The Kidney Biopsy in Lupus

N e p h r i t i s : Is It Still Relevant?
Brad H. Rovin,

MD*,

Samir V. Parikh,

MD,

Anthony Alvarado,

MD

KEYWORDS
 Lupus nephritis  Kidney biopsy  Systemic lupus erythematosus
KEY POINTS
 The kidney biopsy is the standard of care for diagnosis of lupus nephritis and remains
necessary to ensure accurate diagnosis and guide treatment.
 Repeat biopsy should be considered when therapy modifications are necessary, as in
cases with incomplete or no response, or when stopping therapy for those in remission.
 There are several promising biomarkers of kidney disorders; however, these markers
needed to be validated in a prospective clinical trial before being applied clinically.
 Molecular analysis may provide the information presently lacking from current evaluation
of kidney disorders and may better inform prognosis and treatment considerations.

INTRODUCTION

The percutaneous kidney biopsy was introduced in the 1940s and incorporated into
clinical practice in the 1950s.1,2 This procedure, along with advances in the histopathologic examination of kidney tissue such as immunofluorescence and electron
microscopy, greatly enhanced understanding of the pathogenesis of human glomerulonephritis. Perhaps more than for any of the other glomerular diseases, biopsy findings have been used to classify and subgroup lupus nephritis (LN) in order to inform
treatment decisions and predict prognosis. Several LN classification schemes have
been applied clinically, the most recent being from collaboration between the International Society of Nephrology and the Renal Pathology Society in 2004.3 In an effort to
forecast kidney outcomes, pathologic findings in LN have also been combined into
composite indices of active and chronic disease independent of LN class.4,5
The role of the kidney biopsy in LN has recently been challenged on several fronts:
(1) the widespread clinical practice of using mycophenolate mofetil (MMF) for

Disclosures: None.
Nephrology Division, Department of Internal Medicine, Ohio State University Wexner Medical
Center, 395 West 12th Avenue, Columbus, OH 43210, USA
* Corresponding author. Medical Office Tower, Ground Floor, 395 West 12th Avenue, Columbus, OH 43210.
E-mail address: [email protected]
Rheum Dis Clin N Am 40 (2014) 537552
http://dx.doi.org/10.1016/j.rdc.2014.04.004
rheumatic.theclinics.com
0889-857X/14/$ see front matter 2014 Elsevier Inc. All rights reserved.

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induction treatment of proliferative and membranous LN has led many clinicians to

question the need for diagnostic biopsies to guide therapy. (2) Diagnostic biopsies
do not predict treatment response or long-term kidney outcomes in LN, and the added
value of serial biopsies remains unclear. (3) Noninvasive biomarkers of renal disorders
are actively being sought to substitute for invasive kidney biopsies. (4) The kidney biopsy is still only analyzed histologically, whereas molecular diagnostics are being
applied to biopsies of other diseases to personalize therapy.
In light of these challenges, this article reexamines the kidney biopsy in LN in order
to assess its ongoing relevance to disease management, and considers how molecular analyses can increase the information derived from the kidney biopsy.
WHEN AND HOW OFTEN DOES A PATIENT WITH LN NEED A KIDNEY BIOPSY?

An evidence-based literature has developed around this question, but to date there is
no consensus answer. Three possible responses are considered here.
1. A kidney biopsy should routinely be obtained to confirm the diagnosis of LN before
treatment is started.
This traditional approach is used when patients with systemic lupus erythematosus
(SLE) develop clinical evidence that is consistent with renal involvement by lupus and
that cannot be explained by other conditions. These clinical findings include hematuria, pyuria, red and white blood cell casts, declining kidney function, and/or proteinuria.6 Dysmorphic red blood cells, specifically acanthocytes (Fig. 1), indicate
glomerular hematuria and are often seen in the urine sediment of patients with LN
with active nephritis. Red blood cell casts (see Fig. 1) also indicate glomerular hematuria, but are found less commonly. Urine white blood cells and white blood cell casts,
in the absence of kidney or urinary tract infection, are consistent with kidney inflammation caused by LN. A kidney biopsy is generally not indicated for hematuria or pyuria
alone, but patients who develop active urine sediment require close follow-up for signs
of worsening kidney injury such as proteinuria and an increase in serum creatinine.
Besides LN, the differential diagnosis of renal dysfunction in patients with SLE includes tubular injury caused by systemic infection or nephrotoxins. Patients with lupus
are susceptible to infection because they are routinely immunosuppressed, and often

Fig. 1. Urine findings of glomerular hematuria in LN. (A) Acanthocytes are a type of dysmorphic red blood cell that is specific for glomerular hematuria. The arrow indicates a bleb that
distorts the normal biconcave disc appearance of the red blood cell. (B) Red blood cell casts
also indicate glomerular bleeding.

