Chapter 35. Glomerular Disorders due to Infections

Essentials of Diagnosis

• HIV-associated nephropathy (HIVAN) is a distinct and separate pathology with a unique combination of collapsing focal segmental glomerulosclerosis (FSGS), tubuloreticular inclusion bodies, microcystic dilation of renal tubules, and interstitial inflammation found in HIV-infected individuals.
• While HIVAN should be suspected in any HIV-infected person of African descent with proteinuria and decreased glomerular filtration rate (GFR), definitive diagnosis can be made only by renal biopsy.
• At-risk HIV-positive patients should be tested for proteinuria or decreased GFR to allow earlier detection of HIVAN.
• HIVAN is caused by expression of HIV-1 genes in renal epithelial cells.
• Highly active antiretroviral therapy (HAART) is the primary mode of treatment to retard progression of glomerular filtration decline and proteinuria.
• The diagnosis of HIVAN is an indication to begin HAART regardless of CD4 or viral counts.
• Angiotensin-converting enzyme (ACE) inhibitors and steroids may be of benefit in selected patients.

General Considerations

At the onset of the HIV epidemic, little was known concerning the ability of the virus to cause organ-specific pathology. By 1984, it became apparent that there was a renal syndrome associated with HIV infection that was characterized by severe proteinuria and rapidly progressive renal failure. Proteinuria was frequently severe with levels of protein excretion as high as 10 g daily. Patients were often asymptomatic until features of renal failure became manifest. As opposed to similar glomerulopathies, the nephropathy associated with HIV infection did not often result in severe hypertension or edema. On ultrasound imaging of the kidneys, nephromegaly was a common finding with increased kidney weight seen on autopsy. Renal biopsy of these patients revealed the presence of focal glomerulosclerosis, tubular microcystic dilation, and tubulointerstitial inflammation and fibrosis.

Now widely accepted to be a distinct renal disease, HIVAN does not affect all populations equally. In the United States, HIVAN is the third leading cause of end-stage renal disease (ESRD) in African-Americans between the ages of 25 and 64 years. International studies evaluating HIV-infected patients with proteinuria corroborate the U.S. data. Two studies in Thailand and Italy did not detect any cases of HIVAN among HIV-infected patients who underwent renal biopsy to evaluate the cause of proteinuria. Neither of these studies included any patients of African descent. A South African study, however, evaluated 30 patients with proteinuria and 7 patients with microalbuminuria. Renal biopsies revealed that 83% of the patients had pathology consistent with HIVAN, including six out of the seven patients with microalbuminuria. Moreover, ESRD due to HIVAN is 12.2 times more likely to occur in African-Americans than in whites and has the strongest racial predisposition of any form of acquired renal disease leading to ESRD.

Prior to the availability of HAART, the clinical course of HIVAN was characterized by a rapid decline of renal function resulting in the need for renal replacement therapy within weeks to months after diagnosis. The prognosis of these patients was dismal, approximately 1 year once on dialysis. However, since HAART became widely available, the outcome of HIV-infected patients has improved significantly. Similarly, the prognosis of HIV-infected patients with ESRD has improved. After the introduction of HAART in 1995, the mortality rate fell from about 72% to 24% for patients with both HIV and ESRD. Unfortunately, despite the improvements in the efficacy of medical treatment, the prevalence of ESRD in persons living with HIV/AIDS has continued to increase. This increase is closely paralleled by the increased prevalence of HIV/AIDS in African-Americans, the group at highest risk of developing HIVAN (Figure 35–1).

Patients with biopsy-proven HIVAN most often present with obvious signs of renal involvement. Severe proteinuria and impaired GFR are common indications for biopsy in patients with renal disease. However, even the presence of microalbuminuria may herald the presence of HIVAN. In recent years, as HAART has become widely used, the classic clinical presentation of HIVAN has become less common. However, the incidence of ESRD due to HIVAN has not changed appreciably in the past 10 years. While there are no large recent biopsy series of patients with renal disease while on HAART, it is likely that HAART usage has transformed HIVAN from a rapidly progressive disease with severe proteinuria into an indolent disease with lower levels of proteinuria.

Acute kidney injury (AKI) and chronic kidney disease (CKD) are common problems in patients living with HIV/AIDS. Both outpatient and inpatient populations have been observed to have increased rates of AKI that are correlated with higher viral loads and lower CD4 counts; the most important predisposing factor to AKI is the presence of CKD. Interestingly, black race is not associated with increased risk for AKI in patients with HIV/AIDS, suggesting that HIVAN is not the underlying cause of CKD in most cases of AKI in HIV-infected patients. Analysis of over 2 million patients in the Veterans Administration (VA) database for a median time period of 3.7 years yielded a risk of ESRD for black individuals with HIV nearly 10 times that of white individuals with HIV. The most common cause of ESRD in blacks in this study was AIDS nephropathy, the diagnosis that is used by the U.S. Renal Data Service (USRDS) as a surrogate for HIVAN. Strikingly, HIV infection did not increase the risk of ESRD in white patients but did confer an increased risk of ESRD in black patients that was similar to diabetes. A similar study of VA patients with stage III or higher CKD again found a similar risk for ESRD and rate of GFR decline in black patients with HIV as compared to their counterparts with diabetes.

While ESRD is clearly an important problem in patients with HIV/AIDS, the incidence and prevalence figures from the USRDS do not account for persons living with CKD who have not yet reached ESRD. Previous studies have shown that the prevalence of HIVAN in African-Americans with HIV/AIDS is approximately 4–12%, a figure that has not changed appreciably during the past decade. Extrapolating this prevalence to the 224,815 African-Americans known to be living with HIV/AIDS living in the United States, we predict that there are currently 8992–26,977 African-Americans living with HIVAN in the United States. Moreover, 24.7 million of the 39.5 million people living with HIV/AIDS worldwide reside in Sub-Saharan Africa. Recent evidence suggests that the burden of HIV-related renal disease in Africans is similar to that reported in African-Americans. Applying the same prevalence figure leads us to predict that 0.98–3.0 million people currently have HIVAN in Africa. Since most Africans with HIV/AIDS currently receive little or no treatment for their illness, it is likely that many die of opportunistic infections before renal disease becomes clinically apparent. As the health care delivery to Africans with HIV/AIDS improves, particularly with expanding access to antiretroviral therapy (ART), increased survival of patients can be expected. Almost certainly, HIVAN will emerge as a major complication of HIV-1 infection and will become an increasing cause of morbidity and mortality in Africa.

