Diagnostic & monitoring tests for CRF as eGFR, Microalbuminuria & Reticulocyte hemoglobin (for identifying functional Iron deficiency) are important.

1. Update on Diagnostic tests in
Chronic Renal Disease
DR RAJESH V BENDRE
MD(Path), DNB(Path), DPB

2. Chronic Renal Disease- Introduction
CKD is defined as - Structural or functional abnormalities of the kidneys for >3 months, as manifested by
either:
1. Kidney damage, with or without decreased GFR, as defined by
◦ Pathologic Abnormalities
◦ Markers of kidney damage, including abnormalities in the composition of the blood or urine or abnormalities in imaging tests
2. GFR <60 ml/min/1.73 m2, with or without kidney damage
In India, there is a rising incidence of chronic kidney disease that is likely to pose major problems for both
healthcare and the economy in future years. The prevalence of CKD was observed to be 17.2% with ~6%
have CKD stage 3 or worse. Recently estimated age-adjusted incidence rate of ESRD is 229 per million
population (pmp), and >100,000 new patients enter renal replacement programs annually.
Epidemiology & risk factors of chronic kidney disease in India – results from the SEEK (Screening & Early Evaluation of Kidney
Disease) study.
Singh et al. BMC Nephrology 2013, 14:114

3. Classification of CKD by Diagnosis
Diabetic Kidney Disease
Glomerular diseases (autoimmune diseases, systemic
infections, drugs, neoplasia)
Vascular diseases (renal artery disease, hypertension,
microangiopathy)
Tubulointerstitial diseases (urinary tract infection,
stones, obstruction, drug toxicity)
Cystic diseases (polycystic kidney disease)
Diseases in the transplant (Allograft nephropathy, drug
toxicity, recurrent diseases, transplant glomerulopathy)

4. Progression of CKD after Diagnosis

5. CKD – Evaluation- Markers for Kidney Damage
Basic Screening-
Complete blood count (CBC), basic biochemistry panel with calculation of renal function, urinalysis, Urine
protein/creatinine ratio
Serum phosphate, 25-(OH) vitamin D, alkaline phosphatase, & intact PTH to screen for evidence of renal
bone disease.
Underlying Pathologic Disease specific Screening-
• Serum and Urine protein electrophoresis, serum and urine free light chains: Screen for a monoclonal
protein possibly representing multiple myeloma
• Antinuclear antibodies (ANA), dsDNA antibody: Screen for systemic lupus erythematosus
• Serum complement,C3,C4: Results may be depressed with some glomerulonephritides
• C-ANCA and P-ANCA: Positive findings helpful in diagnosis of Wegener granulomatosis &
microscopic polyangitis
• Anti–GBM antibodies: screen for underlying Goodpasture syndrome
• Hepatitis B & C, HIV, VDRL serology: for associated glomerulonephritides
• Renal Biopsy– for Evidence of glomerular disease without diabetes & Sudden onset of nephrotic
syndrome or glomerular hematuria

6. Basic Biochemistry- Creatinine & eGFR
The medical care of patients with CKD focuses on the following:
◦ Delaying or halting the progression of CKD through early diagnosing and treatment of underlying
pathologic disease & the manifestations of CKD
◦ Timely planning for long-term renal replacement therapy
In February 2014, the Canadian Society of Nephrology released new guidelines with following
recommendations-
◦ Delaying dialysis in CKD patients without symptoms until their glomerular filtration rate (eGFR)
drops to 6 mL/min/1.73 m2 or until the first onset of a clinical indication (which includes symptoms of
uremia, fluid overload, and refractory hyperkalemia or acidemia).
◦ Close monitoring should begin when eGFR reaches 15 mL/min/1.73 m2. Additional factors that may
affect dialysis initiation include patient education and modality selection, the severity of existing
uremic symptoms, and the rate of renal function decline

7. Creatinine & eGFR
One of the most common reasons for a nephrology consult is an elevated creatinine.
An elevation in the serum creatinine concentration usually reflects a reduction in the
glomerular filtration rate (GFR).
There are various causes of factitious elevation of creatinine. They can be broadly
grouped into increased production of creatinine, interference with the assay and
decreased tubular secretion of creatinine.