Kidney Biopsy in Lupus Nephritis

receive medications that are potentially nephrotoxic. Renal insufficiency caused by LN

is usually accompanied by proteinuria and/or hematuria.
The most common clinical finding that triggers a request for kidney biopsy in patients with SLE is proteinuria of at least 500 to 1000 mg/d, in the presence or absence
of renal insufficiency or active urine sediment. Although there is no set proteinuria
threshold for biopsy, patients with proteinuria of 500 mg/d or less can have severe
LN.7 In a pilot study of 38 patients with lupus undergoing a kidney biopsy for the first
time, and who had glomerular hematuria with less than 500 mg/d proteinuria, 95% of
the patients had class III, IV, or V LN, and only 5% had class II.8 These data show the
difficulty of estimating kidney involvement in SLE using clinical data without histologic
examination of the kidney.
2. A kidney biopsy is not routinely needed before starting therapy for LN.
This nontraditional approach is mainly a response to the overwhelming acceptance
of MMF as first-line therapy for all of the serious forms of LN (class III, IV, and V),9 theoretically eliminating the need to differentiate between classes before starting therapy.
Furthermore, it has been difficult to show that the pathologic findings at diagnostic kidney biopsy prognosticate how the kidney will do in the long term or how it will respond
to therapy in the short term.10,11 However, it has been shown that a kidney biopsy
done after completion of induction therapy does provide prognostic information on
renal outcomes.10,11 It may therefore be advantageous to delay biopsy until induction
treatment is finished.
In contrast, we suggest a diagnostic kidney biopsy before therapy is necessary for
the following practical (13) and theoretic reasons (4): (1) patients with SLE may also
develop glomerular diseases that are not the classic immune complexmediated
glomerulonephritis that is defined as LN. Most commonly these are histologically
similar to minimal change disease or focal segmental glomerulosclerosis, and have
been termed lupus podocytopathies, but other disorders can also be seen.1215 The
incidence of nonimmune-complex glomerulonephritis in patients with SLE is hard to
estimate, but was 5% in a series of more than 200 patients with SLE.13 These glomerulopathies cannot be distinguished from LN clinically; a biopsy diagnosis is required.
Furthermore, the treatment of nonimmune-complex nephritis is not necessarily the
same as for LN. For example, the lupus podocytopathies often respond to short
courses of corticosteroids alone, and do not need the addition of a cytotoxic agent.16
(2) Renal thrombotic microangiopathy caused by antiphospholipid syndrome is found
in about 30% of patients with lupus, and can occur alone or with classic immunecomplex LN.1719 Renal thrombotic microangiopathy cannot be diagnosed without a
biopsy. It is an important finding because treatment is anticoagulation, and failure to
treat may lead to insidious loss of kidney function despite adequately addressing
immune-complex LN with immunosuppression.20 (3) As discussed previously, it is difficult to predict the extent of renal histologic activity or chronicity using only clinical information such as serum creatinine, level of proteinuria, or urine analysis.21,22 The balance
between activity (glomerular neutrophils, necrosis, endocapillary hypercellularity,
cellular crescents, interstitial inflammation) and chronicity (glomerulosclerosis, fibrous
crescents, interstitial fibrosis, tubular atrophy) dictates whether to immunosuppress or
to use kidney-protective therapies such as strict blood pressure control, sodium restriction, and inhibitors of the renin-angiotensin-aldosterone system.23 (4) Several
novel biologics have been tested and failed as therapies for LN, and more are in development.24 A factor contributing to these failures may be the heterogeneity of LN. Success with the new, highly specific agents may be limited to certain subsets of patients
with LN, and the kidney biopsy is likely to be required to identify responsive patients.

