior

It is well documented that the thickness of the normal GBM varies with age, gender and method of tissue preparation; thickness is also influenced by the method of measurement. According to general agreement, the GBM thickness is determined from samples fixed in glutaraldehyde. Recently it has been shown that even the choice of intermediate solvent and type of embedding resin are variables that also affect the thickness of the GBM (Edwards, Griffiths, Morgan, Pitman, & von Ruhland, 2009). Normal GBM thickness has been estimated in several reports (Coleman, Haynes, Dimopoulos, Barratt, & Jarvis, 1986; Das, Pickett, & Tungekar, 1996; Dische et al., 1985; Haas, 2009; Jovanovic GB, 1990). Since there is a lack of a general consensus on diagnostic criteria, it is recommended that a GBM normal range should be established for each laboratory (Dische et al., 1985; Tiebosch et al., 1989). This normal range is a necessary pre-requisite that facilitates the subsequent accurate diagnosis of TBMN. In our department we developed initially a detailed morphometric technique (Marquez et al., 1999) which was subsequently simplified to a more direct method, for measuring GBM thickness in kidney biopsies (Marquez et al., 2003). In this context we are one of a few electron microscopy departments which have been applying a standardized "in house" morphometric method, to diagnose TBMN cases, prospectively for more than 15 years. This method involves surveying the glomerulus and selecting the thinnest 4-5 peripheral glomerular capillary loops. Then the thickness of the GBM is measured in at least 4 different points per loop. If the average (arithmetic mean) of the measurements is less than 300nm, then the case is diagnosed as TBMN. We believe that this approach and practice have contributed to a more accurate estimate of the incidence of

In terms of histopathology, TBMN cases have normal glomeruli but some cases are often associated with mild and often premature glomerular changes. In some reports, the coexistence of thin GBMs with lesions compatible with focal segmental glomerulosclerosis (FSGS), affecting a variable number of glomeruli (up to 25%) was noted (Nieuwhof, de Heer, de Leeuw, & van Breda Vriesman, 1997; van Paassen, van Breda Vriesman, van Rie, & Tervaert, 2004). However, in our experience the occurrence of FSGS lesions in TBMN cases is more frequent and most of the glomeruli in TBMN cases show focal glomerulosclerosis. At the same time the importance of applying ultrastructural morphometry will be highlighted, as this is an essential component of accurate diagnosis. Our department operates as a referral diagnostic centre for the whole of Cyprus so the results are based on 1200 renal biopsies, from an unselected population sample, which is considered to be representative of our population. In this cohort of patients, a total of 75 renal biopsies showed thinning of GBMs, average thickness being less than 300nm and these cases were diagnosed as TBMN. The majority of glomeruli in these patients showed FSGS of different stages (different phases as described in the title), ranging from a mild segmental phenotype to more advance lesions of glomerulosclerosis. Another important aspect of TBMN is its association with other glomerulopathies, as shown by the histological examination of renal biopsy. This simultaneous presentation of TBMN with other glomerulopathies was present in seven of the 75 cases included in our study and gave rise to the selection of the title for this chapter. Consequently this title was chosen to reflect the progressive lesions of FSGS that are associated with TBMN, as well as the

establishment of a normal range of GBM thickness, at each diagnostic centre.

TBMN in our population (Zouvani et al., 2008).

The main aims of the chapter are:

#### **3. Materials and methods**

#### **3.1 Patients**

In the last eighteen years, 1200 renal biopsies from Cypriot patients were investigated in the department of electron microscopy and 75 of these were diagnosed as TBMN. The TBMN group included 35 males and 40 females who at the time of biopsy had microscopic hematuria. Twenty-two patients had hematuria with proteinuria and of these eight had proteinuria in the nephrotic range. The majority of patients had normal renal function (Table 1). Detailed clinical data for some of these patients were described previously (Marquez et al., 2003; Zouvani et al., 2008). In this group of 75 patients, seven patients were diagnosed with TBMN which was associated with other glomerulopathies (Table 2). All patients included in the study were adults, above 18 years old.

#### **3.2 Renal biopsies**

Renal biopsies were obtained using a standard percutaneous technique under local anesthesia. The biopsies were examined and sectioned under an ordinary light microscope to select tissue containing glomeruli for light microscopy, immunofluorescence, and electron microscopy. All biopsies containing at least one glomerulus in the tissue submitted for electron microscopy were included.

#### **3.3 Light microscopy**

Tissue was fixed in 10% phosphate-buffered formalin and embedded in paraffin wax. Paraffin sections were cut at 4μm and stained with hematoxylin and eosin, periodic acid-Schiff (PAS), silver methanamine, Masson's trichrome and Congo red according to standard protocols.

