Histology, Nephron

Issues of Concern

Kidneys are the primary organ in blood filtration, water, and electrolyte balance and maintaining blood pressure in circulation. They are essential to clear bacterial components and cytokines from the blood. In addition, kidney tissue is critically susceptible to immune-mediated disorders. Renal histology is highly specialized in different nephron segments, providing crucial information for diagnosing several kidney diseases.[2]

Structure

Macroscopically, the kidney divides into two sections: the renal cortex, the outer part of the kidney, and the medulla, the inner section. Both contain different structures of the nephron, the functional unit of the kidney. It is crucial to comprehend the nephron’s structure to understand the functioning of the kidney. 

The nephron is comprised of a glomerulus and a complex tubular system (see Image. Histology of the Cortex of a Kidney, Nephron Histology). The glomerulus and the first portion of the tubular system, known as the proximal convoluted tubule (PCT), are located in the renal cortex. Following the PCT, the loop of Henle, a hairpin-like structure, penetrates the medulla and returns to the cortex to connect with the distal convoluted tubule (DCT). Finally, the nephron drains into the collecting duct via connecting tubules.[3]

There are two types of nephrons: (1) superficial nephrons with glomeruli near the cortical surface and short loops of Henle, and (2) juxtamedullary nephrons with glomeruli located near the cortico-medullary junction and long loops of Henle descending deeper into the renal medulla.[4]

The glomerulus filters large amounts of blood, which the tubular system converts into urine through reabsorption and secretion of free water and solutes.[1]

The Glomerulus

The glomerulus forms by a tuft of capillaries surrounded by an impervious capsule denominated Bowman’s capsule.[3] The glomerular capillaries are flanked by two resistance vessels, the afferent and efferent arterioles, regulating intraglomerular pressure. These capillaries have unique characteristics that allow them to filter large volumes of blood. The filtration barrier comprises three structures that provide the support and particular properties needed for forming the primary glomerular filtrate, the ultrafiltrate.

1. Fenestrated endothelium of the glomerular capillaries: this layer confers size selectivity through fenestrae with diameters between 70 to 100 nm. 
2. Glomerular basement membrane (GBM): this is a thick structure composed of extracellular proteins, including proteoglycans, laminin, fibronectin, and type IV collagen. This layer confers charge selectivity to the filtered particles. 
3. Podocytes: a visceral epithelium of specialized cells that lines the GBM and forms the outermost layer of the filtration barrier.[5] These create a slit diaphragm through interdigitating long and thin foot processes.[3] These cells serve to sustain the integrity of the capillary loops.[5]

Once blood is filtered, the ultrafiltrate resides between the visceral epithelium and Bowman’s capsule. From here, the ultrafiltrate flows into the PCT.[3]

The Proximal Convoluted Tubule

Bowman’s capsule gives rise to the PCT, which lies adjacent to the glomerulus in the renal cortex. The PCT forms from a simple cuboidal epithelium that is dedicated to absorbing and transporting water, electrolytes, and other particles. These cells are characterized by a brush border of microvilli designed to increase the surface in contact with the glomerular ultrafiltrate, with abundant long, thin mitochondria lining the basal pole of the cell; and numerous vesicles involved in transcellular transport of 60 to 80% of the ultrafiltrate.[3]

The peritubular capillaries surround the PCT. This capillary network is responsible for the blood supply of the tubules and the recovery of the reabsorbed free water, ions, and other plasma constituents like amino acids and glucose.[3] 

The Loop of Henle

The PCT leaves the renal cortex and turns into the thin descending limb (TDL) of the loop of Henle, penetrating the renal medulla. The tubule becomes narrower, and the cells become smaller, with few mitochondria and short microvilli often unnoticeable on light microscopy.[3]

The tubule then turns upward towards the cortex, turning into the thick ascending limb (TAL). Here the lining cells become more prominent, with more numerous microvilli and mitochondria to engage in the active transport of sodium to dilute the urine.[3]

The Juxtaglomerular Apparatus and the Distal Convoluted Tubule

The juxtaglomerular apparatus is the region regulating the glomerular filtration through the tubuloglomerular feedback.[6] Histologically, this region is near the vascular pole of the glomerulus. It comprises the macula densa cells of the cortical TAL and the granular smooth muscle cells of the afferent arteriole of the glomerulus, functionally and structurally connected by glomerular mesangial cells.[7] The macula densa cells are morphologically distinct tubule cells characterized by a dense region of tall cells.[3]

The TAL turns into the DCT after returning to the renal cortex near its glomerulus of origin.[3] The DCT comprises the nephron segment between the macula densa and the cortical collecting tubule (CCT).[4]

