Primary focal segmental glomerulosclerosis

1. AIMS
To assess the expression of CD44 in MCNS and FSGS and to evaluate its
association with the known clinical and histopathological prognostic
factors.

2. A statistical significance was noted between serum creatinine and CD44 positivity in the
current study.
Hunt et al. proposed that an elevated plasma creatinine, either at onset or at 1 year and
heavy urinary protein loss persisting at 1 year were the factors most significantly related
to eventual ESRD, thus indicating the prognostic significance.
Korbet et al. could demonstrate that increased baseline creatinine value of >1.3 mg/dl
as a poor prognostic factor, and also 30% of patients with renal insufficiency (serum
creatinine ≥3.5 mg/dl) ended up with ESRD.

3. Estimated GFR (eGFR)
GFR represents the flow of plasma into the Bowman's space over a specified period. It
is a chief measure of kidney function.
 A decrease in GFR is considered a poor prognostic factor in NS.
There was a statistical association between eGFR and CD44 positivity in the present
study which correlated with other studies.
These studies demonstrated that CD44 positive cases had lower baseline eGFR, and
on follow up had a significant fall in eGFR.
Martinelli et al. could not demonstrate any prognostic significance for eGFR. However,
a reduced baseline GFR, indeed predicts the progression of the disease..

4. Histopathology
Mesangial hypercellularity
Mesangial changes following a glomerular injury include the production of
chemoattractants for inflammatory cells, proliferation of mesangial cells, and increased
ECM production.
Mesangial expansion with or without hypercellularity can cause compression of
glomerular capillaries causing decreased GFR which results in disease progression
The lack of association between mesangial hypercellularity and CD44 positivity as in
other studies could be due to a smaller sample size.

5. Podocyte hypercellularity
Six of the 30 FSGS patients (20.0%) had podocyte hypercellularity.
There was no statistical association between CD44 positivity and podocyte hypercellularity.
This is not at all surprising because glomerulomegaly in FSGS following a podocyte injury causes an
increase in GBM surface area.
In order to maintain coverage, podocyte tries to hypertrophy initially but gets arrested in the G1 phase
of the cell cycle leading to actin rearrangement, detachment, and finally cell death.
Podocytes may die by apoptosis, necrosis, necroptosis, and death due to decreased autophagy.
Podocyte depletion is well documented in both experimental animals and human kidneys.
Thus the six cases with podocyte hypercellularity in the current study may be due to the reparative
mechanisms in play.

6. Segmental sclerosis
Segmental sclerosis can occur due to post compensatory hemodynamic changes
following nephron loss or by primary podocyte damage due to mediators released from
mesangial cells following a glomerular injury.
The association between segmental sclerosis and CD44 positivity was in line with the
findings by Paik et al.
The more advanced the injury to the podocytes, the more decline in the renal function
and progression to ESRD ensues.

7. Tubular atrophy/interstitial fibrosis
Regardless of the etiology, TA is defined as the disappearance of either individual tubular
epithelial cells or entire tubules, often in conjunction with interstitial fibrosis.
TA is found to be a better predictor of the progression of renal failureThe association
between segmental sclerosis and CD44 positivity was in line with the findings by Paik et al.
Fibrosis on renal biopsy is an index of functional renal impairment and a predictor of
disease progression.
Studies have shown that IF > 50% are poor candidates for therapy effectiveness.
There was a statistical association between TA/IF with CD44 positivity (p = 0.027). This was
correlating with other studies.
Studies have shown that TA/IF > 20/30% is associated with a poor prognosis.
However, some studies failed to demonstrate any prognostic significance.
TA/IF due to any disease process carries a worse prognosis.

8. Histological diagnosis
Several possible explanations are given for the decreased yield in histological diagnosis of
FSGS:
(1) if glomerular scars are uniformly distributed in a biopsy with 10–30 glomeruli, the diagnostic
accuracy for the detection of at least one scarred glomerulus will be 80% when at least 10% of
juxtamedullary glomeruli or 20% of other cortical glomeruli are scarred.
 (2) With an average of 20 glomeruli with 20%–60% glomerular sclerosis, the predicte error rate is ±
50% for the extent of glomerular involvement.
Thus the distinction of MCD versus early FSGS may be difficult, particularly when biopsies
are small and diagnostic segmental lesions are not adequately sampled or when the
glomerular injury is in an early pre-sclerotic stage.
This is where activated PECs come into the picture. Activated PECs show de novo
expression of CD44 and thus help pick up early FSGS lesions surpassing the limitations
mentioned above
A statistical association was established between CD44 positivity and histological diagnosis.
This was corroborated by Hunt et al.
However, Martinelli et al. could not establish any prognostic significance with morphological
diagnosis.