The Many Faces of Thin Basement Membrane Nephropathy; A Population Based Study 97

b. Presence of thin GBMs having an arithmetic mean of less than 300 nm, or having more

c. Presence of diffuse GBM thinning in a minimum number of four peripheral capillaries

Seventy-five renal biopsies were selected for the present morphometric analysis based on the light microscopical observations and inclusion criteria outlined above (Table 1). On histology, 14 cases exhibited normal glomeruli. In 26 cases, up to 10% of glomeruli were affected by glomerulosclerosis (Figs. 1a and 1b) and in 27 cases glomerular sclerosis was present in between 10-50% of glomeruli (Figs. 2a and 2b). Focal glomerulosclerosis was characterized most often by an increase in mesangial matrix and very rarely this also included an increase in mesangial cells. In the remaining eight cases, glomerulosclerosis was evident in more than 50% of the total glomeruli identified in each biopsy (Fig. 3 and Table 1). These 75 patients included seven cases with the diagnosis of incidental TBMN which was

In all cases the immunofluorescence results were essentially negative except in the three cases that had a diagnosis of IgA nephropathy (IgAN), where moderate mesangial staining for IgA was present. In ten cases, there was mild and focal mesangial staining for IgM and

All biopsy specimens, especially the ones obtained from patients with proteinuria showed a variable degree of epithelial cell foot process fusion. In all 75 cases foot process effacement was segmental and never global. In the selection process, renal biopsies showing global foot process fusion were excluded from the TBMN group, in order to eliminate inclusion of cases with primary FSGS. In terms of GBM thickness, 14 out of 75 patients who exhibited histologically normal glomeruli showed widespread thinning of the GBM. In the remaining patients, that showed glomerulosclerosis affecting between 10-50% of glomeruli, thinning of the GBM was more obvious in the glomerular segments that were unaffected by increased mesangial matrix and glomerulosclerosis. In all patients examined attenuation of GBM was the predominant finding. Thin GBMs were present in normal-looking capillary loops (Fig. 4) as well as in areas showing mild expansion of the mesangial matrix (Fig. 5). In some biopsies that exhibited a more marked increase in mesangial matrix, thin GBMs could still be distinguished even in severely affected areas, especially in peripheral glomerular capillaries (Fig. 6). In some patients, the thickness of the GBM was not uniform, and this was not related to orientation or differences in sectioning (Fig. 7). In such cases morphometry (see

For classifying our own patients, the normal adult mean GBM thickness range of 300–400nm was adopted. This is our ''in-house'' range of normal adult GBM thickness that was

than 50% of the points measured below 300 nm upon direct measurement.

e. Absence of diffuse fusion of podocytes in order to exclude primary FSGS.

per glomerulus.

**4.2 Light microscopy**

**4.3 Immunofluorescence**

**4.4 Electron microscopy**

**4.5 Morphometry**

two cases showed staining for C3.

d. Absence of splitting or lamellation of the GBM.

associated with other glomerulopathies (Table 2).

below) provided more accurate estimates of the GBM thickness.

#### **3.4 Immunofluorescence**

A small piece from each biopsy was embedded in optimal cutting temperature (OCT) compound and frozen directly in liquid nitrogen. Cryostat sections were cut at 4μm and incubated with a series of flourescein isothiocyanate (FITC)-linked mouse antibodies against human immunoglobulin A (IgA), IgG, IgM, C1q, C3 and fibrin, for direct immunofluorescence.

#### **3.5 Routine electron microscopy**

Specimens were fixed for a minimum of 4 h in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, postfixed for 1 h in 1 % osmium tetroxide, dehydrated in a series of graded ethanols, cleared in propylene oxide, and embedded in an Epon/Araldite mixture. Blocks were sectioned with a Recheirt Jung Ultracut, ultramicrotome (Vienna, Austria). Semithin sections, 1μm thick, were stained with toluidine blue to locate glomeruli. Ultrathin sections of gold interference color were mounted on 200-mesh copper grids and stained with uranyl acetate and lead citrate. Electron microscopy was carried out in a JEM -1010 (JEOL-Tokyo) transmission electron microscope.

#### **3.6 Morphometry**

For measuring the thickness of the GBMs two approaches were used as our electron microscope was recently fitted with a digital camera. Consequently for the years spanning 1991-2008 the following methodology was used.

Each available block from a biopsy was serially sectioned to obtain the maximum number of glomeruli. In each glomerulus, each patent peripheral loop was photographed and this sometimes required a maximum of three separate micrographs per loop. Micrographs were taken at a magnification of x4,000 on 6.5 x 9cm Kodak electron microscope film. A grating replica with 2160 cross lines/mm was used for calibration (Electron Microscope Sciences, Washington, PA 19034). Prints were made to a final magnification of x12,000 and morphometric measurements were taken as described previously, using the simplified method (Marquez et al., 2003).

Since 2008 the software tool of the MEGAVIEM, Olympus Soft Imaging, system is being used to measure GBM thickness. In each biopsy every available glomerulus is scanned and at least four thin glomerular loops that are situated peripherally, are measured at a magnification of ×8000. For measurement, a square grid, size 2000x2000 nm, is superimposed over the glomerular capillaries and point measurements are taken at regions where the grid intersects the capillary loops exactly at right angles. Each point measures the distance from the cell membrane of the epithelial cell, as it attaches on the lamina rara externa, to the inner aspect of the endothelial cell membrane. In this manner a minimum of 16-20 separate points were measured in each peripheral glomerulus which represented 4-5 points per individual loop (Figs. 8 and 9b). In each patient the arithmetic mean of the GBM thickness was calculated, averaging measurements at 16 points, which is the minimum number of points measured per glomerulus. This approach was very similar to the direct method that was used previously (Marquez et al., 2003).

#### **4. Results**

#### **4.1 Inclusion criteria**

The selection criteria of biopsies for this study included:

a. Normal histology or minimal to moderate histological glomerular abnormalities.

#### **4.2 Light microscopy**

96 Topics in Renal Biopsy and Pathology