Investigation of Nephrotic Syndrome

1. Urine Investigations

Urine Dipstick and Microscopy

• Dipstick typically shows 3+ to 4+ proteinuria
• Urine microscopy: look for oval fat bodies, fatty casts (Maltese cross pattern under polarized light), and lipiduria - hallmarks of nephrotic syndrome
• Hematuria (RBCs or RBC casts) suggests a nephritic component; their presence should prompt consideration of lupus nephritis, MPGN, or IgA nephropathy

Quantification of Proteinuria

• 24-hour urine protein collection: gold standard; nephrotic range = >3.5 g/day in adults (>40 mg/m²/hr in children)
• Spot urine protein:creatinine ratio (PCR): correlates well with 24-hour collection; PCR >3.0 is consistent with nephrotic syndrome. First-morning void sample is most accurate. This is now preferred in clinical practice, especially in children
• Normal values: >2 years old PCR <0.2; 6 months-2 years PCR <0.5

Urine Protein Electrophoresis (UPEP)

• Distinguishes selective (predominantly albumin - suggests MCD) from non-selective proteinuria (mixed - suggests FSGS, membranous nephropathy)

2. Blood Tests

Routine Bloods

Immunological / Serological Screen

In cases with nephritic or nephrotic-nephritic syndrome, a low serum complement strongly suggests immune complex-mediated disease. In pure nephrotic syndrome, complement levels are usually normal. - Goldman-Cecil Medicine

3. Imaging

Renal Ultrasound

• Usually the first-line imaging
• In nephrotic syndrome the kidneys are often normal in size or enlarged (echogenic kidneys suggest chronicity or infiltrative disease)
• Can detect structural abnormalities (polycystic kidney disease, reflux nephropathy), ascites, and obstruction
• Generally non-specific but useful for pre-biopsy assessment

Chest X-Ray

• Can demonstrate pulmonary edema, pleural effusion (from fluid overload)

Doppler Ultrasound / CT Venography

• If renal vein thrombosis is suspected (especially in membranous nephropathy with severe proteinuria >10 g/day and albumin <2 g/dL)
• Pulmonary embolism workup (CTPA) if indicated by symptoms

4. Kidney Biopsy

• Light microscopy (H&E, PAS, silver, Masson trichrome stains)
• Immunofluorescence (IgG, IgA, IgM, C3, C4, C1q deposits)
• Electron microscopy (foot process effacement, deposit location)

When is biopsy indicated?

• All adults with nephrotic syndrome (biopsy changes management in most cases)
• Children: NOT routinely required in first-onset typical nephrotic syndrome (assumed MCD and treated empirically with steroids). Biopsy is indicated in children if:
  • Age <1 year or >8 years (lower prior probability of MCD)
  • Hematuria, hypertension, or elevated creatinine (suggesting nephritic component)
  • Steroid-resistant disease (no remission after 4-8 weeks)
  • Steroid-dependent or frequently relapsing

Key biopsy findings:

• Light microscopy: normal glomeruli (hence the name)
• Electron microscopy: widespread podocyte foot process effacement - the hallmark finding
• No immune deposits on IF

5. Additional/Targeted Investigations

6. Summary Algorithm

Suspected Nephrotic Syndrome
       ↓
Urinalysis + urine PCR (>3.0 = nephrotic range)
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Bloods: albumin, lipids, renal function, glucose/HbA1c
       ↓
Secondary cause screen:
  ANA/dsDNA → Lupus
  HBV/HCV/HIV → Viral
  Complement (C3, C4)
  SPEP/immunofixation → Myeloma/amyloid
  Anti-PLA2R → Primary membranous
       ↓
Renal ultrasound
       ↓
Adults: Kidney biopsy (almost always)
Children (typical NS): Empirical steroids → biopsy if steroid-resistant