8. M Thorp. An Approach To The Evaluation Of An Elevated Serum Creatinine. The Internet Journal of
Internal Medicine. 2004 Volume 5 Number 2.
Creatinine & eGFR

9. Creatinine & eGFR
Renal function calculation in adult patients-
Modification of Diet in Renal Disease (MDRD) Study equation used to
calculate GFR. This equation does not require a patient's weight. However,
underestimates the measured GFR at levels > 60 mL/min/1.73 m2.
Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation is
more accurate than the MDRD Study equation overall and across most
subgroups and that it can report estimated GFRs that are at or > 60
mL/min/1.73 m2.
Renal function calculation in pediatric patients-
GFR in children is calculated using the Schwartz formula. An updated
equation includes cystatin C is also available.
Limitations-
These formulas may overestimate GFR, with change in methods used to
measure creatinine, hence following consistently with one laboartory &
method(IDMS traceable) is important.
Both the CKD-EPI and MDRD equations underestimated GFR in patients
with type 2 diabetes.Accuracy was 67% for the CKD-EPI equation and 64%
for the MDRD equation.

11. Urine SedimentAnalysis-
Akin to Liquid Renal Biopsy
The urine sediment examined by bright field microscopy under both low and high
power without staining. For accurate characterisation of cellular elements in
urine several staining techniques were developed. Dinda et al developed a
simple supravital stain using crystal violet and safranin O, which is effective
in differentiating various cell type including eosinophils and various casts. This
Supravital stain overcomes artefactual changes due to drying & fixation as with
other stains, definitely useful in doubtful cases routinely.
Active urinary sediment is defined as >5 RBCs & >5 WBCs per HPF and/or
cellular casts, which where not existing previously. These findings further
correlated with degree of proteinuria becomes an important diagnostic &
prognostic tool in patients with chronic renal disease, in both primary(eg.
glomerulonephritis) & secondary(eg. Lupus nephritis).

12. Hematuria- normal individuals do occasionally excrete RBC’s in the urine.
Widely accepted 3-5 RBC’s/hpf as upper limit of normal. Microscopic examination
for RBC’s is used as a diagnostic test, distinguishing between haematuria of
glomerular origin and haematuria of urinary tract is of importance.
• Using phase contrast microscopy (PCM)- Dysmorphic RBCs look different
from normal RBCs since they can have holes through them, protrusions, or both
& when present often associated with presence of RBC casts. By definition
there should be atleast two different populations of RBCs identified.
• Method to identify dysmorphic RBC’s described by Dinda et al, using
supravital staining and looking for G1 cells (RBC’s which are doughnut shaped
with one or more blebs). Once stained these cells can be identified by both
bright field microscopy and PCM with equal sensitivity and specificity. There is
a wide range of cut off values for dysmorphic red cells reported in the literature
to be used to distinguish between glomerular and non-glomerular pathology.
However most widely accepted cut off point is > 20% of dysmorphic red
cells for diagnosing glomerular haematuria with supravital stain and
bright field microscopy, a specificity of 82-92.8% and sensitivity of 88-
91.1%.
Urine Sediment Analysis-

13. Future- Urinary Sediment- New Biomarkers- Proteins, chemokines profiling

14. Clinical scenario Study mRNA in urinary cells Key findings
Primary Glomerular
disease and Chronic
kidney disease
Szeto et al. Nephrin/podocin Both increase in proteinuric glomerulonephritis
Szeto et al. TGF-β1 Correlation with GFR and the degree of tubulointerstitial scarring
MCP-1 Correlation with the degree of glomerulosclerosis
Lupus Nephritis Chan et al. TGF-β1 High expression in active disease; correlation with SLEDAI score and with
the histologic activity index
MCP-1 High expression in active disease, correlation with the degree of
glomerulosclerosis and the SLEDAI score
Chan et al. IFNγ High expression in active disease; correlation with SLEDAI score and the
SLEDAI renal score
IL-2 High expression in active disease
Avihingsanon et al. IP-10 CXCR3 TGF-β VEGF High expression in class IV LN; reduction with therapy
Renal Allograft Li et al. Perforin Granzyme B High expression in allograft acute rejection
Kotsch et al. Granulysin High expression in allograft acute rejection
Ding et al. CD103 High expression in allograft acute rejection
Tatapudi et al. IP-10 CXCR3 High expression in allograft acute rejection
Muthukumar et al. FOXP3 High expression in allograft acute rejection
Mas et al. TGF-β1 High expression in CAN
The urinary sediment beyond light microscopic examination
Studies investigating the cellular content of specific mRNAs in the urinary sediment
Future- Urinary Sediment- immune profiling