Pathogenesis

Mouse Models

The mechanisms through which infection with HIV-1 results in the HIVAN phenotype are not completely known. However, much research has been done to elucidate important aspects of disease pathogenesis mainly through the use of animal models that develop a phenotype analogous to HIVAN.

Initially, controversy existed as to whether HIVAN resulted from viral infection of the renal parenchyma or from the cytokine milieu created by infection and activation of lymphocytes throughout the body. An HIV-transgenic mouse model was created in which the mice express an HIV-1 provirus under control of the endogenous HIV viral promoter with deletions in the gag and pol genes, rendering the virus noninfectious. A subsequent experiment performed with this transgenic mouse involved reciprocal transplantation with a mouse not possessing the HIV transgene. Wild-type mice receiving a kidney with an HIV transgene developed proteinuria, glomerular, and tubular changes similar to HIVAN. HIV-transgenic mice, receiving a wild-type kidney, did not develop the diseased status. These data suggest that expression of HIV gene products within the kidney is necessary for the development of HIVAN.

Molecular Basis of Pathogenesis

The HIV-1 genome is a 9 kb RNA that encodes nine genes, including three structural genes (env, gag, and pol) two regulatory genes (tat and rev), and four accessory genes (vif, vpr, vpu, and nef) (Figure 35–2). After processing, these nine genes yield 15 respective proteins, each of which plays a role in the life cycle of the virus. Of the 15 proteins, not all have been shown to be involved in the pathogenesis of HIVAN (Figure 35–3). The gag and pol genes (accounting for seven HIV proteins after proteolytic cleavage), for example, are not functional in the Tg26 mouse model of HIVAN, suggesting that their presence is not necessary for the development of disease. Further research has indicated two genes whose function is most important for the development of the HIVAN phenotype: nef and vpr.

Nef Gene

Several studies have attempted to determine the HIV-1 genes that are responsible for inducing HIVAN. In vitro studies have demonstrated that the nef gene is necessary and sufficient to induce the podocyte proliferation and dedifferentiation that are characteristic of HIVAN. Several transgenic murine models have also been developed to further delineate which viral genes are pathogenic to the kidney. HIV-transgenic mice that lack gag, pol, and nef develop FSGS. However, the breeding of these mice with nef-transgenic mice worsened the renal phenotype as compared to nef-mutant mice, suggesting that nef has deleterious effects in the kidney when other HIV gene(s) are present. In addition, transgenic mice expressing nef selectively in expression podocytes expression have increased podocyte proliferation and dedifferentiation, which are characteristic of HIVAN. However, these mice do not develop proteinuria or histologic glomerular disease, providing further evidence that while podocyte expression of nef is an important component of HIVAN pathogenesis, other HIV genes are necessary to induce the full phenotype.

Vpr Gene

Vpr has been demonstrated to have a number of pathogenic effects upon infected cells. These include induction of cell cycle arrest in the G2 phase, nuclear import of the HIV preintegration complex, transactivation of the viral long terminal repeat (LTR) promoter, and apoptosis. The ability of vpr to induce apoptosis has been linked to its ability to increase mitochondrial permeability. In vitro studies have demonstrated the rapid release of apoptogenic proteins (cytochrome-c and apoptosis-inducing factor) from mitochondria when exposed to a carboxy-terminal construct of vpr. Vpr also promotes apoptosis by selectively increasing expression of proapoptotic molecules including caspase-9. Studies using a series of transgenic mice expressing HIV genes demonstrated that deletion of vpr eliminated the renal disease phenotype, further supporting the critical role of vpr in HIVAN pathogenesis.

Other HIV Genes

Though several published in vitro studies have suggested that expression of HIV-1 genes other than nef and vpr may affect renal cells, in vivo animal studies have not supported a role for these genes in HIVAN pathogenesis. Several transgenic lines were created, each expressing one HIV gene (vif, vpr, vpu, nef, rev, tat) except for gag and pol, under the control of the podocyte-specific Nphs1 promoter. Expression of the HIV transgene in the kidney was confirmed by real time polymerase chain reaction (PCR). No mouse expressing the transgenic product of rev, tat, vif, or vpu developed proteinuria or histologic glomerular lesions. Supporting the findings of previous studies, only transgenic mice expressing nef and/or vpr developed proteinuria and glomerular abnormalities, including FSGS. Furthermore, double-transgenic mice with kidney-specific expression of both vpr and nef developed proteinuria 1 week, as compared to 4 weeks, in mice transgenic for vpr or nef alone. Nef–vpr transgenic mice also developed markedly worsened renal function as compared to vpr transgenic and nef single transgenic mice by 4 weeks.

Taken together, the preponderance of published studies support the role of HIV-1 vpr and nef as the most important viral genes in the pathogenesis of HIVAN. While expression of either gene in the kidney can reproduce stereotypical features of HIVAN histology, coexpression results in the most severe phenotype, indicating an additive or synergistic effect of these genes.

Clinical Findings

The most common pathologic finding in HIVAN biopsy specimens is focal glomerulosclerosis (FGS). This FSGS is often associated with retraction of the glomerular capillary walls resulting in narrowing of the lumen and wrinkling of the glomerular basement membrane causing the glomerulus to appear collapsed and is therefore described as “collapsing focal glomerulosclerosis.”

The visceral epithelial cells of the glomerulus, known as podocytes, undergo characteristic phenotypic changes in HIVAN. Podocytes are usually terminally differentiated, quiescent cells, however, in HIVAN, they often become hypertrophic and proliferate. Prominent tubulointerstitial disease is present, the severity of which may exceed the glomerular pathology. Interstitial infiltrates are pleomorphic and may consist of lymphocytes, plasma cells, and macrophages. The tubular abnormalities include flattening and atrophy of tubular epithelial cells and dilation of the tubular lumen, which can be filled with proteinaceous casts. This “microcystic” dilation of the tubules is characteristic of HIVAN and can take place among multiple segments of the nephron.