Key Complications to Screen For at Presentation

1. Hypercoagulability: DVT, pulmonary embolism, renal vein thrombosis - due to urinary loss of antithrombin III, protein C, and protein S. Risk highest in membranous nephropathy (especially with albumin <2 g/dL and proteinuria >10 g/day)
2. Infections: Hypogammaglobulinaemia (IgG lost in urine) + T-cell dysfunction - especially pneumococcal peritonitis in children
3. AKI: From volume depletion (low oncotic pressure) or acute tubular necrosis
4. Metabolic: Vitamin D deficiency, hypothyroidism, iron deficiency anaemia

Role of Electron Microscopy (EM) and Immunofluorescence (IF) in Diagnosis of Glomerular Lesions

Principles

Immunofluorescence (IF)

• Granular ("lumpy-bumpy"): Indicates immune complex deposition - either from circulating complexes deposited in the GBM or from in situ immune complex formation. Seen in most glomerulonephritides.
• Linear: Indicates antibody binding to continuously distributed GBM antigens. Classic of anti-GBM disease (Goodpasture syndrome). The antibodies are directed against type IV collagen (α3 chain).
• Negative / No deposits: Seen in MCD, FSGS (primary), diabetic nephropathy, and thrombotic microangiopathies.

Electron Microscopy (EM)

1. Localizing immune deposits by their ultrastructural position relative to the GBM:
   • Subendothelial (between endothelium and GBM inner lamina): associated with greater inflammation (accessible to complement/leukocytes from blood)
   • Subepithelial (between GBM outer lamina and podocyte): often produces proteinuria without overt inflammation - typical of membranous nephropathy
   • Intramembranous (within GBM): characteristic of dense deposit disease (DDD)
   • Mesangial: associated with IgA nephropathy and early lupus nephritis classes
2. Characterizing foot process effacement: The hallmark of all causes of nephrotic syndrome. Foot process effacement (fusion) results from disruption of slit diaphragms
3. Identifying specific structures: Fibrils in amyloidosis; tubuloreticular inclusions (TRI) in lupus nephritis; organized microtubular deposits in fibrillary/immunotactoid GN

EM and IF Findings in Specific Glomerular Diseases

1. Minimal Change Disease (MCD)

• The absence of IF deposits distinguishes MCD from all immune complex diseases
• EM foot process effacement is the only abnormality and is the primary pathological finding
• Steroid therapy reverses the foot process changes concomitantly with clinical remission

2. Focal Segmental Glomerulosclerosis (FSGS)

• EM helps distinguish FSGS from other causes of focal scarring (e.g., healing post-infectious GN, diabetic nodules) - these may show GBM deposits or structural changes not seen in primary FSGS
• The collapsing variant shows podocyte detachment and retraction on EM

3. Membranous Nephropathy (MN)

• In primary MN: anti-PLA2R antibodies bind to PLA2R antigen on podocytes → in situ subepithelial immune complex formation → granular IF
• EM stages of MN (Ehrenreich-Churg staging): Stage I = subepithelial deposits; Stage II = spikes; Stage III = deposits engulfed by GBM; Stage IV = irregular scarred GBM

4. IgA Nephropathy (Berger's Disease)

• IF is the most critical investigation - IgA in the mesangium is pathognomonic
• Without IF, IgA nephropathy cannot be reliably distinguished from other mesangial GN on LM alone
• Oxford MEST-C classification scores mesangial hypercellularity (M), endocapillary hypercellularity (E), segmental glomerulosclerosis (S), tubular atrophy/interstitial fibrosis (T), and crescents (C)

5. Membranoproliferative Glomerulonephritis (MPGN)

6. Poststreptococcal GN (Diffuse Proliferative GN)

7. Anti-GBM Disease (Goodpasture Syndrome)

• Serum anti-GBM antibodies correlate with IF and confirm the diagnosis without biopsy in many cases