9. Immunohistochemistry
The 90% CD44 positivity in FSGS biopsies makes it a sensitive marker. But we did
encounter CD44 negative FSGS cases.
This has decreased the specificity of the marker to 76.67%.
This could be explained by the fact that the sclerotic lesions could have been lost on
serial sectioning of the biopsies used for immuno-histological assessment.
Regarding the absence of sclerotic lesions in CD44 positive histologically diagnosed
MCNS cases, several explanations are possible.
Studies have shown that the small sclerotic lesions in CD44 positive MCNS cases were
not detectable in consecutive PAS-stained sections, prepared after the immunohistologic
analysis.

10. Thus, the cells expressing CD44 on the glomerular tuft could be an earlier marker than overt
sclerosis; whether these patients follow a course indicative of FSGS could not be evaluated
in those studies due to the lack of follow up.
In the present study, the original diagnosis was made on the sections of the same biopsy
analyzed for immunohistochemistry.
Although we examined only those biopsies with eight or more glomeruli, the sclerotic lesions
could still be present in other glomeruli or in different planes of sectioning.
We could not demonstrate any sclerotic lesions in these CD44 positive MCNS cases even
after reviewing the slides.
Surprisingly, the original diagnosis is normally based on 32 serial sections of a single
biopsy, thereby examining several planes of sectioning.
We stained and analyzed only one biopsy section per PEC marker staining, and still, it could
pick up these CD 44 positive cells which were missed in the earlier 32 sections.
This highlights the sensitivity of CD44. Immunohistologic staining for PEC markers could
detect sclerotic lesions in 25% of the biopsies originally diagnosed as MCNS in a recent
study,[1] which was reflected in the present study too.

11. The differentiation of MCNS from primary FSGS relies exclusively on the histologic
findings in the biopsy, emphasizing the relevance of the pathologic analysis.
In general, the lesions detected by PEC markers were small and often located close to
the glomerular tip.
The focal and segmental nature of FSGS, especially for these small lesions, may
explain why the lesions were missed in the original sections.
In fact, it is a well-known fact that small lesions particularly at the glomerular tip may be
missed in biopsies initially diagnosed as MCNS.
Another explanation is that CD44 could stain migrated PECs even before overt
sclerosis develops, reiterating that diagnosis made by light microscopy alone may be
insufficient.

12. The positivity of CD44 favors the view that MCNS and FSGS are a continuum of the
same entity and that in steroid responsive MCNS too prognosis can be uncertain
because of morphological transition to FSGS on repeat biopsy and subsequent renal
impairment develops later.
A hypothesis that a greater number of glomeruli present in the renal biopsies could be a
reason why CD44 positivity could be demonstrated in those histologically diagnosed
MCNS cases, has been disproved by several studies.
Present study also failed to demonstrate any statistical significance between these
variables (p = 0.131).
In fact, CD44 positivity in MCNS was seen more in biopsies with less than 25 glomeruli
in them.

13. Next, we compared the proven poor prognostic factors with CD44 positivity in the
MCNS group to speculate possible poor prognosis and thus indirectly to a diagnosis of
FSGS.
The mean serum creatinine among the CD44 positive MCNS cases was 1.466 ± 0.641
mg/dl, which is not significantly high.
Even other parameters both clinical and laboratory parameters could not give a hint
towards FSGS.
Five biopsies did not show any TA or IF. Even the remaining two cases also had TA/IF <
15%.
However, the glomeruli in the vicinity of TA/IF were histologically unremarkable.
There was no podocyte hypercellularity in any of these cases, again ruling out another
histological clue that could have made the pathologist think of an alternative diagnosis.
This further underscores the fact that CD44 can prove to be a game-changer to detect
activated PECs when light microscopy does not show any sclerotic lesion.