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3. A diagnostic kidney biopsy and a follow-up biopsy during treatment should be

routinely done in patients with LN.
The data on repeat biopsies for LN come from studies that have been done for clinical indications (ie, for patients with LN who did not respond to therapy as expected)
and from protocol biopsy studies in which the repeat biopsy was done after induction
or maintenance therapy to determine the effect of treatment on kidney histology.
These investigations have provided important information on the prognostic value of
the kidney biopsy for long-term renal health, and a time line of the renal histologic
response to treatment.
Protocol biopsies performed after 6 to 9 months of induction therapy in adults and
children have shown that the second biopsy is more predictive of long-term patient
and kidney outcomes than the initial biopsy.10,11,25 In adults, the findings at the
6-month biopsy that predicted a doubling of serum creatinine (a surrogate for endstage kidney disease) after a mean follow-up of 7.6 years were ongoing glomerular
and interstitial inflammation, ongoing presence of glomerular capillary immune complexes, and the presence of macrophages in tubular lumens.10,25 The extent of chronicity on the second biopsy did not predict long-term outcome. Other studies reported
on the relationship between repeat kidney biopsies a year or more after completion of
induction therapy and kidney outcomes 7 to 8 years later.26,27 The activity index4 on
the repeat biopsy, persistent glomerular and tubulointerstitial inflammation, and
persistent or worsening of subendothelial immune-complex deposits were predictive
of poor long-term outcomes such as doubling of serum creatinine, renal impairment,
or death.
One potential confounding issue in all of these studies is that treatment after the
second biopsy was not standardized and/or not described. Therefore it is not possible
to determine the impact of treatment decisions on long-term kidney outcomes, or how
treatment affects the predictive value of these pathologic findings. Nonetheless, it is
reassuring that different cohorts, undergoing second biopsies at different intervals,
found similar pathologic predictors of renal deterioration.
Protocol repeat biopsy studies also show how the kidney responds to treatment.
Second biopsies done directly after induction therapy with corticosteroids, plus a cytotoxic agent or antimetabolite, generally show a decline in active lesions, but an increase in scarring lesions.28 There is seldom complete histologic remission after
induction therapy. However, some patients did not show active lesions after induction,
which raises the question of whether they need further maintenance immunosuppression.10,28 There are no data to answer this question, and focal active lesions could have
been missed on the biopsy, but, if studied prospectively and such patients did well
without ongoing treatment, there would be a strong argument for performing a second
biopsy after initial treatment to see who could immediately stop further toxic therapy.
Repeat biopsy studies in which the second biopsy was done after several years
(1.54.7 years) of treatment are even more informative than repeat biopsies done after
induction.26,27,2931 After prolonged standard-of-care treatment, 30% to 77% of patients still had persistent proliferative or class V LN. More concerning, 30% to 60%
of patients with complete clinical renal response had persistent histologic activity,
including glomerular and tubulointerstitial inflammation and subendothelial immune
deposits.26,31 As discussed previously, these histologic findings at repeat biopsy
have been associated with poor long-term kidney outcomes. These data reinforce
the known discordance between clinical measures of remission and histologic remission, and raise the question of whether a repeat biopsy is needed to define LN remission. The concept of histologic remission is also applicable to patients who have been

Kidney Biopsy in Lupus Nephritis

treated aggressively but have persistent proteinuria. A recent repeat biopsy investigation showed that several such patients had no residual histologic activity, and,
although not considered to be complete responders on clinical grounds, seemed to
be complete responders histologically.32
Repeat biopsies for clinical indications have been done to assess LN flares or to
determine why patients have not responded to treatment.30,33,34 Some investigators
have suggested that the decision to repeat a biopsy at LN flare should be based on
the LN class on the initial biopsy.35 Class switch in LN is common,22,35 but a class
switch from a proliferative lesion to a pure nonproliferative class is less common.35
Thus patients who have a nonproliferative LN class at baseline biopsy are more likely
to benefit from a repeat biopsy. In general, most reports conclude that the information
gathered from repeat biopsies in patients doing poorly has been useful in deciding
further management.
Synthesizing all of these data, Fig. 2 presents our suggested algorithm of when to
do a kidney biopsy in patients with LN.
BIOMARKERS OF KIDNEY DISORDERS