8. Lupus Nephritis

9. Diabetic Nephropathy (Diabetic Kidney Disease)

10. Amyloidosis

Summary Table: EM and IF Patterns by Disease

Clinical Significance

1. IF is often the single most discriminating test in biopsy interpretation - the IgA predominance in IgA nephropathy, the linear IgG of anti-GBM, and the full house of lupus cannot be inferred from LM alone
2. EM confirms and characterizes what IF localizes - it identifies the precise substructural position of deposits, characterizes their internal architecture (fibrils vs. amorphous), and provides a complete picture of podocyte health
3. In diseases where IF and LM are both negative (MCD, FSGS, diabetic nephropathy), EM of the foot processes is the only modality that reveals the pathological change driving proteinuria
4. Together with LM, all three modalities provide a definitive pathological diagnosis guiding immunosuppressive treatment, prognosis, and genetic counseling

Part 1: Chemical Pathology in Acute Renal Failure (Acute Kidney Injury)

A. Azotaemia (Elevated BUN and Creatinine)

• Serum creatinine: The most widely used marker of renal function. In AKI, it rises due to decreased GFR (reduced filtration of creatinine) and increased muscle breakdown. Important caveat: fluid resuscitation can artificially lower creatinine by dilution; diuresis can raise it.
• BUN: A product of protein catabolism. Normal BUN:creatinine ratio is ~10:1.
  • BUN:Cr ratio >15-20 → suggests prerenal azotaemia (volume depletion, effective underfilling, GI bleeding, hypercatabolic states, or steroid use)
  • BUN:Cr ratio ~10 → suggests intrinsic renal (tubular) injury (ATN) where tubular function is damaged and BUN cannot be differentially reabsorbed
  • Ratio may be spuriously low in malnutrition, liver disease, or low protein intake
• BUN goal for RRT: Keep BUN <100 mg/dL (37.7 mmol/L), though no absolute threshold

B. Sodium and Water Imbalance

• Hyponatremia is common in AKI, primarily dilutional, due to impaired free water excretion from the failed tubules
• Oliguria (<400 mL/day) is the hallmark of severe AKI - reduced GFR limits the ability to excrete filtered water
• Fluid overload/oedema arises from inability to excrete sodium and water, especially if IV fluids are continued

Fractional Excretion of Sodium (FENa)

• FEurea <35% → prerenal; >50% → intrinsic

C. Metabolic Acidosis

• The kidney normally excretes ~50-70 mEq of H⁺/day as NH₄⁺ and titratable acid
• In AKI, this excretion fails, leading to H⁺ retention
• Early AKI: Primarily hyperchloraemic metabolic acidosis (normal anion gap) - Cl⁻ accumulates as NH₄⁺ excretion falls
• Later/severe AKI: High anion gap metabolic acidosis develops as unmeasured anions accumulate (phosphate, sulfate, urate, organic acids). 50% of AKI in critical illness has a normal anion gap; early in AKI, hyperchloremia is the principal source of acidosis; subsequently, 50-60% is caused by unmeasured anions and up to 30% is associated with hyperphosphatemia.
• Indications for RRT: Metabolic acidosis with pH <7.2 is an indication for urgent renal replacement therapy (RRT)

D. Hyperkalaemia

• Potassium is primarily excreted by the kidneys; in AKI, filtered load decreases and tubular secretion fails
• Compounded by:
  • Acidosis: H⁺/K⁺ exchange shifts K⁺ out of cells (~K⁺ rises 0.6 mEq/L per 0.1 fall in pH)
  • Catabolism/cell lysis: Rhabdomyolysis, haemolysis, tissue necrosis release intracellular K⁺
  • Inadequate excretion: Oliguria or anuria prevents K⁺ clearance
• Serum K⁺ >6.5 mEq/L with ECG changes is an emergency indication for dialysis
• ECG changes of hyperkalaemia: peaked T waves → prolonged PR → wide QRS → sine wave → VF