On electron microscopy, several findings characterize the HIVAN lesion. There is almost complete effacement of podocyte foot processes and the visceral epithelial cytoplasm can have large, electron-dense resorption droplets. The endothelium has large tubuloreticular inclusions located within the cisternae of the endoplasmic reticulum or the Golgi bodies. This can also be seen in patients with lupus nephritis. However, is often not present in patients with the idiopathic collapsing variant of FSGS and therefore should alert the clinician to the possibility of HIVAN if the patient is HIV positive or if the HIV status of the patient is unknown.

Treatment

Strategies for the prevention and/or treatment of HIVAN have never been evaluated in prospective randomized controlled studies and most studies on the treatment of HIVAN have been retrospective and/or lack proper controls. Three types of medical therapy for HIVAN have been studied in humans: Antiretroviral therapy, corticosteroids, and ACE inhibitors.

HAART Therapy

Studies demonstrating that HIV infection of kidney parenchymal cells is necessary for the development of HIVAN support the hypothesis that antiretroviral medications should be beneficial for treating and/or preventing HIVAN. Early papers on the use of antiretroviral therapies were mainly case reports involving zidovudine. The first of multiple reports of HIVAN regression in response to zidovudine therapy was published in 1989. A retrospective study from France of patients with biopsy-proven HIVAN found that past use of antiretroviral therapy was associated with a relative risk of 1.9 for progression to ESRD. It was not clear whether use of antiretroviral medications worsened the outcome or if patients who progressed while on therapy were a treatment-resistant group and therefore at higher risk of worsening renal function.

Two of the largest studies looking at the effect of antiretroviral therapy in patients with HIVAN were both retrospective. The first study, published in 2004, assessed 3976 patients in a Baltimore AIDS clinic. Over the 12-year course of the study, HIVAN was diagnosed by biopsy or a conservative clinical protocol in 94 of the patients. Patients with HIVAN were evaluated in groups based on their treatment. The incidence of HIVAN was found to be 26.4, 14.4, and 6.8 per 100 patient-years in groups receiving no treatment, treatment with nucleoside analog only, or treatment with HAART, respectively. In addition, the study demonstrated a significant decrease in the incidence of HIVAN in the years 1998–2001 as compared to 1995–1997. The use of HAART appeared to be superior to nucleoside analog treatment alone, and appeared to account for the decreased incidence of HIVAN diagnosis. The decreasing incidence of HIVAN in this study contrasts with the relatively unchanged incidence of ESRD due to HIVAN over the past decade. This may reflect a change in the HIVAN phenotype in patients treated with HAART resulting in a more indolent disease that is less likely to be evaluated by diagnostic renal biopsy.

The second study, published in 2006, involved a cohort of 263 consecutive HIV-infected patients referred to the renal clinic at Johns Hopkins University. Of the 263 patients, 53 had biopsy-documented HIVAN. Seventeen were excluded because they required dialysis within a month of diagnosis. The characteristics of the remaining 36 were evaluated and compared with regard to their outcomes. Multivariate analysis demonstrated a significantly lower progression of disease with ART (hazard ratio of 0.30). The median renal survival was found to be 552 days in the treated group and 117 in the untreated group. Since this study included patients treated with nucleoside analog solo therapy (prior to the availability of HAART) and those treated with HAART, these results may underestimate the effect of modern ART upon renal outcomes.

Unfortunately, there have been no randomized controlled trials to evaluate the use of HAART in treating HIVAN. Since most patients with HIVAN have other indications for treatment with HAART, randomized placebo-controlled trials are not ethically feasible. Fortunately, the published literature continues to support the use of HAART in patients with HIVAN. As a result, the Infectious Diseases Society of America guidelines now state that a diagnosis of HIVAN is a strong indication for initiating HAART regardless of viral load or CD4 count.

The effect of stopping ART in patients previously diagnosed with HIVAN has not been studied. However, a case report of a woman who developed recurrent HIVAN after stopping HAART suggests that cessation of treatment may not be safe once a diagnosis of HIVAN is made and if antiretroviral medications must be withheld, patients should be monitored for worsening renal function or proteinuria.

Decades have passed since zidovudine was the sole antiretroviral available to treat chronic HIV infection. Available antiretroviral medications are increasingly diverse in their mechanisms of action and include nucleoside and nonnucleoside reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, and viral entry inhibitors. The ability to control viral replication and enact immune reconstitution therefore continues to improve. Hopefully, these advances in the treatment of HIV/AIDS will eventually translate into a decrease in the morbidity and mortality from HIV-mediated renal disease in these patients.

Angiotensin-Converting Enzyme Inhibitors and Receptor Blockers

Blockade of the renin–angiotensin system (RAS) has been shown to attenuate proteinuria in various glomerular diseases. There are also data from studies in HIV-transgenic models of HIVAN that suggest that RAS blockade using ACE inhibitors or angiotensin receptor blockers can decrease proteinuria and improve histologic and functional renal outcomes.

ACE inhibitors have been evaluated in several small studies. Initially, case reports in the early 1990s supported a beneficial role of ACE inhibitors in decreasing proteinuria and preserving renal function in HIVAN, but conclusions regarding the effect of these medications were made difficult by the concomitant use of ART. In 1996, a retrospective case–control study found that use of captopril was associated with slower progression to ESRD.

In 1997, the course of 20 patients with biopsy-proven HIVAN was documented. Seven of the 11 patients with nonnephrotic-range proteinuria and five of the nine patients with nephrotic-range proteinuria were treated with 10 mg of fosinopril daily. In the patients with initial nonnephrotic- range proteinuria, the mean serum creatinine went from 1.3 mg/dL and 1.0 mg/dL to 1.5 mg/dL and 4.9 mg/dL in the treated and untreated groups, respectively. Similarly, in the group with nephrotic-range proteinuria, the mean serum creatinine went from 1.7 mg/dL and 1.9 mg/dL to 2.0 mg/dL and 9.2 mg/dL in the treated and untreated groups. Proteinuria followed a similar trend. Diminished proteinuria was observed in the treated patients and proteinuria progressively worsened in the untreated patients. A subsequent study from the same group also suggested a beneficial effect of ACE inhibition upon renal outcomes; however, a confounding role for concomitant ART could not be ruled out.

