Front Pediatr 7: 341

PMC: PMC6700319

PMID: 31456999

Analysis of 14 Patients With Congenital Nephrotic Syndrome

Ethical Statement

This study was approved by the Peking University First Hospital Ethics Committee. Written informed consent (for the publication of the cases reported and any potentially identifying information) was obtained from all participants or their parents. All data were analyzed anonymously.

Patients

From January 1995 to June 2018, 14 patients (8 boys and 6 girls) were diagnosed with CNS at the Department of Pediatrics, Peking University First Hospital. The onset of the symptoms of all patients occurred within the first 3 months of life.

The patients were included based on the following diagnostic criteria for CNS:

Proteinuria: qualitative examination ≥ +++, quantitative ≥50 mg/kg/day, urine protein/creatinine (random) >2.0 mg/mg;
Hypoalbuminemia: serum albumin <30 g/L;
Hypercholesterolemia: total cholesterol >5.72 mmol/L (220 mg/day l);
Edema;
Onset age <3 months.
Criteria 1, 2, and 5 are prerequisites (1–3).

Clinical Data

Clinical data, including sex, age of onset, amniotic fluid volume, and size of placenta were collected. The medical histories of CMV, syphilis, toxoplasmosis, rubella virus, HBV, and HIV infection during pregnancy were also collected.

In addition, information regarding medication use during pregnancy (including steroid use), suspected mercury poisoning, family medical history (especially family members with kidney disease, unexplained edema, and death), and pet exposure was obtained.

Laboratory Tests

The following three panels of laboratory tests were performed:

To confirm the diagnosis of CNS: urine routine test (sterile urine bag or catheterization sampling), 24-h urinary protein (urethral catheterization sampling) or urine protein/creatinine (sterile urine bag or catheterization sampling), serum albumin, and serum cholesterol;
To determine the structure and function of the kidney: renal ultrasound; renal function (serum creatinine and creatinine clearance), early kidney injury biomarkers, and urine protein electrophoresis;
To explore the possible pathogens: serum CMV IgM and IgG, toxoplasmosis, and rubella virus were examined in 14 cases. HBV, hepatitis C virus (HCV), herpes simplex virus, syphilis, and HIV were also examined. serum and urine CMV-DNA levels were examined in 14 cases.

Gene Analysis

Informed consent was obtained from the parents of the patients for genetic analysis. The NPHS1, NPHS2, WT1, PLCEI, and LAMB2 genes, which have been previously reported to cause CNS, were analyzed from 1998 to 2014. Genomic DNA was extracted from the peripheral blood lymphocytes of both patients and their parents using the TIANamp Blood DNA Kit (Tiangen Biotech, China). Sanger sequencing was applied. Positive polymerase chain reaction (PCR) products (with specific expected bands) were sequenced with technical help from MyGenostics Co. Ltd. Abnormal results were re-amplified and sequenced, and the parents' DNA samples were also examined. From 2015 to present, the second-generation sequencing method is used for genetic PANEL testing of hereditary kidney disease.

The judging criteria for pathogenicity of unreported variation is family separation analysis (the number of patients who received parental and/or sibling DNA and analyzed).

Renal Biopsy Pathology

Renal biopsy was performed upon parents' agreement. Histopathological changes were analyzed. CMV inclusion bodies in the kidney tissue were examined using immunohistochemical methods at the Capital Institute of Pediatrics Medical University. Kidney tissue slices were also examined for CMV-DNA by using molecular probes at the Department of Pathology, Peking University Health Science Center. All methods were performed in accordance with the relevant guidelines and regulations.