E. Hyponatraemia and Hyperphosphataemia

• Hyperphosphataemia: Phosphate (normally excreted by the kidney) accumulates in AKI. Contributes to metabolic acidosis (~30% of the anion excess in renal acidosis)
• Hypocalcaemia: Secondary to hyperphosphataemia (Ca × PO₄ product precipitation) and impaired renal conversion of 25-OH Vitamin D to active 1,25-(OH)₂ Vitamin D. Hypocalcaemia worsens the cardiac toxicity of hyperkalaemia

F. Hyperuricaemia

• Uric acid (end product of purine metabolism) is normally excreted by the kidney
• In AKI, uric acid accumulates - can worsen tubular injury (urate crystal deposition) and is especially severe in tumour lysis syndrome

G. Urine Chemistry Findings in AKI

H. Biomarkers of AKI (Beyond Creatinine)

I. Summary - AEIOU Indications for Urgent Dialysis in AKI

Part 2: Pathophysiology of Chronic Renal Failure (CKD)

A. Causes of Initial Nephron Loss

1. Diabetic kidney disease (DKD) - most common in developed countries
2. Hypertensive nephrosclerosis
3. Glomerulonephritis (primary and secondary)
4. Polycystic kidney disease
5. Recurrent pyelonephritis/obstructive nephropathy
6. Amyloidosis, sickle cell nephropathy, others

B. The Intact Nephron Hypothesis and Single Nephron Hyperfiltration

• Loss of nephrons → reduction in total renal mass
• Surviving nephrons undergo hypertrophy (glomerular enlargement)
• Increased afferent arteriolar vasodilatation (relatively more than efferent vasoconstriction) → increased intraglomerular hydrostatic pressure
• GFR per nephron rises (single-nephron hyperfiltration)
• This is initially adaptive (maintains solute excretion) but is ultimately maladaptive:
  • Increased transcapillary filtration pressure damages glomerular capillary endothelium
  • Promotes proteinuria (disruption of size and charge selectivity barriers)
  • Activates TGF-β → mesangial expansion → glomerulosclerosis
  • Loss of additional nephrons → more hyperfiltration in remaining nephrons → progressive loss → end-stage kidney disease (ESKD)

C. Proteinuria as a Driver of Progression

• Glomerular hypertension increases protein leak across the damaged filtration barrier
• Proteinuria itself is nephrotoxic: filtered proteins (especially albumin, transferrin, complement) are taken up by proximal tubular cells, activating NF-κB, releasing cytokines (MCP-1, RANTES), and recruiting interstitial macrophages and T cells
• This drives tubulointerstitial inflammation and fibrosis - the final common pathway of CKD regardless of initial aetiology
• Hypertension accelerates this process by further raising intraglomerular pressure

D. Tubulo-interstitial Fibrosis - The Final Common Pathway

1. TGF-β1 upregulation - the master fibrogenic cytokine
2. Epithelial-to-mesenchymal transition (EMT): tubular epithelial cells acquire fibroblast phenotype
3. Myofibroblast activation: produce collagen I, III, IV → replace functional parenchyma with scar
4. Complement activation: C3a/C5a released locally amplify inflammation
5. Renin-angiotensin system (RAS) activation: angiotensin II is directly fibrogenic (stimulates TGF-β, MCP-1) in addition to causing hypertension

E. Systemic Consequences of Reduced GFR - The Uraemic Syndrome

1. Fluid and Electrolyte Disturbances

2. Acid-Base Disorder

• Uremic acidosis (most common): Decreased ammonia production from reduced nephron mass → inability to excrete H⁺ → metabolic acidosis
• Mechanism: ↓ NH₃ synthesis → ↓ urinary buffering → H⁺ accumulation
• Initially normal anion gap (hyperchloraemic)
• At GFR <25 mL/min (Stage G4-G5): elevated anion gap as phosphate, sulfate, and organic anions accumulate
• Serum bicarbonate typically stabilises at 12-18 mEq/L (bone acts as buffer)
• Causes of early-stage acidosis:
  • Type 1 RTA (distal: impaired H⁺ secretion, non-acid urine, hypokalemia)
  • Type 2 RTA (proximal: impaired HCO₃⁻ reabsorption, hypokalemia)
  • Type 4 RTA (hyporeninemic hypoaldosteronism: acid urine, hyperkalemia - most common in diabetic nephropathy)