LN is so intrinsically heterogeneous it is difficult to adequately convey what is seen on

kidney biopsy using only standard classification terminology. LN classes are divided
into active, active plus chronic, and chronic, and lesions into global and segmental.
Active raises a spectrum of lesions including endocapillary hypercellularity, subendothelial immune complexes, crescents, and/or glomerular capillary necrosis. Likewise,

SLE Paent Develops New:

Proteinuria ( 500 mg/d)
Glomerular Hematuria (especially in
combinaon with proteinuria)
Renal Insuciency (aributable to SLE)
PERFORM KIDNEY BIOPSY
Inducon and
Maintenance Treatment

No Renal
Response
REPEAT KIDNEY
BIOPSY

Paral Renal
Response

Complete
Renal Response

Stop Therapy?
REPEAT KIDNEY BIOPSY

Modify Therapy?
REPEAT KIDNEY
BIOPSY

CONSIDER REPEAT KIDNEY

LN Flare BIOPSY

Fig. 2. Algorithm for kidney biopsy in LN. A diagnostic kidney biopsy should be done to
guide therapy when a patient with lupus presents with clinical evidence of new kidney
injury. A repeat biopsy should be done to confirm complete histologic remission in patients
who have achieved complete clinical renal response so maintenance immunosuppression
may be stopped. A repeat biopsy should be done to guide changes in therapy for patients
who have incompletely responded. A repeat kidney biopsy should be considered at LN flare
if there is suspicion that histology has changed and therapy may need to be modified.

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chronic connotes glomerulosclerosis, fibrous crescents, interstitial fibrosis, and/or

tubular atrophy. Thus a pathologic diagnosis of class IV-G (A/C) must be accompanied
by a description of what was seen under the microscope. Complicating the heterogeneity of LN is that the lesions evolve over time, classes change, and patients are not
uniformly identified at a specific stage. Therefore, biomarkers that noninvasively identify the presence (or absence) and severity of specific pathologic lesions are likely to
be more useful for prognostic and treatment decisions than biomarkers that identify a
specific LN class. Such biomarkers are applicable across LN classes and individual
patients. Nonetheless, many investigators have focused on identifying biomarkers
to noninvasively diagnose LN class (Table 1).
For all the reasons outlined previously, lesion-specific biomarkers are not intended
to preclude a diagnostic kidney biopsy when LN is first suspected. Rather, lesionspecific biomarkers are expected to be used to monitor changes in kidney injury in
real time, as a noninvasive equivalent of serial kidney biopsies (Fig. 3). The prospective
assessment of kidney histology during treatment would allow therapy to be modified
to account for tissue response, facilitating personalized titration of immunosuppressive agents. Improved kidney outcomes and less therapeutic toxicity would be
expected to result from such personalization. Furthermore, a biomarker-based treatment platform could help refine the definition of complete and partial renal response
beyond the arbitrary levels of proteinuria and serum creatinine used currently (see
Fig. 3). In addition, it is conceivable that following pathology biomarkers prospectively
after a renal response could provide an early warning of increasing kidney inflammation, allowing preemptive intervention to avoid a full-blown LN flare (see Fig. 3).
Because the percutaneous needle biopsy is the gold standard for renal pathology,
biomarkers of disorders will have to approach the same level of accuracy to be
accepted clinically. To estimate accuracy, needle biopsies were done at the time of
autopsy in patients with and without kidney disease (n 5 103).36 Overall, the needle
biopsy agreed with the autopsy findings 84% of the time in diffuse disease, but only
51% of the time in focal disease. With respect to specific compartments, there was
96% agreement for glomerular lesions and 31% to 87.5% agreement for interstitial lesions. However, only 7 patients in this autopsy study had a glomerular disease, and
none of these were focal lesions. Because accuracy diminishes with focal disease,
glomerular accuracy may be less than 96% for LN. Other investigators estimated biopsy accuracy as the theoretic misclassification rate assuming a binomial distribution
of abnormal glomeruli in the kidneys.37 This calculated misclassification rate was 25%
with 10 glomeruli and 15% with 20 glomeruli, but does not account for interstitial lesions. The misclassification rate of chronic allograft nephropathy, a tubulointerstitial
disease found in transplanted kidneys, was not influenced by number of glomeruli,
and was 12% in one study.38
In summary, a misclassification rate of 10% to 20% may be acceptable for biomarkers of kidney disorders in LN, especially when considering focal lesions and interstitial disease. It is conceivable that urine-based biomarkers are more accurate than
needle biopsies, because urine analytes presumably represent lesions throughout
the kidneys and are therefore not subject to sampling error or inadequate sample
size. However, this is difficult to prove because biomarkers can only be directly
compared with needle biopsies.
A representative selection of putative LN kidney pathology biomarkers reported to
date is illustrated in Table 1. These include biomarkers that differentiate proliferative
and nonproliferative LN, biomarkers that differentiate between global levels of activity
and chronicity in the kidney, and biomarkers that identify the presence, absence, or
severity of specific pathologic lesions. Most of these biomarkers are serum or urine