16. Interpretation Explanation
Marker of kidney
damage
Spot urine albumin-to-creatinine ratio >30 mg/g or
spot urine total protein-to-creatinine ratio >200 mg/g
for >3 months defines CKD
Clue to the type
(diagnosis) of CKD
Spot urine total protein-to-creatinine ratio >500-
1000 mg/g suggests diabetic kidney disease,
glomerular diseases, or transplant glomerulopathy.
Risk factor for adverse
outcomes
Higher proteinuria predicts faster progression of
kidney disease and increased risk of CVD.
Effect modifier for
interventions
Strict blood pressure control and ACE inhibitors are
more effective in slowing kidney disease
progression in patients with higher baseline
proteinuria.
Hypothesized
surrogate outcomes
and target for
interventions
If validated, then lowering proteinuria would be a
goal of therapy.
Importance of Proteinuria in CKD

17. Early Diagnosis & Monitoring for CKD-
GFR & Albuminuria

18. Kidney failure risk - Prediction Equations
The kidney failure risk equations were developed in patients
with CKD stages G3-G5 has now been validated in more
than 700,000 individuals spanning 30 + countries
worldwide.
The four and eight variable equations accurately predict the
2 and 5 year probability of treated kidney failure (dialysis
or transplantation) for a potential patient with CKD Stage 3
to 5.
Determining the probability of kidney failure may be useful
for patient triage, management of disease with timely
dialysis and counselling for kidney transplant. Prospective
trials evaluating the utility of this instrument for clinical
decision making are in progress.
Tangri N, Grams ME, Levey AS et al
Multinational Assessment of Accuracy of Equations for Predicting Risk of Kidney Failure: A Meta-analysis. JAMA.
2016;315(2):1-11.
Equations include following parameters-
• Four variable (age, gender, eGFR, Urine
albumin) &
• Eight variable (age, gender, eGFR, Urine
albumin, serum albumin, serum phosphorus,
serum bicarbonate, serum calcium) equations
For patients with CKD Stage 3,
predicted 5-year risk of kidney failure
• 0-5 % as low risk,
• 5-15 % as intermediate risk, &
• > 15 % as high risk

19. Feedback Loops in Secondary Hyperparathyroidism-
 Ca++
Decreased Vitamin D Receptors
and Ca-Sensing Receptors
 PTH
Bone Disease
Fractures
Bone pain
Marrow fibrosis
Erythropoietin resistance
 Serum P
 PTH
Systemic Toxicity
CVD
Hypertension
Inflammation
Calcification
Immunological
25D
1,25D
Calcitriol
Renal Failure

20. Pathology and Diagnosis of Bone Turnover in CKD
From the Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, 7th Edition., 2008 American Society for Bone and Mineral Research
Laboratory
values
KDOQI
Recommen
d.
Grading
KDIGO
Recommend.
Grading
iPTH (pg/mL) 150 to 300 Evidence Suggested range
2 to 9 x ULN 2C
Corrected Ca
(mg/dL) 8.4 to 9.5 Opinion
Suggested to
maintain in the
normal range
2D
P (mg/dL) 3.5 to 5.5 Evidence
Suggested to
lower toward
the normal
range
2C
CaxP (mg2/dL2) <55 Evidence
Not suggested
to direct clinical
practice
N/A

21. Pathology and Diagnosis of Bone Turnover in CKD
Phosphorus
Goal = Normal
Calcium
Calcification represents the
highest risk
Detect with x-ray/ultrasound
Restrict Calcium in
1. Hypercalcemia
2. Calcification
3. Low PTH
4. ADBD
PTH
Evaluate PTH in context of
hyperP, hypoCa, vitamin D
deficiency
Marked changes should trigger
treatment changes
Especially, in event of
hypocalcemia
Treat the trends: Treat P and Ca to normal, PTH to Goal
KDIGO International Clinical Practice Guidelines
KDIGO. Kidney Int. 2009; 76 (Suppl 113):S1-S130

22. Diagnostic assessment of anaemia [NICE2011]
Consider investigating and managing anaemia
in people with CKD if:
− Hb level falls to 11 g/dl or less (or 10.5 g/dl
or less if younger than 2 years)
or
− develop symptoms attributable to
anaemia (such as tiredness, shortness of
breath, lethargy and palpitations)
• The correction to normal levels of Hb with
ESAs is not usually recommended in people
with anaemia of CKD
• Typically maintain the aspirational Hb
range between:
Population Hb range
Adults, young people &
children > 2 yrs
10 and 12 g/dl
Children < 2 yrs 9.5 and 11.5 g/dl
– Do not wait until Hb levels are outside the
aspirational range before adjusting treatment,
for example, take action when Hb levels are
within 0.5 g/dl of the range’s limits