3. Calcium, Phosphate, and Bone Disease (CKD-MBD)

1. ↓ GFR → phosphate retention (phosphate cannot be excreted)
2. ↑ Serum phosphate → directly stimulates PTH secretion
3. ↓ Renal 1α-hydroxylase activity → ↓ 1,25-(OH)₂ Vitamin D (calcitriol)
4. ↓ Calcitriol → reduced intestinal Ca²⁺ absorption → hypocalcaemia
5. Hypocalcaemia + hyperphosphataemia → further stimulate secondary hyperparathyroidism
6. PTH causes bone resorption (osteitis fibrosa cystica), but also vitamin D deficiency causes defective mineralisation (osteomalacia)
7. Fibroblast Growth Factor-23 (FGF-23) rises very early in CKD (before PTH rises) as a phosphaturic factor; elevated FGF-23 is independently associated with left ventricular hypertrophy and cardiovascular mortality
8. Ca × PO₄ product elevation → vascular and soft tissue calcification (metastatic calcification)
9. In ESKD: autonomous PTH secretion = tertiary hyperparathyroidism

4. Anaemia of CKD

• Mechanism: ↓ erythropoietin (EPO) production by peritubular fibroblasts of the kidney (main driver)
• Compounded by: ↓ red cell survival, iron deficiency (absolute or functional - elevated hepcidin blocks iron absorption and release from macrophages), deficiency of folate/B12, blood loss from dialysis
• Normochromic, normocytic (or mildly microcytic if iron-deficient)
• Appears at stage G3a-G3b; becomes significant (Hb <10 g/dL) at stage G4
• Contributes to: fatigue, reduced exercise tolerance, left ventricular hypertrophy (compensatory for tissue hypoxia)

5. Cardiovascular Complications

6. Uraemic Neurotoxicity

• At GFR <15 mL/min (stage G5): accumulation of uraemic toxins (particularly middle molecules, phenols, indoles, and guanidino compounds) causes:
  • Peripheral neuropathy (glove-and-stocking sensorimotor; "restless legs syndrome")
  • Uraemic encephalopathy (asterixis, confusion, seizures, coma)
  • Autonomic neuropathy
• Elevated prolactin levels (normally cleared by kidney) → sexual and reproductive dysfunction

7. Uraemic Bleeding Tendency

• Platelet dysfunction (qualitative defect): uraemic toxins impair platelet-vWF binding (GPIb-vWF interaction) and platelet degranulation
• Anaemia worsens bleeding (RBCs normally promote platelet margination to vessel wall)
• Results in prolonged bleeding time despite normal platelet count, PT, and aPTT

8. Immunodeficiency

• Uraemia is an immunocompromised state
• Impaired neutrophil and T-cell function → increased susceptibility to infections (especially encapsulated organisms, TB)
• Hypogammaglobulinaemia in nephrotic states

F. CKD Staging and Biochemical Manifestations by Stage

G. Summary of Pathophysiological Mechanisms

PRIMARY INSULT (diabetes, hypertension, GN, etc.)
         ↓
Nephron loss
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Surviving nephrons: HYPERFILTRATION (SNGFR ↑, glomerular HTN)
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Proteinuria → tubular injury → TGF-β ↑ → tubulointerstitial fibrosis
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Progressive nephron loss (self-perpetuating cycle)
         ↓
↓ GFR: Retention of H⁺, K⁺, PO₄³⁻, Na⁺, H₂O, uraemic toxins
         ↓
Metabolic acidosis | Hyperkalaemia | CKD-MBD | Anaemia | CVD | Uraemia