Clinical Features

From January 1995 to June 2018, 2,583 patients were diagnosed with nephrotic syndrome in the Department of Pediatrics, Peking University First Hospital. Of these patients, only 14 had confirmed CNS. Of the 14 patients, 8 were boys, and 6 were girls. The gestational age (GA) was between 35 + 6 weeks and 42 weeks, and the mean GA was 38+1 weeks. The birth weight (BW) ranged from 1,860 to 4,000 g, with a mean of 2,987 g. The onset age was between 1 day and 3 months of life, and the mean was 27.4 days. The mean onset age for patients with Finnish type CNS was 8.4 days. The main symptom for 12 patients was edema. Two patients (cases 6 and 11) had no obvious symptoms except for proteinuria in laboratory tests (Table 1). Three patients (cases 2, 5, and 7) had hypertension. Three patients (cases 1, 8, and 12) were delivered with placentas >800 g, and 2 patients (cases 1 and 12) had less amniotic fluid (amniotic fluid index, AFI <5) (4). All mothers denied a history of SLE. No family histories of unexplained death were found. No families had pets or evidence of mercury poisoning. The families also did not show evidence of syphilis or HBV infection.

Table 1

Onset conditions of 14 patients.

No.	Sex	Age of onset	Chief complaint	Plasma albumin level (40–55 g/L)	Urinary protein	Cholesterol (3.4–5.2 mmol/L)
1	M	27 days	Edema	8.4	4+	9.65
2	F	2 months 27 days	Ascites	10.3	3+	–
3	M	2 days	Edema	9.9	4+	7.71
4	F	20 days	Ascites	12.8	4+	8.18
5	F	2 months 24 days	Edema	21.7	3+	10.54
6	M	15 days	Asymptomatic	21.7	3+	3.33
7	M	3 months	Edema	11.9	4+	10.02
8	M	19 days	Edema	9.4	4+	5.82
9	M	16 days	Edema	16.6	3+	7.99
10	M	13 days	Edema	16.6	3+	6.26
11	F	1 days	Asymptomatic	28.4	2+	6.02
12	M	1 days	Edema	11.6	4+	5.09
13	F	2 days	Edema	14.2	3+	7.83
14	F	7 days	Edema	14	4+	7.61

Case 14 is an abandoned baby.

All children were delivered uneventfully.

Family history: All 14 families were non-consanguineous. Only 1 patient (case 11) had a family history of renal disease. Eight individuals in the family had proteinuria among three generations. The mother of patient 11 had proteinuria and hypoalbuminemia at 8 months of age. The mother did not undergo renal biopsy or any treatment. The cousin of the mother had proteinuria at 1 year of age and died at 12 years of age.

Laboratory Test Results

Of the 14 patients, seven patients' parents agreed to indwell urethral catheterization and we got the urine protein of 24 h, seven patients had random urine collected by sterile urine bags. All the patients met the diagnostic criteria of the nephrotic syndrome.

Of the 14 patients, only 1 patient (case 5) had elevated serum creatinine (91 μmol/L; normal range, 30–40 μmol/L) and blood urea nitrogen (BUN) levels (10.6 mmol/L; normal range, 1.8–7.1 mmol/L).

All patients underwent renal ultrasound examination. In case 3, the results revealed renal pelvis separation with an anteroposterior diameter of 1.04 cm. However, this is not a characteristic manifestation of CNS. This may be the process of kidney development. The kidney sizes and structures in the other children were normal.

CMV infection: Of the 14 patients, 8 patients had evidence of CMV infection. Seven patients had active CMV infection (CMV-IgM positive and/or serum CMV-DNA positive). One patient (case 2) had positive urinary CMV-DNA results. He was hospitalized in critically ill condition and died 2 days after admission before antivirus therapy was initiated.

Pathogenic test results for syphilis, HIV, HBV, HCV, toxoplasma, rubella, and herpes simplex virus were all negative.

Gene Analysis Results

Eight patients underwent nephropathy-related genetic testing. Seven patients had NPHS1 gene mutations, and 1 (case 6) had both NPHS1 and COL4A5 (the gene that causes Alport syndrome) mutations. One patient (case 11) had negative genetic test results. Three patients (cases 3, 6, and 8) had both NPHS1 gene mutations and CMV infection (Table 2). Other patients did not undergo such testing due to the undeveloped medical condition at the time the testing was offered or lack of consent from their parents. Analysis of variant sites of NPHS1 gene is shown in Table 3.