Taken together, the strong evidence in the literature on the protective effect of RAS blockade in a variety of proteinuric renal diseases and the suggestive retrospective studies discussed above support a role for these medications in slowing the decline in renal function and in decreasing the rates of proteinuria in patients with HIVAN.

Prednisone

Tubulointerstitial inflammation is one of the most prominent histopathologic findings in HIVAN and the inflammatory response of renal parenchymal cells to HIV infection is an important component of HIVAN pathogenesis. It is therefore plausible that anti-inflammatory agents may be useful in the treatment of HIVAN. However, since patients with HIV/AIDS already have suppressed immune systems, use of immunosuppressive medications may expose these patients to excess risk of infection and/or malignancy.

In the early 1990s, case reports demonstrated improvements in proteinuria and renal function in patients with HIVAN who were treated with steroids for other indications. In 1996, a prospective case series of 20 consecutive patients with mostly biopsy-proven HIVAN (17 of 20) who were treated with oral prednisone was documented. Patients were treated with 60 mg/day for varying periods of time followed by a tapering course. Seventeen of the patients experienced a decrease in their serum creatinines from an average of 8.1 mg/dL to 3.0 mg/dL. Five patients who had an increase in their creatinine after cessation of treatment were then retreated. These patients again responded to the prednisone with subsequent decreases in serum creatinine measurements. There was, however, no control group in this study and six patients developed serious opportunistic infections while being treated with prednisone. Several other retrospective studies have also found a striking association between steroid treatment and improved renal outcomes in patients with HIVAN.

A beneficial effect of steroid treatment upon tubulointerstitial inflammation is suggested by a case report of a patient with biopsy-proven HIVAN who was rebiopsied when his creatinine began to increase as steroids were being weaned. The repeat biopsy revealed marked reductions in interstitial lymphocytes and macrophages after the steroid treatment, suggesting that prednisone reduced the interstitial inflammation in this patient.

Since there are still no prospective controlled studies demonstrating a benefit of corticosteroids in patients with HIVAN, we believe that they should be considered for use only as a short-term adjuvant while antiretroviral medications and ACE inhibitors are being titrated or in cases in which antiretroviral therapy and angiotensin blockade have failed.

Hepatitis C Virus-Associated Renal Diseases

Essentials of Diagnosis

• The presence of circulating hepatitis C viral RNA and antibody.
• Type I membranoproliferative glomerulonephritis.
• Circulating type II mixed cryoglobulins [polyclonal immunoglobulin (Ig)G and monoclonal IgMκ rheumatoid factor].
• Strong association with hepatitis C virus (HCV) infection chronicity.
• Very low serum C4, C1q, and CH50, but normal C3 levels.

General Considerations

Historic Perspective

The clinical manifestations associated with “mixed” cryoglobulinemia were first described in 1966. They included a constellation of clinical features that consisted of the triad of palpable purpura, arthralgias, and weakness plus variable degrees of glomerulonephritis, lymphadenopathy, and hepatosplenomegaly in some patients. Cryoglobulinemia in these patients had a “mixed” composition of IgG and IgM rheumatoid factor (RF). The cause of this disease was unknown in those days, and its association with hepatitis C virus (HCV) infection became increasingly apparent only after its discovery in 1989. It is now apparent that the classic clinical triad occurs in a minority of patients and that the main characteristic of the disease is the markedly heterogeneous manifestations of a systemic vasculitis, with purpuric skin lesions that show leukocytoclastic vasculitis on biopsy being an almost constant and predominant feature.

Hepatitis C Virus Virology

HCV is a small RNA virus that is included in the Flaviviridae family and has recently been classified as the sole member of the genus Hepacivirus. HCV is a small double-shelled virus consisting of a lipid envelope (E) with virally encoded glycoproteins (E1 and E2) and an inner nucleocapsid (core) that contains a positive-sense single-stranded RNA genome of 9500 nucleotides. It has well-defined structural (core, E1, and E2) as well as several nonstructural (from NS2 to NS5) proteins. The nonstructural proteins encode several proteases, a virus-specific helicase, and an RNA-dependent RNA polymerase responsible for replication of the genome. The evolution of HCV has been characterized by the emergence of six major genotypes based on sequence homology, and more than 50 subtypes.

Epidemiology

To date, around 170–200 million individuals worldwide are estimated by the World Health Organization to have chronic HCV infection. Although HCV infection appears to be primarily a disease that is almost exclusively confined to the liver, a wide variety of extrahepatic disease manifestations have been reported to be associated with HCV infection. The prevalence of extrahepatic diseases is not known with certainty, but it suggests that HCV is involved in nonhepatic pathologic processes.

Two immunologic features of HCV may predispose patients to manifestations of extrahepatic disease. First, HCV is known to evade immune elimination and lead to chronic infection and accumulation of circulating immune complexes. Membranoproliferative glomerulonephritis (MPGN) associated with HCV infection may be the result of this phenomenon. The second feature is that HCV stimulates production of monoclonal rheumatoid factors (mRF). This feature causes type II cryoglobulinemia that is responsible for most of the symptomatic cryoglobulinemic vasculitis. Although this manifestation occurs relatively infrequently, as do all the extrahepatic disease manifestations, it is an important extrahepatic involvement of chronic HCV infection responsible for much of the increased morbidity and mortality accompanying the disease.

The prevalence of mixed cryoglobulinemia increases with the duration of the hepatitis. Patients with chronic hepatitis C who have mixed cryoglobulinemia have an apparent duration of disease that is almost twice as long as those without cryoglobulinemia. A high prevalence of mixed cryoglobulinemia (35–90%) has been reported for patients with HCV infection. However, the prevalence of mixed cryoglobulinemia has not been assessed in populations of unselected HCV-infected patients. Hence, reports of high prevalence of mixed cryoglobulinemia may be influenced by selection bias, eg, studies on cirrhotic patients with long-standing HCV infection from gastroenterology centers. Frank symptomatic cryoglobulinemia occurs in 1% or less of patients and is usually associated with high levels of RF and cryoglobulins. Testing unselected patients with cryoglobulinemia has shown that up to 90% have anti-HCV antibody. Type I MPGN has long been regarded as idiopathic, but a considerable proportion of patients has concomitant chronic infection with HCV. The exact proportion of patients with type I MPGN who are anti-HCV-antibody positive is unknown.