The DCT cells are tall cells notable for containing the largest number of mitochondria among other cells in the nephron. They have an extensive basolateral amplification enclosing multiple mitochondria, creating a palisading appearance in the basal part of the cells.[4][8] Intercalated cells appear in the latter segment of the DCT and remain throughout the connecting and collecting tubules.[4]

Connecting and Collecting Tubules

The final part of the nephron is the connecting tubules, where the last fine-tuning of the urine occurs. These tubules have two types of cells; the intercalated cells and the connecting tubule (CNT) cells. The intercalated cells appear dense on electron microscopy and do not have the basolateral amplification characteristic of the DCT cells. These cells regulate hydrogen and bicarbonate secretion. The connecting tubule cells also have basolateral amplification but possess fewer mitochondria than DCT cells.[4]

The appearance of principal cells marks the transition into the collecting tubules and the end of the nephron. In cortical nephrons, the CNT leads to the collecting tubule, which drains to a collecting duct. The connecting tubules of juxtamedullary nephrons join and form an arcade that drains into a shared collecting duct.[4][8]

Function

The kidneys are responsible for several vital functions, including electrolyte and volume regulation, excretion of waste products, acid-base balance, synthesis of hormones such as erythropoietin, and metabolism of low molecular weight proteins. 

The Glomerulus

The kidneys receive from 20 to 25% of the cardiac output, approximately 1,200 ml/min of renal blood flow or 600 ml/min of renal plasma flow (RPF). The filtration fraction (FF) represents the proportion of the RPF that passes into the renal tubules and usually is 20%; this means that the glomerular filtration rate (GFR) is 120 ml/min (180 L per day) in an average of 60 kg person.[5]

The GFR is the product of the Ultrafiltration Coefficient (Kf) and the net filtration pressure (the change in P),

GFR  = Kf (ΔP)

where ΔP represents the sum of the Starling forces across all capillary beds, and Kf is determined by the surface area available for filtration and the hydraulic conductivity of the glomerular capillary wall. Variation in any of the mentioned components may alter the GFR. 

In short, filtration at a single glomerulus occurs because of four major components: (1) Kf; (2) hydraulic pressure gradient, favoring passage of water and molecules; (3) transcapillary oncotic pressure, favoring intravascular maintenance of free water and solutes; and (4) the glomerular flow rate.[5]

The Proximal Convoluted Tubule

From the 160 to 180 L of ultrafiltrate produced per day, only 1.5 to 2 L of urine is excreted. Reabsorption of 60 to 65% of free water and NaCl occurs in the PCT. Additionally, most potassium, phosphate, and HCO3, and nearly all nutrients, such as glucose and amino acids, are reabsorbed in this segment. The solute and water reabsorption in the proximal tubule is isotonic, with a minimum change in luminal osmolarity. This nephron site is also responsible for active solute secretion, hormone production, and renal gluconeogenesis.[9]

The Loop of Henle

Reabsorption of 30 to 40% of sodium occurs in this segment with essential changes in urine osmolarity. The loop of Henle divides into three parts: (1) the thin descending limb (TDL), (2)  the thin ascending limb (ATL), and (3) the thick ascending limb (TAL).[10]

The TDL is permeable to water and small solutes. In contrast, the ATL and TAL are impervious to water but permeable to solutes. The furosemide-sensitive Na+-K+-2CL- cotransporter (NKCC2) is located in the apical membrane of the TAL cells of juxtamedullary nephrons. These solutes are reabsorbed from the tubular fluid into the interstitium, increasing its osmolarity. This hypertonicity contributes to free water flow from the TDL into the renal interstitium. This process is known as the countercurrent mechanism. Urine becomes hypertonic as it passes through the TDL and hypotonic in the TAL, the diluting segment of the nephron. The reabsorbed water returns to circulation along the renal vasa recta.[11]

The Distal Nephron

The DCT is responsible for the fine-tuning of urine. It contributes 5 to 10% to the reabsorption of filtered sodium and chloride, as well as with K+ secretion. Just like the loop of Henle, the DCT is water-impermeable, further diluting the urine.[8] The following cluster of transporters accomplishes solute reabsorption:  