Table 1
Biomarkers of kidney disorders in LN
N

Histologic Lesion

AUC

Sensitivity (%)

Specificity (%)

PPV/NPV (%)

Reference

Panel: serum C1q,

albuminuria

18

Presence of ongoing proliferative

LN after induction

100

100

100

28

Urine CD41 cells

147

Presence of proliferative LN

0.99

100

98

49

Urine taurine
Urine citrate

14

Presence of class V LN

1
0.88

100
86

100
100

39

Panel: urine MCP-1,

ceruloplasmin, a1-acid
glycoprotein

76

Presence of high activity

0.85

72

60

40

Panel: urine NGAL, MCP-1,

and GFR

76

Presence of high chronicity

0.83

73

67

40

Anti-C1q autoantibodies

52

Correlates with glomerular:

Endocapillary proliferation
Inflammation
Necrosis

0.3
0.42
0.3

49

Anti-C1q autoantibodies

52

Correlates with:
Glomerular sclerosis
Interstitial fibrosis

0.32
0.39

49

Anti-C1q autoantibodies

63

Presence of glomerular necrosis

100

31

37/100

44

Anti-C1q autoantibodies

63

Presence of glomerular crescents

92

31

45/86

44

Panel: urine MCP-1, serum

creatinine

61

Differentiates between levels of

interstitial inflammation

0.92

100

83

68/100

41

Abbreviations: AUC, area under the receiver-operating characteristic curve; C1q, fragment of the first component of complement; GFR, glomerular filtration rate;
MCP-1, monocyte chemotactic protein-1; NGAL, neutrophil gelatinase-associated lipocalin; NPV, negative predictive value; PPV, positive predictive value; R, correlation coefficient.

Kidney Biopsy in Lupus Nephritis

Biomarker

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Biomarkers of kidney pathology

Resolve
discordance
between renal
pathology and
clinical measures

Adjust
medicaons
connuously

Refine renal
ena
response criteria;
prevent ongoing
kidney injury and
new flares

Follow response
ponse
connuously and respond
in real me to decrease
duraon of kidney injury

Surveillance for
inflammaon preflare

Iniate trea
treatment
pre-empvely to
prevent or shorten
flare duraon

Prevent CKD/ESRD
Fig. 3. Application of biomarkers of kidney pathology in LN. The rationale for identifying
biomarkers of disorders and how these biomarkers are expected to improve the management of LN are shown. In this schema the biomarkers of kidney disorders reflect the
presence and severity of specific lesions such as glomerular crescents, interstitial inflammation, or interstitial fibrosis. Lesion-specific biomarkers measured serially and frequently provide an understanding of how kidney injury evolves in real time during and after LN
treatment. CKD, chronic kidney disease; ESRD, end-stage renal disease.

proteins, although 1 study used urine metabolomic profiling to differentiate between

pure class V and proliferative LN,39 and another study used urine T-cell counts to
determine the presence of proliferative LN. Biomarkers can be single analytes or a
panel of analytes, although several investigations suggest that accuracy improves
when analyte panels are used.28,4043 Some of the panels include nonspecific indicators of kidney injury such as serum creatinine, glomerular filtration rate, and proteinuria/albuminuria. Novel biomarkers are expected to bring added value to these
clinical measurements, and such combinations may yield robust composite biomarkers. In addition, in multibiomarker panels, individual biomarkers most likely
contribute differentially to the composite biomarker. We have used linear discriminant
analysis to weight and combine individual biomarkers that are biologically plausible or
that have been identified as relevant to LN using unbiased screening techniques such
as urine proteomics.41 In this approach, the discriminant function is the composite
biomarker and takes the form of an equation in which urine and serum analyte measurements are input, and the resulting calculated value differentiates between levels
of severity of specific histologic findings in LN kidneys.41 Such composite biomarkers
can readily be adapted to the clinical laboratory.
The clinical performance metrics of sensitivity, specificity, and area under the
receiver-operating characteristic curve are encouraging for several of the pathology
markers (see Table 1). The positive predictive values of the biomarkers are variable
but generally low, whereas the negative predictive values are uniformly good, suggesting that these biomarkers may be most useful for excluding a particular disorder.
Misclassification rate is a key performance metric for novel biomarkers, and for these
markers it ranges from 0% to 49%. However, several markers have an accuracy of
80% to 90% in relation to the needle kidney biopsy.39,41,44