Table 2

Clinical features of 14 patients.

No.	Plasma creatinine level μmol/L	eGFR^a	Gene type	CMV infection	Proteinuria at present	Outcomes
1	21	106.7	-	Y	2+	-
2	47.3	43.9	-	Y		Death (3 months, 12 days)
3	12	166.7	NPHS1	Y		Death (10 months)
4	16	113.7	-	Y	Negative	9 years
5	91	26.4	-	Y	4+	-
6	39	55.4	NPHS1 COL4A5	Y	3+	13 months
7	38	70.5	-	Y	Negative	9 years
8	17	115.3	NPHS1	Y		Death (2 months)
9	21.7	92.2	-	N	3+	-
10	17.4	108.1	NPHS1	N		Death (2 months)
11	53	36.2	Negative	N	3+	5 years
12	14.4	129.2	NPHS1	N	4+	Death (3 months)
13	16	120	NPHS1	N	4+	Death (4 months)
14	11.2	196.4	NPHS1	N	4+	Death (4 months)

Y indicates patients with CMV infection, N indicates patients without CMV infection, - indicates that the test was not completed.

^{Schwartz's formula: eGFR = k × height (cm)/plasma creatinine}.

Case 14 is an abandoned baby. Cases 4 and 7: follow-up in December 2016; case 6: follow-up in April 2016; case 11: follow-up in September 2015; cases 2, 3, 8, 10, 12, 13, and 14: follow-up in June 2018; cases 1, 5, and 9: lost contact.

Table 3

Variant locus analysis in patients with NPHS1 mutation.

No.	Variation	Amino acid change	Mutation status	Mutation type
3	c.2663G>A	p. Arg888Lys	Het	Missense
	c.3286+5G>A	-	Het
6	c.2396G>T	p. Gly799Val	Het	Missense
	c.1339G>A	p. Glu477Lys	Het	Missense
8	c.3027C>G	p. Tyr1009^*	Het	Nonsense
	c.3478C>T	p. Ary1160^*	Het	Nonsense
10	c.1740G>T	p. Trp580Cys	Het	Missense
	c.2042G>A	p. Trp681^*	Het	Nonsense
12	c.713-1G>C	-	Het	Missense
	c.1760T>G	p. Leu587Arg	Het
13	c.2506+5G>T	-	Het	Missense
	c.1135C>T	p. Ala379Thr	Het
14	c.313G>A	p. Asp105Asn	Het	Missense
	c.2386G>C	p. Gly796Arg	Het	Missense

^{Mutations detected in NPHS1 gene}.

Case 14 is an abandoned baby, and no parental verification was performed.

Renal Pathology Results

Only 1 patient (case 6) underwent renal biopsy at the age of 2 months, and the findings were as follows:

Light microscopy: The mesangial cells and matrix showed mildly diffused hyperplasia. Multiple podocytes and immature glomeruli could be seen with cellular crescent formation. Vacuolar and granular degeneration were found in the tubular epithelia. Small focal atrophy, focal lymphoid interstitial mononuclear cells, and eosinophil infiltration with fibrosis could be seen along with thickening of the small artery walls. These changes represented mild mesangial proliferative glomerulonephritis disease (Figure 1).
Figure 1
Light microscopy (LM) of the kidney biopsy specimen with periodic acid–silver methenamine (PASM) staining. The mesangial cells and matrix had mild diffuse hyperplasia. One cellular crescent formation can be seen (white arrow).
Immunofluorescence demonstrated mesangial deposits of IgM and C3 (IgM ++, C3 +) (Figure 2).
Figure 2
Results of immunofluorescence analysis during renal biopsy. (a) Immunofluorescence demonstrated mesangial deposits of C3 (C3+); (b) Mesangial deposits of IgM (IgM ++).
Electron microscopy confirmed the light microscopy findings: The mesangial cells and matrix showed mild hyperplasia. The glomerular basement membrane was normal. The epithelial foot process fused together with no electron dense deposits. The tubular epithelia had degenerated, and the lysosomes were increased. The renal interstitium had no obvious lesions (Figure 3).
Figure 3
Electron microscopy (EM) of the kidney biopsy specimen. The mesangial cells and matrix exhibited mild hyperplasia. The epithelial foot processes were fused with no electron dense deposits.