• Na+-K+ ATPase: it expresses at the basolateral membrane of the distal nephron. It contributes to Na+ reabsorption in two ways: (1) it maintains the intracellular Na+ concentration low and K+ concentration high, and (2) it generates an electronegative gradient towards the inside of the cell. The DCT is the nephron segment expressing the highest Na+ -K+ ATPase activity.[4]
• Thiazide-sensitive Na-Cl cotransporter (NCC) is a cotransporter that mediates the majority of Na+ and Cl- reabsorption. The expression of NCC is limited to the DCT.[12]
• Amiloride-sensitive Na+ transporter (ENaC) and Renal outer medullary potassium channel (ROMK): ENaC generates an electrogenic gradient that mediates potassium secretion through ROMK. The more sodium is reabsorbed through ENaC; the more potassium is excreted through ROMK. Aldosterone, an adrenal hormone stimulated by hyperkalemia and hypovolemia, favors this process.[8]
• Acid-base ionic channels: H+ and HCO3 are secreted through intercalated cells type A and B, respectively, located in the collecting duct.[13]

Tissue Preparation

A kidney biopsy is a gold standard for diagnosing and managing multiple diseases. Since renal diseases may be secondary to evident causes and renal biopsy is an invasive test, its indications are limited. Ultrasound-guided percutaneous renal biopsy (PRB) is the most accepted and commonly used technique to perform a renal biopsy.[14]

The ideal sample for microscopy should contain 20 glomeruli for a native kidney biopsy and at least ten glomeruli in a transplant kidney biopsy for diagnosis. The kidney cortex contains glomeruli, and the medulla primarily has tubules. Hence it is essential to obtain renal cortical tissue for analysis. However, in rare circumstances, medullary tissue is helpful in diagnoses like BK virus nephropathy and antibody-mediated rejection in a transplanted kidney.[15]

After obtaining kidney tissue, it is fixed and processed with a microtome into thin sections and processed for light microscopy (LM), immunofluorescence (IF), and electron microscopy (EM).[16]

Microscopy, Light

There are three primary microscopy modalities of clinical relevance light microscopy (LM), immunofluorescence (IF), and electron microscopy (EM).[16]

• Light microscopy: It is the essential modality used on all tissue samples and provides descriptive information regarding existing lesions in different renal parenchyma segments—this aids clinicians in determining differential diagnoses, particularly in pathologies affecting renal glomeruli. Histologic description of glomerular pathologies includes terms such as “proliferative” when there is an increase in the number of cells, “sclerosing” when there is scarring, and “necrotizing” when there are areas of cellular death. Lesions are further described as diffuse or focal if more or less than 50% of all glomeruli are involved, respectively. In an individual glomerulus, the process is considered global or segmental if more or less than 50% of the glomerular tuft is involved. LM can be further characterized based on the stains used; below is a brief description of various LM stains.

1. Haematoxylin-eosin staining for general evaluation.
2. Periodic Acid-Schiff stain (PAS) is widely used to evaluate glycogen storage disorders after kidney transplants to display tissue rejection (see Image. Normal Glomerulus, Periodic Acid-Schiff Stain).[17] 
3. Masson’s trichrome stain for the determination of renal fibrosis.[18]
4. Methenamine-silver stain (Jones) for better visualization of glomerular basement membranes.[19]

• Fluorescence microscopy (IF): As fluorescent dye-associated antibodies developed IF has revolutionized clinical nephrology and is particularly useful in determining the primary physiopathological mechanism generating a given renal lesion. This modality has helped guide the diagnosis of immune-mediated pathologies, as mentioned in other sections.

Microscopy, Electron

Electron microscopy (EM) has been widely used for diagnosing many common and uncommon diseases in nephrology, including minimal change disease, hereditary nephritis, fibrillary glomerulonephritis, and certain classes of lupus nephritis. This is one of the few medical disciplines in which EM has an active role in clinical practice, and findings from EM are necessary to make the final diagnosis. Therefore, it is generally recommended for renal biopsies to preserve a piece of specimen in an appropriate fixative for EM examination.[20][21]

Pathophysiology

Nephron pathologies are as complex as their structure. Each section of the nephron is susceptible to different forms of damage; for instance, glomerular diseases are often immunologically mediated, whereas tubular and interstitial disorders are more likely to be caused by toxic or infectious agents. However, a sole disease can affect more than one structure, and the structure interdependence in the kidney affects other components when only one part is damaged. Immune disorders affecting glomeruli can be either 1) mediated by antibodies against glomerular antigens, 2) mediated by complement, or 3) pauci-immune. The clinical manifestations and the microscopic appearance of the glomerulus will depend on the mechanism of damage.[22][2]

Clinical Significance

Diseases affecting the glomerulus generally divide into two different entities according to the clinical presentation: 