23. Anaemia Pathway: diagnosis
Is anaemia due to CKD?
• Consider other causes if eGFR ≥60 ml/min/1.73m2
Consider treating anaemia when:
• Hb falls to ≤ 11 g/dl (or ≤10.5 g/dl if younger than 2 years), or
• symptoms attributable to anaemia develop
Determine iron status:
Iron deficiency anaemia:
• diagnosed when serum ferritin < 100 μg/l
in stage 3 CKD & above.
•Functional iron deficiency defined by:
• serum ferritin > 100 μg/l and either %
hypochromic red cells > 6% (if test
available), or transferrin saturation < 20%
Iron correction should maintain:
• serum ferritin > 200 μg/l
• transferrin saturation > 20% (unless ferritin> 800 μg/l)
• % hypochromic red cells < 6% (unless ferritin>800 μg/l)
Review iron dose:
• when serum ferritin reaches 500 μg/l (should not rise
above 800 μg/l)
Optimise iron status:
• before or when starting ESAs, before deciding whether
to use ESAs in non-dialysis patients

24. Functional iron deficiency (FID)
Functional iron deficiency (FID) is a state in which there is insufficient iron incorporation into
erythroid precursors in the face of apparently adequate body iron stores, as defined by the
presence of stainable iron in the bone marrow together with a serum ferritin value within
normal limits
R e t i c u l o c y t e h e m o g l o b i n content (CHr) is a measurement of hemoglobin inside the
reticulocyte. It correlates directly with the functional availability of iron in the marrow
CHr constitutes the most valuable screening tool for identifying iron deficiency (ID) with or
without anaemia. Decreased CHr (cutoff value of 25 pg/cell) stands for accurate d i a g n o s i s o
f f u n c t i o n a l i r o n deficiency with sensitivity of 94% & specificity of 80%

25. CKD - Management

26. Genetic Testing in Nephrology
Nephrology Syndrome Panel Target genes- NGS, Multiplex PCR, Microarray Technologies
aHUS/TMA/C3 Glomerulopathy (13 genes) ADAMTS13, C3, CD46, CFB, CFH, CFHR1, CFHR2, CFHR3, CFHR4, CFHR5, CFI, DGKE ,THBD
Alport Syndrome (3 genes) COL4A3, COL4A4, COL4A5
Bartter Syndrome (5 genes) ATP6V1B1, BSND, CA2, CASR, CLCNKA
Nephrotic Syndrome & Focal Segmental
Glomerulosclerosis (34 genes)
ACTN4, ADCK4 (COQ8B), ANLN, APOL1, ARHGAP24, ARHGDIA, CD2AP, COL4A3, COL4A4, COL4A5, COQ2, COQ6,
CRB2, CUBN, EMP2, INF2, ITGA3, ITGB4, LAMB2, LMX1B, MEFV, MYH9, MYO1E, NEIL1, NPHS1, NPHS2, PDSS2,
PLCE1, PTPRO, SCARB2, SMARCAL1, TRPC6, TTC21B, WT1
Cystic Disease and Nephronopthisis (23 genes) AHI1, CEP290, GLIS2, INVS, IQCB1, NEK8, NPHP1, NPHP3, NPHP4, RPGRIP1L, TMEM67, TTC21B, XPNPEP3,
BICC1, CRB2, EYA1, HNF1B,PAX2, PKD1, PKD2, PKHD1, SIX5, UMOD
Polycystic kidney (5 genes) BICC1, PKD1, PKD2, NOTCH2, PKHD1
Renal tubular acidosis (5 genes) ATP6V0A4, ATP6V1B1, CA2, EHHADH, HNF4A
Inherited renal diseases are estimated to cause 50% of pediatric chronic renal failure (CRF) and 20% of adult CRF
A significant proportion of these patients with nephrotic syndrome (NS) suffer from steroidresistant NS.
Establishing the underlying genetic defect and inheritance pattern also allows family member testing to identify at-risk relatives.

27. CKD - Summary
CKD patients are asymptomatic for long periods, hence basic simple
tests like serrum creatinine with eGFR, Albuminuria & Urinary
sediment analysis are the key in early diagnosis & followup of CRF
patients
Associated Diabetes & Hypertension are major risk factor for rapid
progression to disease, hence early therapeutic intervention will
prolong the adverse outcome.