Renal biopsy sections: immunohistochemistry (IHC): CMV inclusion bodies (-); Molecular Probes: CMV-DNA (-).

The other patients did not undergo renal biopsy due to a lack of consent from their parents, or because their condition was critical after admission to our hospital.

Treatment

Anti-CMV Therapy

In 8 patients with CMV infection, 1 patient (case 2) was hospitalized in critically ill condition and died 2 days after admission.

The other 7 patients were administered ganciclovir as antiviral therapy (5 mg/kg, given twice daily, 12 h apart for 4 weeks). We did not observe any side effects related to ganciclovir therapy.

The urinary protein results became negative after antiviral therapy in 2 patients (cases 4 and 7) and have remained negative up to the follow-up.

Two patients (cases 3 and 8) had NPHS1 gene mutations combined with CMV infection. After the antiviral therapy, the clinical symptom did not show improvement, and urinary proteinuria remained at 4+. The patients died at 10 months and 2 months, respectively.

The urinary proteinuria test results in case 6 became negative after antiviral therapy was administered at 3 months of age. The proteinuria levels then increased to 3+ at the 8-months follow-up due to infection and have remained unchanged to date.

Notably, patient 6 had both NPHS1 mutations (CNS) and COL4A5 gene mutations (Alport syndrome) along with active CMV infection. At onset, the urinary protein/creatinine ratio, urine protein, serum albumin, and CMV IgM were 24.97, 4+, 21 g/L, and 27.7 U/mL, respectively. After ganciclovir therapy, the urine protein/creatinine ratio decreased to 1.63; the urine protein results were negative; serum albumin (without albumin infusion) increased to 30.4 g/L; and no virus was detected in the blood. The CMV IgM was 5.8 U/mL (negative) at 3 months. However, at the 8-months follow-up, the urinary protein increased to 3+ due to infection and has remained stable to date.

The urinary protein results in 2 patients (cases 1 and 5) decreased from 4+ to 2+ after ganciclovir therapy, but these patients were lost to follow-up.

Corticosteroid Treatment

In 8 patients with CMV infection, 3 patients (cases 1, 3, and 5) were treated with corticosteroids (prednisone acetate, 1.5–2 mg/kg daily for 1–4 weeks). The dose was gradually tapered and then stopped. The corticosteroids showed no effect on CNS.

Other Therapies

One patient (case 1) was administered with cyclosporin 1 mg/kg daily for 2 weeks. One patient (case 3) was administered gamma globulin 1 g/kg. Ten patients (cases 1, 2, 3, 4, 8, 9, 10, 12, 13, and 14) were administered albumin 1 g/kg but not daily albumin infusions in conventional hospitalization.

None of the patients underwent early bilateral nephrectomy, dialysis, and early kidney transplantation.

Outcomes (Table 2).

In all 14 patients, only 2 patients (cases 4 and 7, active CMV infection) showed negative urine protein results at 9 years of age.

One patient (case 6, active CMV infection + NPHS1 + COL4A5 gene mutations) was followed up with 3+ gross proteinuria and is still being monitored.

One patient (case 11) had 2+ proteinuria and is now 5 years old without edema.

The urinary protein levels in 3 patients fluctuated between 2+ and 4+ (cases 1 and 5 with active CMV infection and case 9 without CMV infection; the parents of these 3 patients refused genetic testing).