Kidney Biopsy in Lupus Nephritis

The pathology biomarkers presented in Table 1 seem to be diverse and unrelated,

but several are mediators of inflammation. It is informative to discuss a few of these
proposed markers. The first component of complement, C1q, and antibodies to
C1q are heavily represented in Table 1. The complement system plays a key role in
the pathogenesis of SLE, and virtually all patients with complete C1q deficiency
develop lupus or a lupuslike disease, and often severe LN.45,46 This tendency may
be caused by loss of the protective effect of C1q in mediating clearance of apoptotic
debris and immune complexes.46 However, most patients with SLE do not have C1q
deficiency and although the complement system can be protective it is also proinflammatory. C1q is almost always present in the glomeruli of patients with active LN.47
Glomerular C1qanti-C1q immune complexes may focus complement activation to
glomeruli, amplifying kidney inflammation and injury,48 which is consistent with C1q
consumption and C1q autoantibodies as surrogate markers for proliferative (inflammatory) LN classes and inflammatory glomerular lesions (crescents, necrosis), and
the negative correlation of anti-C1q with chronic lesions (glomerulosclerosis, interstitial fibrosis).28,44,49
Monocyte chemotactic protein-1 (MCP-1) is a proinflammatory chemokine that has
been shown to recruit inflammatory cells to the kidney in several types of nephritis
including LN.50,51 Blocking the activity of MCP-1 in animal models of LN ameliorates
kidney injury.52 In humans, MCP-1 is a specific biomarker of LN flare.53 It is therefore
expected that MCP-1 may be a marker of inflammatory disorders in LN.40,41 It may be
surprising that MCP-1 also contributes to a panel of biomarkers that is a surrogate for
chronic disorders,40 but macrophage infiltration into the kidney is necessary for generation of fibrosis. In addition, MCP-1 stimulates fibroblast and monocyte collagen
production, and monocyte expression of the profibrotic cytokine transforming growth
factor beta-1.54,55
Neutrophil gelatinase-associated lipocalin (NGAL) is a small, glycosylated protein
that acts as an epithelial growth and differentiation factor and belongs to a family of
proteins that are involved in cellular iron transport.56 Other iron regulatory proteins
such as transferrin and hepcidin have been identified as potential biomarkers of LN
activity, raising the possibility that iron availability may be involved in the pathogenesis
of kidney injury in LN, perhaps through redox signaling.57 NGAL is minimally
expressed in healthy kidneys, but in acute kidney injury caused by ischemia or inflammation it increases in segments of the distal nephron.58 In a longitudinal pediatric LN
cohort, urine NGAL levels increased 3 to 6 months before LN flare, suggesting that it
may forecast flare.59 At present it is not clear whether NGAL is simply a marker of
tubular injury in LN and other kidney diseases or is directly involved in tissue injury.
There are some animal data suggesting that NGAL may even be tubuloprotective.56
Nonetheless, NGAL seems to be mainly associated with acute processes, which
seems inconsistent with NGAL being on a biomarker panel for biopsy chronicity.40
However, the primary data show that NGAL decreases in the chronicity panel.40
Although many of the biomarkers listed in Table 1 seem promising, none are ready
for clinical practice. Most were identified in small cross-sectional cohorts and have not
yet been verified in independent patient populations. After verifying reproducibility, it
would be ideal to clinically validate pathology biomarkers by testing how well they prospectively track changes in kidney histology over time. The most direct way to do this
would be with serial kidney biopsies, but such a study would be excessively invasive.
An alternative trial design for clinical validation is shown in Fig. 4. This design tests
whether altering duration and intensity of standard induction and maintenance medications provides an advantage compared with usual management in terms of complete response rate, LN flare rate, safety, and cumulative drug exposure.