The most frequent form of renal involvement in HCV infection is MPGN, described mainly in the United States and Japan. The real prevalence of MPGN without detectable cryoglobulinemia is difficult to assess. Such cases might represent a subclinical form of cryoglobulinemia because of failure to detect circulating cryoglobulins by standard laboratory techniques or inadequate methods. In addition, the production of IgM antibodies with anti-IgG activity might induce immune complexes without cryoprecipitable properties. Finally, these patients may develop detectable circulating cryoglobulinemia only later in the course of the disease.

Pathogenesis

Cryoglobulinemia is defined as the presence in serum of immunoglobulins that precipitate at reduced temperatures (Figure 35–4). Therefore, blood samples obtained from patients for detection of cryoglobulins must be stored and transported at 37°C.

Cryoglobulins were classified based on their Ig composition, and three types were defined. Type I consists of a single monoclonal Ig without antibody activity, and can be found in patients with multiple myeloma, Waldenström's macroglobulinemia, or idiopathic monoclonal gammopathy. Types II and III, or mixed cryoglobulins, consist of polyclonal IgG and monoclonal IgMκ (type II) or polyclonal IgM (type III) with RF activity (Table 35–1). When no definite disease association is found, the condition is referred to as essential mixed cryoglobulinemia. The observation that up to 90% of unselected patients with cryoglobulinemia have anti-HCV antibody indicates that the disease is not genuinely “essential,” but more likely is related to HCV infection. Hence, the term “essential” may be a misnomer and can no longer be used for the majority of cases, where cryoglobulins consist of complexes of RF, IgG, anti-HCV antibody, and HCV virions. The pathogenesis of cryoglobulinemia due to HCV infection is not well understood, but it appears to be related to excessive proliferation of B cells as a result of the chronic antigenic stimulation of HCV infection.

Clinical Findings

Symptoms and Signs

Hepatitis C Virus-Related Cryoglobulinemia

Full-blown symptomatic cryoglobulinemia occurs infrequently, and the typical symptoms are fatigue and palpable purpura, which histologically consists of a leukocytoclastic vasculitis (with complexes of anti-HCV antibody and HCV in injured tissue). These lesions are usually found on the lower limbs (Figure 35–5), although they can occur anywhere, and represent small vessel vasculitis. A smaller proportion of patients has fever, arthritis, Raynaud's phenomenon, and neuropathy. Peripheral neuropathy is usually characterized by paresthesias and variable degrees of motor deficits. Abdominal pain arises from mesenteric vasculitis, and may mimic an acute abdomen during disease flare. Hepatosplenomegaly is due to chronic liver disease as a result of HCV. Cryoglobulinemia is more common in women than men and typically occurs after a prolonged period, often years or decades, of HCV infection. Although the course of illness tends to wax and wane, occasionally the systemic illness can be severe or even fulminant. For instance, nodular pulmonary infiltrates from deposition of cryoglobulins leading to respiratory failure (Figure 35–6) and non-Hodgkin's B cell and splenic lymphomas have been reported to arise in the setting of cryoglobulinemia. In addition, cryoglobulinemia has also been anecdotally reported in association with adenocarcinoma of the liver and stomach in Chinese.

Cryoglobulinemic Glomerulonephritis

The principal renal manifestation of HCV infection is MPGN type I, usually in the context of cryoglobulinemia. Type II MPGN (eg, dense deposit disease) has not been described in association with HCV infection. From studies in Italy, the United States, and Japan, MPGN associated with type II cryoglobulinemia is the predominant type of glomerulonephritis clinically associated with HCV infection. The prevalence of MPGN in HCV type II cryoglobulinemia is approximately 30%. On the other hand, the prevalence of anti-HCV antibody among patients with MPGN is much lower in Chinese people. MPGN is also occasionally observed in patients with hepatitis C in the absence of cryoglobulinemia.

Renal disease is rare in children and the typical age of disease onset is in the fifth or sixth decade of life after long-standing infection, often in association with mild subclinical liver disease. Clinically, patients may have other symptoms of cryoglobulinemia, such as palpable purpura and arthralgias. Renal manifestations include nephrotic (20%) or nonnephrotic proteinuria and microscopic hematuria. Acute nephritic syndrome is the presenting feature in about 25% of cases. Progression to uremia is associated with male gender and old age. Renal insufficiency, frequently mild, occurs in about 50% of patients. Over 80% of patients have refractory hypertension upon presentation, which may be responsible for a considerable number of cardiovascular deaths.

The natural history of HCV-related cryoglobulinemia remains poorly defined. The clinical course can vary dramatically. The renal disease tends to have an indolent course and does not progress to uremia despite the persistence of urine abnormalities in the majority of patients. Around 15% of patients eventually require dialysis according to an Italian series.

Laboratory Findings

Laboratory testing coupled with renal biopsy establishes the diagnosis of HCV-related MPGN. Most patients will have anti-HCV antibody, as well as HCV RNA, in serum. Serum transaminase levels are elevated in 70% of patients. Cryoglobulins are detected in 50–70% of patients. Serum electrophoresis and immunofixation reveal type II mixed cryoglobulins (Figure 35–4B), in which the monoclonal rheumatoid factor, almost invariably an IgMκ, is a distinguishing feature of cryoglobulinemic glomerulonephritis. Their amount, usually measured as a cryocrit, varies from one patient to another, and varies from time to time in a given patient (ranging between 2% and 70%). Urine κ light chains are also commonly present. The serum complement pattern, which does not change much with clinical activity, is also discriminative. Characteristically, the early complement components (C4 and C1q) and CH50 are at very low, or even undetectable, levels, while the C3 level tends to remain normal or only slightly depressed.