The 7 remaining patients died with severe edema between 2 and 10 months of age (mean, 4 months).

Anti-CMV Therapy

In 8 patients with CMV infection, 1 patient (case 2) was hospitalized in critically ill condition and died 2 days after admission.

The other 7 patients were administered ganciclovir as antiviral therapy (5 mg/kg, given twice daily, 12 h apart for 4 weeks). We did not observe any side effects related to ganciclovir therapy.

The urinary protein results became negative after antiviral therapy in 2 patients (cases 4 and 7) and have remained negative up to the follow-up.

The urinary protein results in 2 patients (cases 1 and 5) decreased from 4+ to 2+ after ganciclovir therapy, but these patients were lost to follow-up.

Corticosteroid Treatment

Other Therapies

None of the patients underwent early bilateral nephrectomy, dialysis, and early kidney transplantation.

Outcomes (Table 2).

In all 14 patients, only 2 patients (cases 4 and 7, active CMV infection) showed negative urine protein results at 9 years of age.

One patient (case 6, active CMV infection + NPHS1 + COL4A5 gene mutations) was followed up with 3+ gross proteinuria and is still being monitored.

One patient (case 11) had 2+ proteinuria and is now 5 years old without edema.

The 7 remaining patients died with severe edema between 2 and 10 months of age (mean, 4 months).

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Department of Pediatrics, Peking University First Hospital, Beijing, China

Edited by: Jakub Zieg, University Hospital in Motol, Czechia

Reviewed by: Mario Giordano, Azienda Ospedaliero Universitaria Consorziale Policlinico di Bari, Italy; Vera Hermina Koch, University of São Paulo, Brazil

*Correspondence: Xinlin Hou ude.uen.yksuh@d.naiq

This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics

Department of Pediatrics, Peking University First Hospital, Beijing, China

Edited by: Jakub Zieg, University Hospital in Motol, Czechia

Reviewed by: Mario Giordano, Azienda Ospedaliero Universitaria Consorziale Policlinico di Bari, Italy; Vera Hermina Koch, University of São Paulo, Brazil

Received 2019 Feb 15; Accepted 2019 Jul 30.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Abstract

From January 1995 to June 2018, 14 patients with congenital nephrotic syndrome (CNS) were diagnosed in the Department of Pediatrics, Peking University First Hospital. The clinical data were retrospectively studied. Eight patients underwent genetic testing; 7 of them had NPHS1 mutations (primary CNS), and 1 did not have a mutation. Of the 7 patients with NPHS1 mutations, 6 died, and 1 had proteinuria. Of the 14 patients, 8 had cytomegalovirus (CMV) infection, and anti-CMV therapy was administered to 7 of them. The other patient was hospitalized in critically ill condition and died before anti-CMV therapy administration. Of the 7 patients who were administered anti-CMV therapy, proteinuria disappeared in 2 patients; 2 patients died; 2 patients were lost to follow up; and 1 patient still had 3+ proteinuria. Three patients had both NPHS1 mutations and CMV infection. After anti-CMV therapy, proteinuria was resolved in 1 patient but relapsed to 3+ proteinuria due to a new infection. The other 2 patients died. Of 14 patients, only 1 patient underwent renal biopsy, with results showing mesangial proliferative glomerulonephritis pathology, negative CMV inclusion body, and CMV-DNA. In this study, genetic defect could play a primary role in CNS, and CMV could play a secondary role. Primary CNS with NPHS1 mutations has a poor prognosis. Primary CNS might be accompanied by CMV infection that responds poorly to antiviral treatment. Secondary CNS caused by CMV infection may be cured with antiviral therapy. However, genetic analysis is necessary to exclude genetic defects.

Keywords: congenital nephrotic syndrome, genetic defect, cytomegalovirus infection, clinical manifestation, prognosis

Abstract

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