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Biomarkerbased strategy

Begin standard
inducon
therapy

Stop maintenance
therapy based on
biomarker response

Compare 12 month outcomes:

Number of CRR, me to CRR, LN
are rate, cumulave
immunosuppression, treatment
toxicies

Randomize
biopsydiagnosed LN
paents
Usual-care
strategy

Adjust length and

intensity of
inducon based on
biomarker response

Standard
inducon
therapy for 6
months

Standard
maintenance
therapy

Fig. 4. Clinical validation of kidney pathology biomarkers. Patients with biopsy-proven LN

are randomized to a usual-care treatment strategy or a biomarker-based treatment strategy.
The usual-care strategy follows the induction and maintenance recommendations of the
2013 ACR treatment guidelines. The biomarker-based arm begins treatment in the same
way, but duration and intensity of treatment are modified monthly based on how biomarkers of active pathologic lesions are responding. For example, if biomarkers are normalizing, induction could be shortened and maintenance started early; if lesions are not
responding well, induction could be lengthened. Maintenance could be discontinued if
the biomarkers indicated a complete histologic remission. All patients are followed for
12 months and at that time outcomes between the arms are compared. The outcomes to
be measured are indicated in the figure. Complete renal response (CRR) is adjudicated clinically from kidney function and proteinuria, as is currently done, and also compared with
complete histologic response.

MOLECULAR DIAGNOSIS: THE FUTURE OF THE PERCUTANEOUS KIDNEY BIOPSY IN LN

As discussed previously, LN is intrinsically heterogeneous. At present all patients diagnosed with a particular class of LN tend to be treated in a similar fashion. It is conceivable that therapy would be more effective and less toxic if it specifically targeted the
pathogenic mechanisms actively involved in kidney injury at the time of diagnosis. This
possibility is especially relevant for novel therapies being trialed or developed for LN.
These new drugs generally work on limited areas of the immune system, so understanding the mechanisms at play in an individual patient with LN should facilitate
appropriate matching of novel therapies to patients likely to derive benefit, improving
outcomes, limiting side effects, and containing costs. Integration of molecular and histologic evaluation of the kidney is feasible by applying-omic techniques to kidney
biopsies in LN.
Molecular analysis of the kidney presents several challenges. Clinical kidney biopsies are small, providing little tissue for evaluation, and are often formalin fixed
and paraffin embedded. Protocols have recently been developed to facilitate transcriptomic and proteomic analysis of the tissue left over from clinical kidney biopsies.6062 The architecture of the kidney adds to the complexity of molecular
analysis. The kidney is divided into glomerular and tubulointerstitial compartments,
and there are several distinct cell types within each compartment, including infiltrating
inflammatory cells in LN. The expression profile of inflammatory mediators may differ
between compartments. For example, glomerular T cells from class III and V LN biopsies expressed Th1, Th2, and Th17 cytokines, whereas isolated interstitial T cells
expressed mainly Th2 and Th17 cytokines.63 Laser-capture microdissection (LCM)