Renal histologic evaluation typically shows evidence of immune complex deposition in glomeruli and changes of MPGN. MPGN refers to a pattern of glomerular injury characterized by diffuse mesangial proliferation and thickening of the capillary wall, hence the synonym of mesangiocapillary glomerulonephritis (Table 35–2). In cryoglobulinemic MPGN, light microscopy reveals an increased number of mesangial cells, expansion of the mesangial matrix, and diffuse accentuation of glomerular tufts, which gives a lobular appearance to the glomeruli (Figure 35–7A). Glomerular capillary walls appear thickened because of the interposition of the mesangial matrix between the glomerular basement membrane (GBM) and the endothelium. Staining of the GBM with periodic acid–Schiff or silver stain shows splitting (“double contour”) or “tram-tracking” due to insertion of the mesangial matrix (Figure 35–7B). Immunofluorescence reveals granular deposits of C3 and IgG in the mesangium and in peripheral capillary loops (Figure 35–7C). A similar morphologic appearance may be seen with infective endocarditis and infected ventriculoatrial shunts (shunt nephritis). In addition, glomerular capillaries may have marked inflammatory cell infiltrates with both mononuclear cells and polymorphonuclear leukocytes (Figure 35–7A), a distinguishing feature from noncryoglobulinemic MPGN. Intracapillary globular accumulations of eosinophilic material representing precipitated immune complexes or cryoglobulins may also be present. Viral HCV-containing antigens had previously been detected in glomerular structures using a three-stage indirect immunohistochemical monoclonal antibody technique but this has not been confirmed by subsequent studies. Electron microscopy shows subendothelial deposits (Figure 35–7D) that may have a tactoid pattern, size, and distribution (Figure 35–7E), suggestive of cryoglobulin deposition. These tend to be of 15–30 μm in size, distinguishing them from the smaller fibrillary deposits (12–25 μm). The presence of immunotactoid glomerulonephritis in a viral disease confirms the association of immunotactoid glomerulonephritis with a systemic disorder, while fibrillary glomerulonephritis is more frequently a “primary” condition. Fibrillogenesis may be favored by circulating paraproteins interacting with matrix proteins in the glomerulus, such as fibronectin. The animal model of membranoproliferative glomerulonephritis derived from induction of mixed cryoglobulinemia strongly suggests a pathogenetic role of cryoglobulins rather than a direct etiologic role of HCV infection. Of interest, however, and again in animal models, both rheumatoid factor and cryoglobulinemic properties may be necessary for the development of skin vasculitis, but cryoglobulin activity alone is sufficient to induce glomerular lesions.

Other forms of glomerular injury have been associated with HCV infection in individual case reports and small series, including membranous glomerulonephritis, IgA nephropathy, focal and segmental glomerulosclerosis, fibrillary glomerulonephritis, immunotactoid glomerulopathy, rapidly progressive glomerulonephritis, exudative–proliferative glomerulonephritis, and lupus nephritis. Membranous nephropathy in HCV carriers is characterized by the absence of cryoglobulins and male predominance.

Treatment

In general, therapy can be directed at two levels: (1) Removal of cryoglobulins by plasmapheresis and (2) inhibition of their synthesis through either attenuation of the immune responses (using corticosteroid or cytotoxic agents) or suppression of viral replication (using interferon and ribavirin).

Before the association between HCV and cryoglobulinemic MPGN was unraveled, corticosteroid and cyclophosphamide were the mainstay of treatment. High-dose pulse methylprednisolone (1 g/day for 3 consecutive days), followed by oral steroids, was used to control the systemic illness. Plasmapheresis may be applied to remove circulating cryoglobulins, thus preventing their deposition in glomeruli and blood vessel walls. Cyclophosphamide ameliorates the vasculitic injury and inhibits the production of monoclonal rheumatoid factors by B-lymphocytes.

Our current understanding of the association between mixed cryoglobulinemia and HCV infection has resulted in a more rational approach to the treatment of this condition. Controlled trials have shown that antiviral therapy with interferon-α is associated with improvements in systemic symptoms of immune complex disease. However, relapse after therapy occurs in a large proportion of patients, particularly with interferon monotherapy given for short durations. Combination therapy with interferon-α2b plus ribavirin represented an important milestone in the treatment of chronic hepatitis C and acute hepatitis C after renal transplantation. Such cocktail therapy has also produced favorable results in mixed cryoglobulinemia, although nonresponses and relapses after initial improvements still occur. In some instances in which sustained viral eradication was unsuccessful, long-term maintenance interferon therapy has led to amelioration of disease. The introduction of pegylated forms of interferon (peginterferon) in 2000 represented another breakthrough in the treatment of chronic hepatitis C. Pegylation refers to the covalent attachment of a large inert molecule of polyethylene glycol (PEG) to a protein to yield a molecule that retains biological activity, but has delayed absorption and clearance, allowing for weekly rather than daily or three times a week administration. Delayed clearance also led to greater, more potent, and longer lasting antiviral effects. Recent data on peginterferon and ribavirin combination therapy in treating HCV infection are encouraging. Furthermore, the higher treatment failure rate of HCV carriers with genotype 1 is recognized.

Despite reports that antiviral therapy can occasionally be associated with the worsening of renal disease or a variable response, there are increasing observational studies suggesting the effectiveness of peginterferon and ribavirin combination therapy in treating HCV-associated cryoglobulinemic membranoproliferative glomerulonephritis. One therapeutic drawback lies in the hemolytic effect that complicates ribavirin therapy, particularly in patients with functional renal impairment. This therapeutic difficulty has been overcome by adjusting the dose according to the glomerular filtration rate instead of body weight alone and utilizing recombinant erythropoietin to overcome anemia. It is necessary to follow ribavirin serum levels when using this approach. Posttreatment renal biopsy showed histologic improvement in two of the three patients who received combination therapy for 12 months. In another study in which the viral genotypes were documented, genotype 1 was again associated with a lower sustained virologic response rate even with combined interferon-α and ribavirin therapy. In severe acute flares of cryoglobulinemia with glomerulonephritis or vasculitis, an appropriate approach is to include corticosteroids and cyclophosphamide as needed to control severe cryoglobulinemic symptoms in addition to combination antiviral therapy. In the most severe cases, plasmapheresis (three to four times weekly exchanges of 3 L of plasma for 2–3 weeks) can be helpful. For refractory cases, monoclonal antibody against the B cell surface antigen CD20 (Rituximab) has been reported to be efficacious with a favorable side-effect profile.