• Nephrotic syndrome: This syndrome presents with proteinuria >3.5g per 24 hours or protein-to-creatinine ratio >3000 mg/g, hypoalbuminemia <3g/dL, edema, and hyperlipidemia.[23]
• Glomerulonephritis or nephritic syndrome occurs when the patient presents with hypertension, hematuria, proteinuria (usually sub-nephrotic), and rapidly progressive azotemia.[24] 

Nephrotic Syndrome

Nephrotic syndrome may be primary or secondary. Primary nephrotic syndrome occurs when the kidney is the primary or sole affected organ. Secondary nephrotic syndrome is when systemic immunologic, metabolic, or vascular diseases affect the glomeruli. Out of these two, secondary nephrotic syndrome is the most common.[25] 

Primary Nephrotic Syndrome

The following are some of the most common causes of primary nephrotic syndrome. 

• Minimal Changes are the most common cause of primary nephrotic syndrome in children. It is idiopathic in most cases, but sometimes it is associated with neoplasias, recent infections, or vaccination. It is characterized by normal glomeruli appearance on H&E stain, negative immunofluorescence (IF) given no immune complex deposits, and effacement of foot processes on electron microscopy (EM).[25] 
• Focal Segmental Glomerulosclerosis (FSGS) is the most common cause of primary nephrotic syndrome in Hispanics and African American adults. It is usually idiopathic (primary)  but can be associated with HIV, sickle cell disease, or heroin use (secondary). It is characterized by focal and segmental sclerosis on H&E stain, effacement of foot processes on EM, and negative IF. This type of glomerulopathy often progresses to chronic renal failure.[25] 
• Membranous nephropathy is the most common etiology of primary nephrotic syndrome in White race adults. It is usually idiopathic (primary) but can be related to hepatitis, rheumatic diseases, neoplasias, or drugs (secondary). Microscopic characteristics are glomerular basement membrane thickening on H&E, subepithelial immune complex deposition with a “spike and dome” appearance on EM, and granular appearance on IF. Similar to FSGS, membranous nephropathy often progresses to chronic renal failure.[25]

Secondary Nephrotic Syndrome

Secondary nephrotic syndrome can result from systemic immunologic diseases, such as systemic lupus erythematosus or vasculitis, metabolic diseases like diabetes, or vascular diseases like hypertension. The most common cause of secondary nephrotic syndrome is diabetes mellitus. In diabetes, hyperglycemia leads to glycosylation of the vascular basement membrane, causing hyaline arteriolosclerosis. The efferent arteriole is most commonly affected, increasing the glomerular filtration pressure and hyperfiltration. This state eventually progresses to albuminuria, one of the first clinical markers of altered renal function. Histologically, it characteristically demonstrates mesangium sclerosis and the formation of Kimmelstiel Wilson nodules.[25]

Nephritic Syndrome

As with nephrotic syndrome, nephritic syndromes can also be primary or secondary. Some of the most common causes of nephritic syndrome are post-infectious glomerulonephritis, IgA nephropathy, and lupus nephritis.

• Poststreptococcal nephritic syndrome arises 2 to 3 weeks after group A B-hemolytic nephritogenic streptococcal infection of the skin (impetigo) or pharynx. It usually occurs in children but can also occur in adults. No characteristic finding on H&E stain but granular IF pattern and subepithelial immune complex deposition (“humps”) on EM.[24] 
• IgA is the most common nephropathy worldwide. It is characterized by the IgA immune complex deposition in the mesangium of glomeruli. It commonly presents as hematuria following mucosal infections, especially gastroenteritis.[24]

A special presentation of the nephritic syndrome is the Rapidly Progressive Nephritic Syndrome. In this clinical scenario, patients progress to renal failure in weeks to months. It presents with the characteristic crescents in the Bowman space on the H&E stain. Crescents are an extra capillary proliferation of macrophages, fibroblasts, and epithelial cells, as well as fibrin deposition due to a rupture of the glomerular membrane, indicating a severe injury to the glomerular capillary wall. The differential diagnosis is possible through histologic immunofluorescence patterns on renal biopsy. Immunofluorescence patterns help identify the etiology. 

• Linear pattern: caused by anti-basement membrane antibodies, as is characteristic of Goodpasture syndrome.
• [24]
• Granular pattern: caused by immune complex deposition. This pattern can occur in post-streptococcal glomerulonephritis or diffuse proliferative glomerulonephritis.[24]
• Negative immunofluorescence: in some diseases, such as Wegener granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome, the tissue is negative to immunofluorescence. This condition is known as pauci-immune glomerulonephritis, which is caused by antineutrophil cytoplasmic antibodies (ANCA).[24]