Kidney Biopsy in Lupus Nephritis

of specific renal lesions from specific renal compartments, followed by RNA or protein
analysis, offers an approach to identify differentially expressed transcripts or proteins
that characterize the pathology and pathogenesis of LN lesions.
In an early application of these techniques, the molecular heterogeneity among patients with LN was convincingly shown. Glomeruli from kidney biopsies were isolated
by LCM and then analyzed by complementary DNA microarray for transcriptional phenotyping.64 Compared with normal kidney, 4 distinct transcriptional patterns emerged,
including increased expression of B-cell genes, myelomonocytic (macrophages and
dendritic cells) genes, type I interferon-inducible genes, and fibrosis-related genes.
The level of transcript expression varied considerably in glomeruli from different patients, but within a kidney transcript expression was surprisingly consistent between
glomeruli despite histologic variability. For example, individual biopsies could be
segregated into 2 broad groups based on the type I interferon-inducible and
fibrosis-related gene clusters. Biopsies with high levels of interferon-inducible genes
had low levels of fibrosis genes, whereas biopsies with high levels of fibrosis genes
had low levels of type I interferon-inducible genes. Although only some kidneys
showed increased glomerular expression of fibrosis-related genes, the expression
of such genes was similar in glomeruli from the same kidney, whether or not they
had histopathologic evidence of glomerulosclerosis. These findings show the difficulty
in personalizing LN treatment using only histologic information.
As discussed previously, diagnostic biopsies are not good predictors of future kidney outcomes in LN. Examination of biopsy gene expression patterns may increase
their predictive value. Diagnostic biopsies from class III and IV LN were segregated
by renal response status an average of 11 months after standard induction and maintenance therapies (cyclophosphamide and MMF; Parikh and colleagues. Molecular
characterization of renal responses in lupus nephritis using serial kidney biopsies.
Poster; American Society of Nephrology, 2013). The expression of 511 immune
response genes was analyzed directly from the kidney biopsy cores by NanoString
technology.65 Using principal component analysis, the samples clustered by whether
the patients were complete responders, partial responders, or nonresponders (Fig. 5).
These results suggest that gene expression analysis of the diagnostic kidney biopsy
may identify patients who are likely to respond to conventional treatment before therapy is started. Alternate therapy may then be considered for those who have an unfavorable transcript response profile.
In addition to profiling differential gene expression, recent work showed that clinical
kidney biopsies could also be analyzed for differential protein expression using unbiased proteomic techniques. Glomeruli and renal interstitium were isolated by LCM and
proteins extracted from these two compartments were identified by mass spectrometry.62 Several differences between normal and LN kidneys were observed. As expected, class IV glomeruli showed increased levels of complement proteins and
decreased levels of antioxidant and podocyte proteins. Tissue proteomics showed
an increase in the protein products of interferon-alfainducible genes, consistent
with the lupus interferon-alfa signature shown by transcript analysis.62 Transcript
and protein analyses of kidney biopsies may thus be considered complimentary,
especially for designing novel drugs for LN. Expression studies focus attention on specific pathways for intervention, and differentially expressed proteins within these pathways may be particularly good therapeutic targets.
The molecular analysis of LN biopsies has generated, and will continue to generate,
large amounts of data. In an effort to organize these data in a mechanistically and therapeutically useful way, disparate and large datasets are now being analyzed using a
systems biology approach. The objective of systems biology is to integrate complex

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Fig. 5. Principal component analysis (PCA) of kidney biopsy transcriptional profiles. RNA was
harvested from archived kidney biopsies performed at LN flare, and for which kidney outcomes after treatment were known. Preimplantation living transplant donor biopsies
were considered normal. Immune gene expression was measured by NanoString technology.
PCA was done on the RNA expression data. PCA shows that, at renal flare, patients clustered
by the type of renal response they had long term. CR, complete renal responder; NR, nonresponder; PR, partial renal responder.

biological information to acquire a comprehensive description of the molecular events

that lead to organ dysfunction.66 With respect to LN, clinical information and molecular
data from kidney biopsies can be combined to identify relevant pathogenic networks
in LN.
To exemplify the potential of a systems approach, transcriptional networks were
compared in murine and human LN.67 Murine models have provided significant insight
into the pathogenesis of LN but do not entirely recapitulate the events that occur in
human LN, which may explain the failure of murine therapies to translate to human
LN. Using LCM, tubulointerstitial regions were isolated from human LN kidney biopsies and their transcriptional profiles determined. Using sophisticated bioinformatic
techniques, these were compared with the transcriptional profiles of whole kidneys
from 3 different murine models of LN. The investigators identified 20 transcriptional
networks that were similarly activated (or repressed) in human LN and all of the LN
mouse models.67 These networks included, but were not limited to, antigen presentation and dendritic cell maturation pathways, and interleukin-17, mitogen-activated
protein kinase, and interferon signaling pathways. Developing and testing novel LN
therapies in the mouse based on these common pathways may increase the likelihood
that the drugs will be effective for human LN.
SUMMARY

The available evidence suggests that the percutaneous kidney biopsy is still relevant to
the clinical management of LN. However, it is also clear that more information resides
within kidney biopsies than is available from histopathology alone. This information

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can be released by molecular analysis of biopsies. Molecular analysis is feasible, and

promises to help personalize therapy and enhance drug discovery for LN.
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