Hepatitis B Virus-Associated Renal Diseases

Essentials of Diagnosis

• Membranous nephropathy is the most frequent association.
• The presence of circulating hepatitis B virus (HBV) or DNA.
• The presence of HBV-specific antigen(s) or viral genome in the glomerulus.
• Serum C3 and C4 levels may be low in 20–50%.

General Considerations

Historic Perspective

Following the landmark discovery in 1965 of the Australian antigen, subsequently renamed the hepatitis B surface antigen (HBsAg), the occurrence of membranous nephropathy (MN) due to glomerular deposition of Australian antigen-containing immune complexes was described in a 53-year-old man in 1971. Different histologic types of glomerular lesions have since been described in association with HBV carriage; however, the most striking is still MN.

Hepatitis B Virus Virology

HBV is a hepatotropic, double-stranded DNA virus belonging to the family Hepadnaviridae. HBV has a double-shelled virion 42–47 nm in diameter, a 27-nm internal core, an excess of incomplete 22-nm spheres, and a circular DNA, with a length varying between 3000 and 3300 base pairs. The DNA genome contains only four genes that encode viral proteins. These include the surface (S) gene, which encodes the three forms of HBsAg, the precore/core (PC/C) gene, which encodes the core protein and hepatitis B e antigen (HBeAg), the X gene, which encodes the X protein, and the polymerase (P) gene, which encodes the viral DNA polymerase. HBV is itself not cytopathic; hepatitis develops as a result of the host's immune reaction toward infected hepatocytes. HBV utilizes a replication strategy closely related to retroviruses, in that transcription of RNA into DNA is a critical step. Unlike retroviruses, HBV DNA is not integrated into host cell DNA during replication. After an HBV particle binds to and enters a hepatocyte, HBV DNA enters the cell nucleus and is converted into covalently closed circular DNA (cccDNA), which is highly stable acting as the intermediate template for transcription of RNA copies. This pregenomic mRNA is transported to the cytoplasm and has the dual functions of acting as a template for synthesis of new HBV DNA and carrying genetic information to direct the synthesis of viral proteins.

Epidemiology

An estimated 350–400 million people worldwide are now infected with HBV. The reported prevalence of HBV-associated nephropathy, particularly MN, closely parallels the geographic patterns of prevalence of HBV. HBV infection occurs throughout the world and is endemic in developing countries, such as Africa, Eastern Europe, the Middle East, Central Asia, China, Southeast Asia, the Pacific Islands, and the Amazon basin of South America (with prevalence rates up to 10% or higher).

In endemic areas, transmission is usually vertical from infected mother to child. Horizontal transmission occurs via direct contact with blood (as in blood transfusions) or mucous membranes (as in sexual contacts), or via the percutaneous route upon contact with blood or body fluids (as in illicit intravenous drug use and needle-sharing practices).

Pathogenesis

The only definitive means to prove that a particular glomerulopathy is etiologically associated with HBV infection is to fulfill the following criteria:

1. The presence of circulating HBV antigen or DNA and HBV-specific antigen(s) or the viral genome in the glomerulus or viral particles identified on ultrastructural examination.

2. The absence of other causes of renal disease.

3. Regression of the pathologic lesion with viral eradication.

4. Reproducibility of the pathology in animal models infected with the virus.

Observations from chronic hepatitis virus infection in woodchucks revealed three types of glomerulonephritis, namely, membranous nephropathy with HBcAg deposits, mesangial proliferative glomerulonephritis with mesangial deposits of HBsAg, and mixed membranous and mesangial proliferative glomerulonephritis with capillary deposits of HBcAg and mesangial deposits of HBsAg. The natural animal model of woodchuck hepatitis reveals pathologic findings similar to those of humans. HBV-associated membranous nephropathy is particularly frequent in male children. Mesangial proliferative forms with IgA deposits appear to be more common in adults. In the woodchuck, the membranous pattern of injury appeared more frequently in the young, whereas the mesangial proliferative pattern of injury tended to appear in older animals. Nevertheless, in humans, it is difficult to rule out the chance occurrence of two common disorders (eg, HBV and IgA nephropathy) presenting in the older population in an endemic area as a cause of this association. The male/female ratio of affected woodchucks was significantly greater than that of the chronic carrier population. One major difference is that the HBeAg system has not been characterized in woodchucks. In clinical practice, regression of the pathology with viral eradication is not easily demonstrable because of ethical concerns involving repeat renal biopsies in human subjects after clinical remission. Hence the diagnosis of HBV-associated renal disease in reality relies heavily upon the demonstration of HBV-specific antigen(s) in the glomeruli.

Clinical Findings

Symptoms and Signs

Pediatric and adult patients tend to have slightly different clinical manifestations of HBV-related MN. In children, there is a strong male preponderance, and the most frequent presentation is nephrotic syndrome together with microscopic hematuria and normal or mildly impaired renal function. Pediatric chronic HBV carriers often do not have overt liver disease, and transaminase levels are usually normal. In adults, proteinuria and the nephrotic syndrome are the most common manifestations, though male predominance is less obvious than that observed in children. In addition, adults are more likely than children to have hypertension, renal dysfunction, and clinical evidence of liver disease.

The prognosis of HBV-associated MN in children is favorable with stable renal function and high rates of spontaneous remission reported in several high prevalence areas, including Hong Kong, South Africa, and Turkey. On the other hand, adults with HBV-associated MN typically develop progressive disease. In Hong Kong, up to 29% of patients had progressive renal failure, and another 10% developed ESRD over 5 years. The prognosis is even worse in patients with nephrotic-range proteinuria and overt hepatitis at presentation, with over 50% of patients requiring renal replacement therapy over 3 years.

Laboratory Findings

Laboratory tests to be followed for diagnostic purposes and also to assess response to treatment include standard liver biochemistries (serum alanine aminotransferase, γ-glutamyltransferase, and bilirubin levels) and HBV serologies (HBsAg, HBeAg, anti-HBe, and anti-HBc antibodies). HBeAg is present in 80% of patients, who may also have high titers of anti-HBc. Subjects with biochemical hepatitis should also be tested for circulating HBV DNA levels and should undergo liver biopsy. In addition, α-fetoprotein assay could be an important adjunct. Serum C3 and C4 levels may be low in 20–50% of patients.

Light microscopic findings are similar to that of idiopathic MN, with some differentiating features. The characteristic glomerular lesion is a diffuse thickening of the glomerular capillary walls to form thick “membranes” (Figure 35–8A). It is now firmly established that this alteration is caused by immune complexes that accumulate subepithelially on the outer aspect of the GBM, which assumes a “membranous” morphology in a stepwise manner. Other pertinent light microscopic findings are reflected in the reactive structural changes of the GBM induced by immune complexes. Therefore special stains highlighting the GBM, such as methenamine silver and periodic acid–Schiff (PASM or silver stain) or trichrome stain, are more useful (Figure 35–8B). The earliest change on silver staining is a mottled appearance best seen on tangential sections and represents slight indentations of the GBM by immune complexes adhering to its surface. The most specific change of the GBM is the so-called “spike” formation (Figure 35–8C). These are projections of GBM material between immune complexes that lead to a saw tooth-like appearance of the GBM. This pattern is pathognomonic of full-blown membranous glomerulonephropathy. Disease progression results in a diffuse thickening of the GBM. The major constituents of the immune complexes are IgG together with C3. IgM, IgA, and C1q may be present. Ultrastructural findings typically consist of both subepithelial and occasional subendothelial deposits. The presence of subendothelial deposits, sometimes referred to as membranoproliferative glomerulonephritis type III changes, favors a secondary case of MN such as HBV associated rather than idiopathic. The presence of mesangial proliferation on light microscopy is helpful in distinguishing this form of secondary MN from idiopathic MN.

Apart from MN, other renal pathologies have also been associated with HBV infection. These include MPGN with or without cryoglobulinemia, mesangial proliferative glomerulonephritis, and IgA nephropathy. Polyarteritis nodosa has also been reported in some patients with HBV and may respond to corticosteroids and interferon-α therapy. Occasionally, overlapping of these pathologic forms leading to double glomerulopathies may be seen. For instance, MN and IgAN have been reported to coexist in an HBV carrier.

Regardless of the pathologic finding, it is important to localize HBV-specific antigens in the biopsy. To document an etiologic association between HBV and MN or other forms of glomerular lesion, the demonstration of HBV-specific antigens by immunofluorescence is indispensable. The three major antigens are HBsAg, HBeAg, and hepatitis B core antigen (HBcAg). Monoclonal antibodies recognize a single antigenic epitope and are in general less sensitive than polyclonal antibodies that bind to more than one epitope. Commercial polyclonal anti-HBc preparations cross-react with both anti-HBc and anti-HBe, as HBeAg is an integral component of HBcAg. HBsAg is characteristically localized in the mesangium while HBeAg is found in the capillary loop. Furthermore, HBV DNA and mRNA have been detected in the glomerulus and tubular epithelia by polymerase chain reaction and in situ hybridization with specific HBV RNA probes.

Treatment

Unlike childhood disease in which there is a high rate of spontaneous remission, adults with HBV-associated MN typically develop progressive disease. Various strategies have been tried, although an ideal agent has yet to be found. Treatment for HBV-associated renal disease should ideally achieve the following objectives:

1. Amelioration of nephrotic syndrome and its complications, such as hyperlipidemia, edema, infection, and venous thrombosis.

2. Preservation of renal function.

3. Normalization of liver function and prevention of hepatic complications of HBV.

4. Permanent eradication of HBV.

In view of the immune complex nature of the disease, immunosuppressive therapy, similar to that applied in the idiopathic form of the disease, was once fashionable. Although it has previously been reported that corticosteroids achieve symptomatic relief in isolated cases, the contemporary view is that steroids and cytotoxic agents may cause deleterious hepatic flares or even fatal decompensation by enhancing viral replication upon treatment withdrawal.

Another approach is treatment with an antiviral agent. Interferon-α is a naturally occurring cytokine produced by B-lymphocytes, null lymphocytes, and macrophages, and possesses antiviral, antiproliferative, and immunomodulatory effects. While reported to be useful in children, interferon-α has produced mixed results in adults with HBV-associated MN.

The introduction of the nucleoside analog lamivudine has revolutionized the treatment of chronic HBV infection. Lamivudine is the (–)-enantiomer of 3′-thiacytidine, and it inhibits DNA synthesis by terminating the nascent proviral DNA chain through interference with the reverse transcriptase activity of HBV. In children and adults with HBV-associated MN, lamivudine has been anecdotally reported to induce remission of nephrotic syndrome and suppress viral replication. In our analysis of 10 adult nephrotic patients with HBV-related MN who received lamivudine treatment versus 12 matched historic control subjects who presented in the prelamivudine era, lamivudine treatment significantly improved proteinuria, ALT levels, and renal outcome over a 3-year period (Figure 35–9). Randomized studies in a larger cohort of patients are needed. A potential limitation of prolonged treatment with lamivudine is the emergence of drug-resistant strains due to the induction and selection of HBV variants with mutations at the YMDD motif of DNA polymerase. One agent that might be considered in case of lamivudine resistance is adefovir dipivoxil, an acyclic nucleotide analogue that is effective against both lamivudine-resistant HBV mutants as well as wild-type HBV. However, this agent is potentially nephrotoxic and there are no clinical data on its efficacy in HBV-related MN that does not respond to lamivudine treatment. There are data to suggest that the recommended dose of 10 mg adefovir dipivoxil is associated with a lower risk of nephrotoxicity.

In the absence of an ideal agent for treatment of HBV-associated glomerulopathy, active immunization remains the most effective measure of immunoprophylaxis. Vaccination for all newborns in some endemic areas such as Hong Kong has dramatically reduced the incidence of chronic HBV infection and its associated complications in children and adolescents. In Taiwan, the introduction of active immunization to all newborns since 1984 has led to a dramatic (10-fold) decline in the incidence of neonatal HBV infection and its subsequent sequelae. In the United States, universal vaccination of infants began in 1991, and a 67% reduction in HBV infection was recorded 10 years later. In 2003, the World Health Organization recommended that all countries provide universal HBV immunization programs for infants and adolescents.